West Coast Worm Meeting Abstracts - Caenorhabditis elegans ...
West Coast Worm Meeting Abstracts - Caenorhabditis elegans ...
West Coast Worm Meeting Abstracts - Caenorhabditis elegans ...
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<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> <strong>Abstracts</strong><br />
1. Meiotic Pairing and Synapsis [p 11]<br />
Amy MacQueen, Anne M. Villeneuve<br />
2. Conditional mitotic spindle mutants in C. <strong>elegans</strong> [p 12]<br />
Danielle R. Hamill, Bruce Bowerman<br />
3. Mitotic Chromosome Segregation by a Conserved Protein Complex [p 13]<br />
K. Hagstrom, R. Chan, D. Pasqualone, B.J. Meyer<br />
4. HIM-10 a Probable Kinetochore Protein Involved in Mitotic and Meiotic Chromosome Segregation [p<br />
14] M. Howe, D G. Albertson, B. J. Meyer<br />
5. A C. <strong>elegans</strong> chromokinesin required for chromosome segregation [p 15]<br />
Jim Powers, Bill Saxton, Susan Strome<br />
6. Nuclear Envelope Dynamics in <strong>Caenorhabditis</strong> <strong>elegans</strong> [p 16]<br />
Kenneth Lee, Yosef Gruenbaum, Katherine L. Wilson<br />
7. The formin protein CYK-1 acts in parallel to an aurora-like kinase/MKLP-1 pathway to execute<br />
cytokinesis in early <strong>Caenorhabditis</strong> <strong>elegans</strong> embryos [p 17]<br />
Aaron F. Severson, Danielle R. Hamill, Bruce Bowerman<br />
8. The L type Cyclin SAG-4 is required for heat-shock induced protein expression [p 18]<br />
Wen J. Chen, Yvonne M. Hajdu-Cronin, Paul W. Sternberg<br />
9. cdl-1 encodes a stem- loop binding protein (SLBP) homolog and may be essential for core histone<br />
expression. [p 19]<br />
Yuki Kodama, Asako Sugimoto, Joel Rothman, Masayuki Yamamoto<br />
10. UNC-23 is a member of the BAG family of chaperone regulators [p 20]<br />
Poupak Rahmani, Donald Moerman<br />
11. Maternal UNC-45 protein co-localizes with NMY-2, a non-muscle myosin at the cleavage furrow of<br />
early embryos [p 21]<br />
Wanyuan Ao, Dave Pilgrim<br />
12. Polyunsaturated fatty acids requirements for proper functioning of the nervous system [p 22]<br />
Jenny Watts, John Browse<br />
13. Testing functions of phagocytosis receptor homologs in cell corpse elimination and gonadal outgrowth<br />
[p 23]<br />
Sambath Chung, Monica Driscoll<br />
14. Regulation of Cell Fusion in C. <strong>elegans</strong> [p 24]<br />
Scott Alper, Cynthia Kenyon<br />
15. Ethanol sensitivity genes in <strong>Caenorhabditis</strong> <strong>elegans</strong> [p 25]<br />
MinGi Hong, JaeYoung Kwon, InYoung Lee, MinSung Choi, Junho Lee<br />
16. State-dependent learning in C. <strong>elegans</strong>. [p 26]<br />
Jill C. Bettinger, Steven L. McIntire<br />
17. The ut236 mutant in C. <strong>elegans</strong> has defects in the interaction of two sensory signals and an<br />
associative learning. [p 27]<br />
Takeshi Ishihara, Yuichi Iino, Isao Katsura<br />
18. Mutation in the LIM homeobox gene lim-6 disrupts asymmetric function of the ASE chemosensory<br />
neurons [p 28]<br />
J.T. Pierce-Shimomura, M.R. Gaston, B.J. Pearson, S.R. Lockery<br />
19. Information Coding in the C. <strong>elegans</strong> Olfactory System [p 29]<br />
PD Wes, A Sagasti, G Jansen, RHA Plasterk, CI Bargmann<br />
20. Roles of osm-9/capsaicin receptor family members in sensory behaviors [p 30]<br />
D. Tobin, D. Madsen, G. Moulder, R. Barstead, A.V. Maricq, M. deBono, C. Bargmann<br />
21. Execution and regulation of male C. <strong>elegans</strong> spicule muscle contractions during mating [p 31]<br />
L. René García, Paul W. Sternberg<br />
22. Genetic Analysis of Nicotine Adaptation in C. <strong>elegans</strong>. [p 32]<br />
Jinah Kim, Laura E. Waggoner, Kari A. Dickinson, Daniel S. Poole, William R. Schafer<br />
23. Neural control of locomotion in C. <strong>elegans</strong> [p 33]<br />
Saleem Mukhtar, Jane Mendel, Jehoshua (Shuki) Bruck, Paul W. Sternberg<br />
24. Analysis of glutamatergic neurotransmission by knockout of glutamate transporter genes. [p 34]<br />
Itzhak Mano, Monica Driscoll<br />
25. Electrophysiological analysis of C. <strong>elegans</strong> ionotropic glutamate receptors [p 35]<br />
Jerry E. Mellem, Penelope J. Brockie, David M. Madsen, Andres V. Maricq<br />
26. Electrophysiological analysis of unc-18 mutants. [p 36]<br />
J. E. Richmond, R. Weimer, W. S. Davis, E. M. Jorgensen<br />
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<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
27. Electrophysiological analysis of serotonin modulation of body wall neuromuscular physiology. [p 37]<br />
Jon Madison, Joshua Kaplan<br />
28. Serotonin signaling in the pharynx [p 38]<br />
Timothy Niacaris, Leon Avery<br />
29. A Muscarinic Contribution to the Regulation of Feeding [p 39]<br />
Kate Steger, Leon Avery<br />
30. <strong>Worm</strong>Base: From ACeDB to a more complete and usable database [p 40]<br />
Paul W. Sternberg, Erich Schwarz, Norma Foltz, <strong>Worm</strong>Base Consortium<br />
31. The C. <strong>elegans</strong> ORFeome project [p 41]<br />
Jerome Reboul, Philippe Vaglio, Cindy Jackson, Troy Moore, Jean Thierry-Mieg, Danielle<br />
Thierry-Mieg, Jim Hartley, Gary Temple, Mike Brasch, Nia Tzellas, Marc Vidal<br />
32. Analysis of splicing and regulatory elements using the Intronerator [p 42]<br />
W. James Kent, Alan M. Zahler<br />
33. A global profile of germ line gene expression using microarrays reveals germ line-specific regulation<br />
of the X chromosome in males and hermaphrodites [p 43]<br />
Valerie Reinke, Harold E. Smith, Jeremy Nance, Abby F. Dernburg, Anne M. Villeneuve, Samuel<br />
Ward, Stuart K. Kim<br />
34. The promise and peril of genomics: sperm development as model system [p 44]<br />
Harold Smith, Marci Millhouse, Sam Ward<br />
35. Functional Analysis of Chromosome I [p 45]<br />
Andrew Fraser, Ravi Kamath, Peder Zipperlen, Maruxa Martinez-Campos, Julie Ahringer<br />
36. Optimizing the mutagenic properties of the mos1 transposon in C. <strong>elegans</strong> [p 46]<br />
Daniel C. Williams, Jean-Louis Bessereau, Erik M. Jorgensen<br />
37. SNAP-25, a protein implicated genetically in C. <strong>elegans</strong> anesthetic mechanisms, binds the general<br />
anesthetic isoflurane [p 47]<br />
Jason Berilgen, Mike Crowder<br />
38. Happy worms: further characterization of fluoxetine (prozac) resistant mutants [p 48]<br />
Robert K.M. Choy, James H. Thomas<br />
39. unc-43 Ca2+/Calmodulin-dependent kinase II (CaMKII) mutant worms have convulsions in response<br />
to the seizure-inducing drug PTZ [p 49]<br />
Elizabeth M. Newton, James H. Thomas<br />
40. Neurotoxin sensitivity of dopaminergic neurons in C. <strong>elegans</strong>: role of the dopamine transporter and<br />
cell death pathways [p 50]<br />
R. Nass, J. Duerr, , J. Rand, D. M. Miller, R. D. Blakely<br />
41. Gene expression in transgenic C. <strong>elegans</strong> animals expressing the human beta amyloid peptide. [p 51]<br />
Chris Link, Carolyn Johnson, Amy Fluet, Kyle K. Duke, Stuart K. Kim<br />
42. A nematode model for mitochondrial diseases [p 52]<br />
William Y. Tsang, Bernard D. Lemire<br />
43. Bacillus toxin (Bt) susceptibility and resistance in C. <strong>elegans</strong> [p 53]<br />
Lisa Marroquin, Dino Elyassnia, Joel Griffitts, Johanna O’Dell, Jerald Feitelson, Raffi Aroian<br />
44. New dauer genes and pathways [p 54]<br />
Michael Ailion, James H. Thomas<br />
45. Temporal regulation of aging in the nematode C. <strong>elegans</strong> [p 55]<br />
Andrew Dillin, Cynthia Kenyon<br />
46. A longitudinal analysis of adult neurons in C. <strong>elegans</strong> [p 56]<br />
Mark I. Snow, Pamela L. Larsen<br />
47. Germ-line cells that regulate aging in C. <strong>elegans</strong> [p 57]<br />
Nuno Arantes-Oliveira, Javier Apfeld, Cynthia Kenyon<br />
48. A screen for genes that control programmed cell death in the germ line [p 58]<br />
S Milstein, A Gartner, M Hengartner<br />
49. C. <strong>elegans</strong> p53: requirement for radiation-induced programmed cell death, stress resistance, and<br />
normal adult lifespan following diapause. [p 59]<br />
W. Brent Derry, Aaron Putzke, Joel H. Rothman<br />
50. Identification of cell-specific regulators of programmed cell death in C. <strong>elegans</strong>. [p 60]<br />
Shai Shaham, Cori Bargmann<br />
51. Biochemical, structural, and genetic analyses of the activation of programmed cell death [p 61]<br />
Jay Parrish, Betsy Metters, Lin Chen, Ding Xue<br />
52. Analysis of RNA associated with P granules in germ cells of C. <strong>elegans</strong> adults [p 62]<br />
Jennifer A. Schisa, Jason N. Pitt, James R. Priess<br />
53. The splicing Sm proteins colocalize with P granules in germ cells and participate in P granule<br />
localization in the early embryo [p 63]<br />
Scott A. Barbee, Alex L. Lublin, Thomas C. Evans<br />
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<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
54. pod-2 defines a new class of mutants required for antero-posterior asymmetry in the early<br />
<strong>Caenorhabditis</strong> <strong>elegans</strong> embryo [p 64]<br />
Akiko Tagawa, Raffi V. Aroian<br />
55. ooc-5 encodes a putative ATPase required for the reestablishment of asymmetric PAR protein<br />
localization in two-cell embryos [p 65]<br />
Stephen E. Basham, Lesilee S. Rose<br />
56. RIC-8 (Synembryn): A novel regulator of G Protein signaling [p 66]<br />
Kenneth G. Miller, Melanie D. Emerson, John R. McManus, James B. Rand<br />
57. MED-1 AND -2 act at the convergence point of SKN-1 and POS-1 to specify MS and E identity [p 67]<br />
Morris F. Maduro, Regina Broitman-Maduro, Joel H. Rothman<br />
58. Mass spectrometric identification of PLP-1 and its role in mesendoderm specification [p 68]<br />
E. Witze, E. Field, D. Hunt, J.H. Rothman<br />
59. The C. <strong>elegans</strong> NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate<br />
specification [p 69]<br />
Steven Hallam, Emily Singer, David Waring, Yishi Jin<br />
60. Left-right asymmetry in C. <strong>elegans</strong> intestinal organogenesis involves a LIN-12/Notch signaling<br />
pathway [p 70]<br />
Greg J. Hermann, Ben Leung, James R. Priess<br />
61. The pho-1 Gene and Three Kinds of Gut Polarity [p 71]<br />
Tetsunari Fukushige, James D. McGhee<br />
62. A role for dishevelled in asymmetric cell division. [p 72]<br />
Nancy Hawkins, Gregory Ellis, Bruce Bowerman, Gian Garriga<br />
63. rho-1, a target of the exchange factor unc-73, is required for cell migrations during C. <strong>elegans</strong><br />
development [p 73]<br />
Andrew G. Spencer, Christian J. Malone, Satoshi Orita, Min Han<br />
64. PTP-1, a LAR-like receptor protein tyrosine phosphatase, may act in parallel with C. <strong>elegans</strong> Eph<br />
signaling to direct morphogenesis [p 74]<br />
Robert J. Harrington, Michael Gutch, Michael Hengartner, Nicholas Tonks, Andrew Chisholm<br />
65. GEX-2 and GEX-3 define a conserved protein complex required for tissue morphogenesis and cell<br />
migrations in C. <strong>elegans</strong> [p 75]<br />
Martha Soto, Katsuhisa Kasuya, Hiroshi Qadota, Kozo Kaibuchi, Craig C. Mello<br />
66. Pharyngeal extension: the short and the long of it [p 76]<br />
MF Portereiko, SE Mango<br />
67. A VAB-8/UNC-51/UNC-14 complex mediates axon outgrowth [p 77]<br />
Tina Lai, Gian Garriga<br />
68. Cytoskeletal Signalling in Response to the UNC-6 Axonal Attractant [p 78]<br />
Zemer Gitai, Erik Lundquist, Marc Tessier-Lavigne, Cori Bargmann<br />
69. Identifying genes involved in axonal branching in C. <strong>elegans</strong> [p 79]<br />
Joe C. Hao, Marc Tessier-Lavigne, Cornelia I. Bargmann<br />
70. UNC-119 suppresses supernumerary branching in C. <strong>elegans</strong> [p 80]<br />
Karla Knobel, Warren Davis, Michael Bastiani, Erik Jorgensen<br />
71. UNC-119 and axon outgrowth: Toward a mechanism [p 81]<br />
Wayne Materi, Dave Pilgrim<br />
72. Three distinct functions of beta-spectrin (UNC-70) [p 82]<br />
Marc Hammarlund, Warren S. Davis, Erik M. Jorgensen<br />
73. RPM-1, a conserved novel protein, regulates presynaptic terminal formation [p 83]<br />
Xun Huang, Mei Zhen, Bruce Bamber, Yishi Jin<br />
74. A C. <strong>elegans</strong> Inositol 5- Phosphatase Homologue Involved In Inositol 1,4,5-triphosphate Signaling and<br />
Ovulation. [p 84]<br />
Yen Kim Bui, Paul W. Sternberg<br />
75. Mechanisms regulating the timing and specificity of anchor cell attachment to the vulval epithelium [p<br />
85] David R. Sherwood, Paul W. Sternberg<br />
76. Mutations in cyclin E reveal coordination between cell-cycle control and vulval development. [p 86]<br />
David S. Fay, Min Han<br />
77. Novel cell-cell interactions during vulva development in Pristionchus pacificus [p 87]<br />
Benno Jungblut, Ralf J Sommer<br />
78. Cellular and genetic analysis of Gq mediated signaling pathways in C. <strong>elegans</strong> [p 88]<br />
C. A. Bastiani, S. Gharib, P.W. Sternberg, M.I. Simon<br />
79. Calcium/calmodulin-dependent protein Kinase II regulates C. <strong>elegans</strong> locomotion in concert with a<br />
G-protein signaling network [p 89]<br />
Merrilee Robatzek, James H. Thomas<br />
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<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
80. A novel lateral signaling pathway determines asymmetric olfactory neuron fates [p 90]<br />
Alvaro Sagasti, Cori Bargmann<br />
81. The search for dosage compensation complex binding sites on X chromosomes [p 91]<br />
Raymond C. Chan, Tammy F. Wu, Barbara J. Meyer<br />
82. Recognition and Assembly of SDC Protein Complexes onto Specific DNA Target Sites [p 92]<br />
Diana Chu, Heather Dawes, Jason Lieb, Annie Kuo, Barbara J. Meyer<br />
83. The TBP-like Factor CeTLF is Required to Activate RNA Polymerase II Transcription in C. <strong>elegans</strong><br />
Embryos [p 93]<br />
Linda S. Kaltenbach, Susan E. Mango<br />
84. The intracellular domain of the feminising receptor TRA-2A interacts directly with the transcription<br />
factor TRA-1A [p 94]<br />
David H. Lum, P. Kuwabara, D. Zarkower, A.M. Spence<br />
85. chw-1 encodes a novel protein that interacts with pha-4 [p 95]<br />
Michael Horner, Linda Kaltenbach, Susan Mango<br />
86. The UNC-4 homeoprotein and its transcriptional co-repressor UNC-37/Groucho regulate<br />
neurotransmitter vesicles in cholinergic motor neurons [p 96]<br />
Kim Lickteig, Janet Duerr, Dennis Frisby, David Hall, Jim Rand, David Miller<br />
87. The components of sensory cilia in C. <strong>elegans</strong> [p 97]<br />
Peter Swoboda, Kerry Bubb, James H. Thomas<br />
88. In vivo imaging of HSN outgrowth [p 98]<br />
Carolyn E. Adler, Cornelia I. Bargmann<br />
89. Temporal and spatial requirement of sensory cilia in the regulation of worm lifespan [p 99]<br />
Joy Alcedo, Javier Apfeld, Bella Albinder, Jennifer Dorman, Honor Hsin, Bernadine Tsung, Cynthia<br />
Kenyon<br />
90. THE TTX-3 LIM HOMEOBOX GENE IS A CENTRAL REGULATOR OF INTERNEURON CELL FATE<br />
[p 100]<br />
Z. Altun-Gultekin, O. Hobert<br />
91. The heterochronic gene pathway: Regulatory interactions and regulatory outputs. [p 101]<br />
Victor Ambros, Marta Hristova, Rosalind Lee, Eric Moss<br />
92. Genetic and phenotypic characterization of evl-14 and evl-20, genes involved in C. <strong>elegans</strong> vulva<br />
development [p 102]<br />
Igor Antoshechkin, Min Han<br />
93. <strong>Caenorhabditis</strong> <strong>elegans</strong> T05H10.5, a homologue of yeast ubiquitin fusion degradation protein<br />
(UDF-2), is expressed throughout the nervous system and in the gut [p 103]<br />
Wanyuan Ao, Dave Pilgrim<br />
94. Genetic analysis of neuroendocrine controls of fat metabolism in C. <strong>elegans</strong> [p 104]<br />
Kaveh Ashrafi, Gary Ruvkun<br />
95. zig genes and the PVT guidepost neuron [p 105]<br />
Oscar Aurelio, Oliver Hobert<br />
96. Analysis of GABA receptor plasticity in C. <strong>elegans</strong> [p 106]<br />
Bruce A. Bamber, Janet E. Richmond, Pierrette K. Danieu<br />
97. Isolation of suppressors of a dominant synapse defective mutant, syd-5(ju89) [p 107]<br />
Renee Baran, Yishi Jin<br />
98. Cargo recognition by synaptic vesicle kinesin [p 108]<br />
Ewa Bednarek, Erik M. Jorgensen<br />
99. The exp-1 locus may encode a subunit of an excitatory GABA receptor [p 109]<br />
Asim A. Beg, Erik M. Jorgensen<br />
100. The life span gene clk-2 is essential for embryonic development [p 110]<br />
Claire Bènard, Brent McCright, Yue Zhang, Stephanie Felkai, Siegfried Hekimi<br />
101. Characterization of <strong>Caenorhabditis</strong> <strong>elegans</strong> gamma-tubulin in dividing cells and differentiated tissues<br />
[p 111]<br />
Yves Bobinnec, Makoto Fukuda, Eisuke Nishida<br />
102. Does CEH-20, an Exd/Pbx homolog in C. <strong>elegans</strong>, play a role in worm embryogenesis? [p 111]<br />
Q.F. Boese, W.B. Wood<br />
103. A-domain-containing protein family in C. <strong>elegans</strong>. [p 112]<br />
Michael Brannan, Joaquin Muriel, Kathryn Taylor, Gordon Lithgow, Danny Tuckwell<br />
104. Distribution and Regulation of Glutamate Receptors in the Locomotory Control Circuit of C. <strong>elegans</strong>.<br />
[p 113]<br />
Penelope J. Brockie, David M. Madsen, Yi Zheng, Jerry E. Mellem, Andres V. Maricq<br />
105. Mutations That Affect Synaptic Localization Of Glr-1 [p 114]<br />
Michelle Burbea, Joshua M. Kaplan<br />
106. Regulation of C. <strong>elegans</strong> dauer formation by an RNA quality control pathway component [p 115]<br />
J Burgess, JC Labbe, S Hekimi<br />
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<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
107. Synaptic vesicle localization is misregulated in unc-16 mutants [p 116]<br />
DT Byrd, Y Jin<br />
108. The egl-21 gene encodes a carboxypeptidase E, which is required for pro-neuropeptide processing<br />
[p 117]<br />
Tija Carey, Joshua M. Kaplan<br />
109. How are anterior cell migrations guided by mig-13? [p 118]<br />
QueeLim Ch’ng, Cynthia Kenyon<br />
110. New Screens for Negative Regulators of let-23 [p 119]<br />
Monica Chan, Marie Tiongsen, Romel C. Castro, Vanessa Lee, Gregg Jongeward<br />
111. C. <strong>elegans</strong> MRE-11 is required for meiotic recombination and DNA repair but not for the meiotic G2<br />
DNA damage checkpoint [p 120]<br />
Gregory Chin, Anne Villeneuve<br />
112. Suppressor Analysis of Eph/Ephrin Defective Signaling in C. <strong>elegans</strong> [p 121]<br />
Ian Chin-Sang, Julie McCleery, Andrew Chisholm<br />
113. RNAi Screen for Components of the C. <strong>elegans</strong> Meiotic Machinery [p 122]<br />
Mónica Colaiácovo, Gillian Stanfield, Kirthi Reddy, Anne Villeneuve<br />
114. Exploring the role of PINCH/UNC-97 in muscle development and focal adhesion assembly in<br />
<strong>Caenorhabditis</strong> <strong>elegans</strong> and mammalian tissue culture cell lines [p 123]<br />
Shaun Cordes, May Dang-Lawson, Poupak Rahmani, Linda Matsuuchi, Donald G. Moerman<br />
115. The SAD-1 kinase regulates presynaptic vesicle clustering in C. <strong>elegans</strong> [p 124]<br />
Justin Gage Crump, Mei Zhen, Kang Shen, Yishi Jin, Cornelia I. Bargmann<br />
116. Mutants with altered sensitivity to the effects of ethanol on locomotion [p 125]<br />
Andrew G. Davies, Tod R. Thiele, Catharine Eastman, Steven L. McIntire<br />
117. A screen for DD/DV axonal morphology defects [p 126]<br />
M. Wayne Davis, Erik M. Jorgensen<br />
118. spn-2 AND spn-3 FUNCTION TO ORIENT THE SPINDLE DURING EARLY CLEAVAGES [p 127]<br />
Leah R. DeBella, Lesilee S. Rose<br />
119. Molecules acting in parallel with UNC-34 to control cell migration [p 128]<br />
Megan Dell, N Chugh, N Hawkins, E Kong, J Hardin, G Garriga<br />
120. Insights into the role of C. <strong>elegans</strong> protein UNC-119 in axonogenesis [p 129]<br />
Chantal Denholm, Wayne Materi, Daniel Gietz, David Pilgrim<br />
121. The defecation gene aex-1 may regulate a retrograde signaling pathway at neuromusclular<br />
junctions. [p 130]<br />
Motomichi Doi, Kouichi Iwasaki<br />
122. Cosuppression in the Germline: Silencing is Golden [p 131]<br />
Abby F. Dernburg, Mónica P. Colaiácovo, Jonathan Zalevsky, Anne M. Villeneuve<br />
123. sur-9 a Suppressor of Activated let-60(n1046) in the C.<strong>elegans</strong> Vulva. [p 132]<br />
Dennis Eastburn, Min Han<br />
124. Knockouts In C. <strong>elegans</strong>: Madness and Methodology [p 133]<br />
Mark Edgley, Erin Gilchrist, Greg Mullen, Bin Shen, Margaret Kotarska, Don Moerman, Steven<br />
Jones, Anil Dsouza, Gary Moulder, Malini Viswanathan, Martin Lansdale, Robert Barstead<br />
125. Using DNA microarrays to identify targets of homeobox genes in C. <strong>elegans</strong> [p 134]<br />
Andreas Eizinger, Tibor Vellai, Fritz Müller, Stuart K. Kim<br />
126. ded Genes Disrupt Cell Division Timing and Patterning in C. <strong>elegans</strong> Embryos [p 135]<br />
Sandra Encalada, Paula Martin, Jennifer Phillips, Rebecca Lyzcak, Danielle Hamill, Kathryn Swan,<br />
Bruce Bowerman<br />
127. Voltage-dependent currents in homologous chemosensory neurons with different functions in C.<br />
<strong>elegans</strong> [p 136]<br />
S Faumont, S.R. Lockery<br />
128. VAV is required for pharyngeal muscle contraction in C. <strong>elegans</strong> [p 137]<br />
R.T. Fazzio, J.E. Mellem, M.C. Beckerle, A.V. Maricq<br />
129. Regulation of C. <strong>elegans</strong> Body Size by Sensory Cues [p 138]<br />
Manabi Fujiwara, Hoan Phan, Steven L. McIntire<br />
130. Regulation of intracellular dynamics of MAPKAPK2 in living C.<strong>elegans</strong> [p 139]<br />
Makoto Fukuda, Yves Bobinnec, Eisuke Nishida<br />
131. Role of cki-1 in terminal embryonic differentiation and cell-cycle arrest [p 140]<br />
Masamitsu Fukuyama, W. Brent Derry, Joel H. Rothman<br />
132. sax-1 and sax-2 act in parallel with unc-34 to Maintain Neuron Polarity. [p 141]<br />
Maria E. Gallegos, Jennifer A. Zallen, Cori Bargmann<br />
133. Identifying pharyngeal targets of PHA-4 using DNA microarrays [p 142]<br />
Jeb Gaudet, Michael Horner, Stuart Kim, Susan E. Mango<br />
134. An overview of predicted cytochrome P450 genes in C. <strong>elegans</strong> [p 143]<br />
Erin Gilchrist<br />
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<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
135. Clues toward understanding EGF/ Wnt signal integration in the specification of P12 fate: analysis of<br />
the egl-5 promoter [p 144]<br />
Lisa Girard, Henrique B. Ferreira, Scott Emmons, Paul Sternberg<br />
136. spn-4: a gene required for mitotic spindle orientation in the 2-cell stage C. <strong>elegans</strong> embryo [p 145]<br />
José E. Gomes, Kathryn A. Swan, Christopher A. Shelton, Bruce Bowerman<br />
137. Ca2+-signalling via the neuron-specific Ca2+ sensor NCS-1 is essential for thermotaxis, a form of<br />
associative learning and memory in C. <strong>elegans</strong> [p 146]<br />
Marie Gomez, Edouard De Castro, Ernesto Guarin, Patrick Nef<br />
138. Characterizing the Neural Circuitry of Chemotaxis to Volatile Odorants [p 147]<br />
Jesse Gray, Maria Gallegos, Tim Yu, Cori Bargmann<br />
139. Studies on the Nematicidal Bacillus thuringiensis Toxins [p 148]<br />
Joel S. Griffitts, Raffi V. Aroian<br />
140. Synaptic localization of the glutamate-gated chloride channel GBR-2 [p 149]<br />
Maria E. Grunwald, Joshua M. Kaplan<br />
141. Regulation and function of lin-11 in C. <strong>elegans</strong> vulval development [p 150]<br />
Bhagwati P Gupta, Paul W. Sternberg<br />
142. sur-7, a gene that suppresses activated ras [p 151]<br />
Eric Hague, Min Han<br />
143. Characterization and Suppression of eat-16; sag-1/dgk-1 lethality [p 152]<br />
Yvonne M. Hajdu-Cronin, Wen J. Chen, Paul W. Sternberg<br />
144. Improved Tissue Preservation Using Metal Mirror Freezing or High Pressure Freezing for TEM [p<br />
153]<br />
David H. Hall, Frank Macaluso, Gloria Stepheney, Marie-Christine Paupard<br />
145. Role of the <strong>Caenorhabditis</strong> <strong>elegans</strong> homologs of cdk5 and p35 in migration and axon outgrowth [p<br />
154]<br />
Thomas Harbaugh, Gian Garriga<br />
146. Regulation of egg-laying by sensory cues [p 155]<br />
Laura Anne Hardaker, William R. Schafer<br />
147. Characterization of the C. <strong>elegans</strong> Serotonin-Synthetic Aromatic Amino Acid Decarboxylase Gene<br />
bas-1 [p 156]<br />
Emily Hare, Curtis M. Loer<br />
148. XOL-1 Files [p 157]<br />
Christian A. Hassig, Barbara J. Meyer<br />
149. Y41G9a.1, the C. <strong>elegans</strong> Homologue of Tg737, is Expressed in Ciliated Neurons [p 158]<br />
Courtney J. Haycraft, Patrick D. Taulman, Stephen M. Krum, Bradley K. Yoder<br />
150. let-381 is a forkhead gene [p 159]<br />
Marika Hellqvist-Greberg, Ann M Rose, David L Baillie<br />
151. Genetic analysis of dynamic search behavior in C. <strong>elegans</strong> [p 160]<br />
T.T. Hills, F. Adler, A. V. Maricq<br />
152. Multiple roles for the Ras-MAPK signal transduction pathway in chemotaxis to odorants? [p 161]<br />
Takaaki Hirotsu, Satoshi Saeki, Yuichi Iino<br />
153. mab-26 encodes the C. <strong>elegans</strong> ephrin EFN-4 [p 162]<br />
Thomas Holcomb, Sean E. George, Ian Chin-Sang, Mei Ding, Andrew Chisholm<br />
154. syd-8, a new player in axon guidance. [p 163]<br />
Xun Huang, Mei Zhen, Yishi Jin<br />
155. Analysis of gcy-31, a putative soluble guanylyl cyclase gene in <strong>Caenorhabditis</strong> <strong>elegans</strong> [p 164]<br />
Martin L Hudson, David S. Karow, Michael A. Marletta, David B. Morton<br />
156. Using C. <strong>elegans</strong> to Determine the Mechanism of Action of Pharmaceuticals and Pesticides [p 165]<br />
Tak Hung, Ben Burley, Emery Dora, Dan Elkes, Steve Gendreau, Denise Jacobus, Rachel Kindt,<br />
Mark Lackner, Lisa Moore, Scott Ogg, Dianne Parry, Roxanna Peng, Ellyn Pham, Jenny Kopczynski<br />
157. In vivo characterization of the effects of the unc-64(md130) mutation on anesthetic sensitivity. [p<br />
166]<br />
Hunt S.J., Mike Crowder<br />
158. Regulation of the C. <strong>elegans</strong> epidermal growth factor homolog LIN-3 [p 167]<br />
Byung Joon Hwang, Paul W. Sternberg<br />
159. Characterization of the regulatory elements required for neuron-specific expression of SNAP-25 in<br />
the nematode [p 168]<br />
Soon Baek Hwang, Junho Lee<br />
160. Analysis of 2° vulval lineage execution [p 169]<br />
Takao Inoue, Paul W. Sternberg<br />
161. Developing a C. briggsae genetic map [p 170]<br />
B. Johnsen, S. Gharib, A. Mah, K. Brown, D. Baillie, P. Sternberg<br />
6
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
162. Coenzyme Q and aging in the nematode <strong>Caenorhabditis</strong> <strong>elegans</strong>. [p 171]<br />
Tanya Jonassen, Pamela L. Larsen, Catherine F. Clarke<br />
163. osm-9 signaling: who’s involved? [p 172]<br />
Amanda H. Kahn, David Tobin, Cornelia I. Bargmann<br />
164. Looking for synergy with PHA-4 on the myo-2 promoter [p 173]<br />
John Kalb, Pete Okkema, Jim McGhee<br />
165. Initial Characterization of Soluble Guanylate Cyclases in C. <strong>elegans</strong> [p 174]<br />
David Karow, Jennifer Chang, Scott Nicholls, Ronald Ellis, Martin Hudson, David Morton, Michael<br />
Marletta<br />
166. Multiple regulatory elements activate end-1 expression in the E lineage [p 175]<br />
Jodie J. Kasmir, Morris Maduro, Joel H. Rothman<br />
167. Mutations that perturb the effect of octopamine/serotonin on pharyngeal activity. [p 176]<br />
John Keane, Leon Avery<br />
168. Pheromone Regulation of Neuroendocrine Outputs in C. <strong>elegans</strong> [p 177]<br />
Scott Kennedy, Gabriel Hayes, Gary Ruvkun<br />
169. Calcium Imaging in Excitable Cells of C. <strong>elegans</strong>. [p 178]<br />
Rex Kerr, Varda Lev-Ram, Roger Y. Tsien, William R. Schafer<br />
170. A genetic analysis of the effects of ethanol on egg laying [p 179]<br />
Hongkyun Kim, M. Christina Yu, James Kim, Steven L. McIntire<br />
171. Genes affecting the activity of nicotinic receptors involved in egg-laying behavior [p 180]<br />
Jinah Kim, Daniel S. Poole, Laura E. Waggoner, Alexandra Treschow, William R. Schafer<br />
172. Sensory axon guidance defects in C. <strong>elegans</strong> [p 181]<br />
Susan Kirch, Gage Crump, Cori Bargmann<br />
173. Isolation of a third lin-4 allele from a lin-3A overexpression line [p 182]<br />
Martha Kirouac, Paul Sternberg<br />
174. elt-5 and elt-6 are essential for development of seam cells, the vulva, and the male tail. [p 183]<br />
Kyunghee Koh, Joel H. Rothman<br />
175. A genetic screen for genes involved in gut development and differentiation in <strong>Caenorhabditis</strong><br />
<strong>elegans</strong> [p 184]<br />
Jay D. Kormish, James D. McGhee<br />
176. An E1-like activating enzyme is involved in cell division processes in the early C. <strong>elegans</strong> embryo. [p<br />
185]<br />
Thimo K. Kurz, Danielle R. Hamill, Bruce Bowerman<br />
177. Olfactory Adaptation [p 186]<br />
Noelle L’Etoile, Cori Bargmann<br />
178. You can’t get there from here: a gene required for pharyngeal extension. [p 187]<br />
SK Lange, JR Saam, SE Mango<br />
179. Signaling by the VAB-1 Eph receptor intracellular domain [p 188]<br />
Kristoffer Larsen,, Sean George, Andrew Chisholm<br />
180. mdf-1 suppressors that may play a role in the metaphase to anaphase checkpoint [p 189]<br />
Elaine Law, Risa Kitagawa, Ann M. Rose<br />
181. Characterization of a C. <strong>elegans</strong> Defecation Mutant [p 190]<br />
Anne Lehtela, Garry Wong<br />
182. Organogenesis of the C. <strong>elegans</strong> Intestine [p 191]<br />
Benjamin Leung, Greg J. Hermann, James R. Priess<br />
183. Expression and regulation of daf-16::gfp constructs [p 192]<br />
Kui Lin, Cynthia Kenyon<br />
184. Identification of novel unc-64 (syntaxin) alleles [p 193]<br />
Christine Liu, C. Michael Crowder<br />
185. Mutations that cause neurite sprouting of the DVB motor neuron [p 194]<br />
Loria, P., Boulin, T., Conte, S., Hobert, O.<br />
186. A b -tubulin gene, tbb-2, functions as an activator of mei-1 and mei-2 in female meiotic spindle<br />
formation in <strong>Caenorhabditis</strong> <strong>elegans</strong>. [p 195]<br />
Chenggang Lu, Martin Srayko, Paul E. Mains<br />
187. Global profile of gene expression during aging [p 196]<br />
James Lund, Pamela Larsen, Pat Tedesco, Thomas Johnson, Stuart Kim<br />
188. Conditional mutations affecting mitotic spindle positioning and polarity in the C. <strong>elegans</strong> embryo [p<br />
197]<br />
Rebecca Lyczak, Bruce Bowerman<br />
189. Role of PDZ domain proteins in establishing gut epithelial polarity [p 198]<br />
Kathleen E. Mach, Stuart K. Kim<br />
190. Genetic analysis of NMDA receptor expression in C. <strong>elegans</strong> [p 199]<br />
David M. Madsen, Chingju Lin, Penelope J. Brockie, Andres V. Maricq<br />
7
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
191. Large Scale Reverse Genetic Approach Using RNAi [p 200]<br />
Sarah Mahoney, Alex Phan, Mark Maxwell, Candace Swimmer, Jonathan Heller, Brett Milash, Kate<br />
McKusick, Monique Nicoll<br />
192. Sequence Confirmation of 182 snps between C. <strong>elegans</strong> N2 and CB4856 Strains and Plans for<br />
Generation of 1000 New snps. [p 201]<br />
Penny Mapa, Kathryn Swan, Mike Ellis<br />
193. Building a dictionary for C. <strong>elegans</strong> promoter sequences [p 202]<br />
Steven McCarroll, Hao Li, Cori Bargmann<br />
194. High Pressure Freezing Methods for C. <strong>elegans</strong> Embryo Ultrastructure and EM Immunolabeling [p<br />
203] Kent L. McDonald, Thomas Mueller-Reichert, Akiko Tagawa, Chad A. Rappleye, Raffi Aroian<br />
195. Molecular Identification of Transcriptional Targets of the DAF-16 Winged Helix Transcription Factor<br />
[p 204]<br />
Joshua J. McElwee, James H. Thomas<br />
196. Functional conservation of C. <strong>elegans</strong> UNC-30 and mouse Pitx2 in GABAergic neuron specification<br />
[p 205]<br />
Jason McEwen, Yishi Jin<br />
197. Genes involved in nicotinic neurotransmission in the pharynx [p 206]<br />
Jim McKay, David Raizen, Leon Avery<br />
198. Genetic analysis of the functions of a GSK-3ß homolog called sgg-1 and a ß-TRCP/slimb homolog<br />
during C. <strong>elegans</strong> embryogenesis [p 207]<br />
Marc Meneghini, Greg Ellis, Ann Schlesinger, Bruce Bowerman<br />
199. The Effect of Nonimmobilizers on C. <strong>elegans</strong> [p 208]<br />
Laura B. Metz, Mike Crowder<br />
200. Isolation and characterization of mutations that enhance let-23(sa62gf) during vulval development [p<br />
209] Nadeem Moghal, Paul W. Sternberg<br />
201. The trampoline assay: A new method for measuring the step response of the chemotaxis<br />
mechanism in C. <strong>elegans</strong>. [p 210]<br />
Moravec, M.L, Cervantes, J., Lockery, S.R.<br />
202. Identification of genes regulating body length in the DBL-1 pathway by differential hybridization of<br />
arrayed cDNAs [p 211]<br />
Kiyokazu Morita, Makoto Mochii, Yukiko Sugihara, Satoru Yoshida, Yo Suzuki, William B. Wood, Yuji<br />
Kohara, Naoto Ueno<br />
203. Mutations in the ephrin mab-26/efn-4 cause defects in closure of the gastrulation cleft and in<br />
epidermal enclosure [p 212]<br />
Sarah L. Moseley, Andrew Chisholm<br />
204. Cellular and developmental events required to generate functional muscle in C. <strong>elegans</strong>. [p 213]<br />
K. Norman, S. Cordes, G. Mullen, P. Rahmani, T. Rogalski, D. Moerman<br />
205. Is the DAG kinase DGK-1 an effector of Go alpha (GOA-1)? [p 214]<br />
Stephen Nurrish, Michael Dybbs, Joshua Kaplan<br />
206. Transforming Nematodes into Insects: Understanding Bt-resistance [p 215]<br />
Johanna O’Dell, Raffi Aroian<br />
207. The cytoskeletal protein zk370.3 may contribute to oocyte development and fertilization [p 216]<br />
Alex Parker, Ann M. Rose<br />
208. Oxidant Stress Responses in C. <strong>elegans</strong> [p 217]<br />
Farhang Payvar, Andrew DeMatteo, Tom Hazinski<br />
209. Pharyngeal pumping defects in unc-103 mutants [p 218]<br />
Christina I. Petersen, David J. Reiner, Elizabeth M. Newton., James H. Thomas, Jeffrey R. Balser<br />
210. A requirement for C. <strong>elegans</strong> Rho-binding kinase in early cleavage [p 219]<br />
Alisa J. Piekny, Paul E. Mains<br />
211. Function of the receptor tyrosine kinase CAM-1/KIN-8 in coordinated movement [p 220]<br />
S. Poulson, D. Madsen, A.V. Maricq<br />
212. The Autosomal Sex Signal in C. <strong>elegans</strong>? [p 221]<br />
Jennifer R. Powell, Barbara J. Meyer<br />
213. Got the blues? Try another genetic screen! [p 222]<br />
Chad Rappleye, Rebecca Lyczak, Bruce Bowerman, Raffi Aroian<br />
214. Identification of Components of the Meiotic Machinery in C. <strong>elegans</strong> [p 223]<br />
Kirthi Reddy, Monica Colaiacovo, Gillian Stanfield, Anne Villeneuve<br />
215. Novel and Atypical Receptor Tyrosine Kinases in Morphogenesis. [p 224]<br />
David J. Reiner, Lewis Leng, Barbara J. Meyer<br />
216. Differential effects of heat shock and cold shock following massed and distributed long-term<br />
habituation training in C. <strong>elegans</strong> [p 225]<br />
Jacqueline Rose, Kenneth Eng, Catharine Rankin<br />
8
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
217. A new en masse training procedure to study long-term habituation in C. <strong>elegans</strong> [p 226]<br />
Jacqueline Rose, Catharine Rankin<br />
218. Global patterns of expression patterns in muscle using mRNA-Tagging [p 227]<br />
Peter J. Roy, Stuart Kim<br />
219. Cooperation between unc-26/synaptojanin and the dynamin-related protein DRP-1 during<br />
mitochondrial division [p 228]<br />
Dan Rube, Todd Harris, Erik Jorgensen, Alexander van der Bliek<br />
220. Calcium dynamics of fertilization in C. <strong>elegans</strong> [p 229]<br />
Aravinthan D.T. Samuel, Venkatesh N. Murthy, Michael O. Hengartner<br />
221. Mutants in Thermosensory Neuron Specification and Function [p 230]<br />
John S. Satterlee, Piali Sengupta<br />
222. Vesicular GABA transport in C. <strong>elegans</strong> requires two proteins UNC-47 and UNC-46 [p 231]<br />
Kim Schuske, Erik M. Jorgensen<br />
223. Utilizing two approaches, genetic and genomic, to identify the vesicular glutamate transporter [p 232]<br />
Kim Schuske, Dan Williams, Erik M. Jorgensen<br />
224. Actin-dependent processes in the early C. <strong>elegans</strong> embryo require the profilin gene pfn-1, the FH<br />
gene cyk-1, and bel-1 [p 233]<br />
Aaron F. Severson, Rebecca Lyczak, David L. Baillie, Bruce Bowerman<br />
225. LIN-12 post-transcriptional downregulation during VPC specification [p 234]<br />
DD Shaye, I Greenwald<br />
226. Distint and redundant functions of mu1 medium chains of AP-1 clathrin-associated protein complex<br />
in the nematode <strong>Caenorhabditis</strong> <strong>elegans</strong> [p 235]<br />
Jaegal Shim, Junho Lee<br />
227. Molecular Mechanisms of Daf-12 Action: Identificationof Response Elements and Functional<br />
Analysis of the Protein [p 236]<br />
Yuriy Shostak, Adam Antebi, Marc R. van Gilst, Kieth R. Yamamoto<br />
228. Serotonin-resistant egg-laying mutants and a receptor knockout in progress [p 237]<br />
Stanley Shyn, William Schafer<br />
229. A novel genetic screen for synaptic transmission genes acting in the diacyglycerol pathway [p 238]<br />
Derek S. Sieburth, Wendy Cham, Josh M. Kaplan<br />
230. Evidence of a Mate-finding Cue in the Free-Living Soil Nematode C. <strong>elegans</strong> [p 239]<br />
Jasper M. Simon, Paul W. Sternberg<br />
231. Understanding C27H5.1: From sequence to sense [p 240]<br />
Jessica Smith, David Pilgrim<br />
232. Genetic screens for novel components involved in blastomere asymmetry in the early C. <strong>elegans</strong><br />
embryo [p 241]<br />
Martha Soto, Craig C. Mello<br />
233. Pax be with you - patterning the pharynx [p 242]<br />
Jeff Stevenson, Andrew Chisholm, Susan E. Mango<br />
234. The evolution and expression of FEM-2 [p 243]<br />
Paul Stothard, Dave Hansen, Tamara Checkland, Dave Pilgrim<br />
235. Forming a gut: the view from an elt and two odds [p 244]<br />
Keith Strohmaier, Morris Maduro, Joel Rothman<br />
236. C. <strong>elegans</strong> homologue of protein phosphatase 4 is required in spermatogenesis [p 245]<br />
Eisuke Sumiyoshi, Asako Sugimoto, Masayuki Yamamoto<br />
237. Transcriptional regulation of the tryptophan hydroxylase gene tph-1 [p 246]<br />
Ji Ying Sze<br />
238. Searching for new genes involved in dosage compensation [p 247]<br />
Chun Tsai, Barbara J. Meyer<br />
239. Characterizing the role of let-99 in spindle orientation [p 248]<br />
Meng-Fu Tsou, Adam Hayashi, Lesilee S. Rose<br />
240. Characterization and cloning of the muscle activation gene unc-58 [p 249]<br />
Monika Tzoneva, James H. Thomas<br />
241. The structure/function relationship of clk-1 in the nematode <strong>Caenorhabditis</strong> <strong>elegans</strong> [p 250]<br />
Antonio Ubach, Siegfried Hekimi<br />
242. Characterization of transcriptional regulation by a class of monomeric nuclear receptors found in C.<br />
<strong>elegans</strong> [p 251]<br />
Marc R. Van Gilst, Keith R. Yamamoto<br />
243. UNC-4 targets ACR-5 and DEL-1: Are they determinants of synaptic choice? [p 252]<br />
Stephen E. Von Stetina, David M. Miller, III<br />
244. Nicotine adaptation: a process involving PKC-dependant regulation of nAChR protein levels. [p 253]<br />
Laura Waggoner, Kari Dickonson, Daniel Poole, Bill Schafer<br />
9
245. Analysis of GLR-7, GLR-5, Ionotropic Glutamate Receptor Subunits [p 254]<br />
Craig S. Walker, David M. Madsen, Penelope J. Brockie, Andres V. Maricq<br />
246. Microarray analysis of gene expression patterns in dauer larvae [p 255]<br />
John Wang, Stuart K. Kim<br />
247. Characterization of CAN cell and excretory canal defects in mig-10(ct41) mutants [p 256]<br />
Nicole Washington, Jim Manser<br />
248. ric-7 encodes a novel presynaptic protein required for neurotransmission [p 257]<br />
Robby M. Weimer, Erik M. Jorgensen<br />
249. The requirement of synaptic vesicle loading for synaptic vesicle exocytosis [p 258]<br />
Robby M. Weimer, Janet E. Richmond, Erik M. Jorgensen<br />
250. Unraveling the biological role of DMWD, a gene close to the unstable CTG-repeat in the myotonic<br />
dystrophy locus. [p 259]<br />
J. <strong>West</strong>erlaken, B. Wieringa, P.E. Mains<br />
251. Calcium imaging of the defecation rhythm in C. <strong>elegans</strong> [p 260]<br />
Jeanna M. Wheeler, James H. Thomas<br />
252. Establishing the left/right asymmetry of Q neuroblast polarisation and migration in C. <strong>elegans</strong> [p 261]<br />
Lisa Williams, Lee Honigberg, Cynthia Kenyon<br />
253. A screen for cell migration and axon outgrowth mutants [p 262]<br />
Jim Withee, Gian Garriga<br />
254. Mapping and Characterization of had-1, an HSN Axon Guidance Gene [p 263]<br />
Lianna Wong, Jim Rader, Gian Garriga<br />
255. Recognition of X-chromosome-enriched DNA elements by dosage compensation proteins [p 264]<br />
Tammy F. Wu, Jason D. Lieb, Barbara J. Meyer<br />
256. Rac-like GTPases and cell migration [p 265]<br />
Yi-Chun Wu, Li-Chun Cheng, Nei-Yin Weng, Ting-Wen Cheng<br />
257. Two new genes regulating neuroblast migration in C. <strong>elegans</strong> [p 266]<br />
Lucie Yang, Mary Sym, Queelim Ch’ng, Cynthia Kenyon<br />
258. Identification and characterization of telomere binding proteins in the nematode C. <strong>elegans</strong> [p 267]<br />
Su Young Yi, Seunghyun Kim, Junho Lee<br />
259. Molecular Analysis of the Dosage Compensation Gene dpy-21 [p 268]<br />
Stephanie Yonker, Edith Cookson, Barbara J. Meyer<br />
260. A search for lethal synaptic function mutants using a sensitized background [p 269]<br />
Karen Yook, Erik Jorgensen<br />
261. Identification of downstream target genes in daf-2 pathway [p 270]<br />
Hui Yu, Pamela L. Larsen<br />
262. Fate specification in male P(9-11).p equivalence group [p 271]<br />
Hui Yu, Paul W. Sternberg<br />
263. Loss of a dynamin related protein MGM-1 causes excessive mitochondrial fragmentation [p 272]<br />
Mauro Zappaterra, Alexander van der Bliek<br />
264. Isolation and phenotypic analysis of syd-7 [p 273]<br />
Mei Zhen, Nikki Alvarez, Yishi Jin<br />
265. A screen to identify genes that regulate the activity of the C. <strong>elegans</strong> glutamate receptor GLR-1. [p<br />
274] Yi Zheng, Heng Xie, Pene J. Brockie, Andres V. Maricq<br />
266. A resource for C. <strong>elegans</strong> microarrays [p 275]<br />
Stuart K. Kim, Min Jiang, Kyle Duke<br />
Leon Avery (Leon@eatworms.swmed.edu)<br />
Last modified: Mon Jul 24 15:24:42 2000<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
10
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MEIOTIC PAIRING AND SYNAPSIS<br />
Amy MacQueen, Anne M. Villeneuve<br />
Department of Developmental Biology, Stanford University, Stanford CA 94305<br />
Successful meiotic chromosome segregation relies upon a prior association between homologous<br />
chromosomes. We want to understand how homologs establish and maintain functional associations<br />
throughout meiotic prophase. Using cytological tools, we screened through a collection of meiotic<br />
chromosome segregation mutants to identify mutations that specifically disrupt homologous pairing and<br />
synapsis.<br />
We have identified mutations in three complementation groups that cause defects in homologous<br />
synapsis. hal-1 (homolog alignment) mutants contain chromosomes that are asynapsed in the pachytene<br />
region of the germline, where partner chromosomes are normally aligned in parallel tracks with one<br />
another. Fluorescence in situ hybridization (FISH) studies indicate that homologous pairing is abolished in<br />
hal-1 germlines. Further, chromatin in hal-1 early meiotic nuclei does not undergo the distinct<br />
morphological transition that normally accompanies the onset of pairing, suggesting that hal-1 disrupts an<br />
early step required for the initiation of pairing.<br />
Chromatin in nuclei entering meiosis in sys-1 or sys-2 (synapsis) mutants do exhibit typical transition<br />
morphology, and homologs initially pair. However, as nuclei progress to later stages of meiotic prophase,<br />
paired associations are lost. Failure to stabilize homolog pairing suggests a role for the sys-1 and sys-2<br />
genes in chromosome synapsis. Further, the sys-1 gene encodes a protein consisting mainly of a<br />
predicted coiled-coil domain, suggesting that it is likely a structural component of the synaptonemal<br />
complex. Interestingly, a timecourse analysis of sys-1 meiotic nuclei using FISH probes near to either end<br />
of chromosome I revealed that the "pairing center" end consistently achieves a higher degree of<br />
association during meiotic prophase compared with the opposite end of the chromosome. This differential<br />
behavior of opposite ends of a chromosome strengthens prior notions that synapsis along a chromosome<br />
initiates at the "pairing center"end, and suggests a role for the sys-1 gene in synapsis.<br />
Our analysis demonstrates that homolog pairing and synapsis can be subdivided into discrete steps: hal-1<br />
gene function is required for the early establishment of homologous associations, while sys-1 and other<br />
genes of its class are required for the maintenance of these pairing interactions.<br />
11
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CONDITIONAL MITOTIC SPINDLE MUTANTS IN C. ELEGANS<br />
Danielle R. Hamill, Bruce Bowerman<br />
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403<br />
The large size of the transparent early embryos and the powerful genetics of C. <strong>elegans</strong>, make it<br />
attractive for studies of cell division. Therefore, we have isolated and observed early embryonic cell<br />
divisions in approximately 600 temperature-sensitive, embryonic-lethal mutants in an ongoing screen in<br />
the lab. About 35 mutants appear defective in microtubule- and/or microfilament-dependent processes,<br />
including pronuclear migration, centrosome function, mitotic spindle assembly or orientation, and<br />
cytokinesis. We isolated ts-alleles of several genes known to affect these processes, including an<br />
aurora-like kinase (air-2) and an MKLP1-like kinesin (zen-4) [see abstract by A.F. Severson], as well as<br />
several previously unidentified genes [see abstracts by J.E. Gomes and R. Lyczak].<br />
Here we describe two temperature-sensitive mutants, spd-4 and spd-5, that are required for mitotic<br />
spindle assembly and function (spd=spindle-defective). spd-4 mutant embryos assemble bipolar mitotic<br />
spindles, but they are shorter than wild type and do not elongate. spd-4 mutant embryos also have<br />
defects in DNA segregation and cytokinesis. Pronuclear migration is defective in spd-5 mutant embryos, a<br />
mitotic spindle does not form, and the first cell division fails. Intriguingly, spd-4 and spd-5 show genetic<br />
interactions that suggest they function together in a complex to regulate mitotic spindle assembly in the<br />
early C. <strong>elegans</strong> embryo.<br />
From the map position and phenotype of spd-4, as well as genetic complementation analysis (in<br />
collaboration with D. Schmidt, S. Strome, and W. Saxton, Indiana University) we believe that spd-4 might<br />
encode a dynein heavy chain gene, although this still needs to be confirmed. Injection of dsRNA<br />
corresponding to the Genefinder locus F56A3.4 phenocopies the spd-5 mutant phenotype. We are<br />
sequencing this locus to determine the molecular nature of the lesion. F56A3.4 encodes a coiled-coil<br />
protein with no significant similarity to other proteins apart from this motif. Therefore, if spd-4 is dynein<br />
heavy chain, spd-5 likely represents a novel dynein regulator.<br />
12
MITOTIC CHROMOSOME SEGREGATION BY A CONSERVED<br />
PROTEIN COMPLEX<br />
K. Hagstrom, R. Chan, D. Pasqualone, B.J. Meyer<br />
HHMI & MCB, UC Berkeley, Berkeley, CA 94720<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
From bacteria to man, the highly conserved SMC (structural maintenance of chromosomes) protein family<br />
is required for chromosome segregation and cell division. In C. <strong>elegans</strong> SMC proteins also direct X<br />
chromosome dosage compensation. We are studying the composition and function of SMC protein<br />
complexes and how individual SMC proteins participate in more than one chromosomal process. For<br />
example, how does the SMC protein MIX-1, essential for both mitosis and dosage compensation, achieve<br />
its dual function within a single cell? MIX-1 requires the SMC protein DPY-27 for its role in dosage<br />
compensation and X localization, but DPY-27 plays no role in mitosis. Thus, it seemed likely that MIX-1<br />
would have a different SMC partner for mitosis. Searching the C. <strong>elegans</strong> genome revealed another SMC<br />
homolog, SLP-2 (SMC-like protein-2.)<br />
The RNAi phenotype and protein localization of SLP-2 suggested its involvement in mitosis. Like MIX-1,<br />
SLP-2 RNAi produces dead embryos with defects such as chromatin bridges and abnormally large nuclei.<br />
Time-lapse microscopy shows a failure in chromosome segregation, and fluorescent in situ hybridization<br />
reveals nuclei with abnormally high DNA content. Thus, loss of SLP-2 prevents chromosome segregation,<br />
but not DNA replication and cell cycle progression. SLP-2 co-localizes with MIX-1 on mitotic<br />
chromosomes in embryos and in the germline. SLP-2 and MIX-1 surround chromosomes as they<br />
condense, then appear on the poleward face of chromosomes aligned at metaphase, where they remain<br />
until they disappear at telophase.<br />
The idea that MIX-1 partners with SLP-2 for mitosis, but with DPY-27 for dosage compensation, is further<br />
supported by immunoprecipitation (IP) results. Co-IP from embryonic extracts is observed between SLP-2<br />
and MIX-1, but not between SLP-2 and DPY-27. Moreover, IPs show that SLP-2 and MIX-1 are part of a<br />
large protein complex. The identities of these subunits are being explored by mass spectrometry. One<br />
subunit (see R. Chan, et al.) shares homology with both a conserved component of the mitotic complex in<br />
other organisms, and a component of the C. <strong>elegans</strong> dosage compensation complex. It will be interesting<br />
to learn if the mitotic and dosage compensation complexes share additional components, and to what<br />
extent the biochemical activities of these complexes are related.<br />
13
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HIM-10 A PROBABLE KINETOCHORE PROTEIN INVOLVED IN<br />
MITOTIC AND MEIOTIC CHROMOSOME SEGREGATION<br />
M. Howe 1 , D G. Albertson 2 , B. J. Meyer 1<br />
1HHMI & Dept. of Mol. Cell Bio. UCB, Berkeley, CA 94720<br />
2CRI, UCSF, San Francisco, CA 94143<br />
The mitotic and meiotic chromosomes of C. <strong>elegans</strong> are atypical. These mitotic chromosomes are<br />
holocentric, that is, the kinetochore (the structure mediating chromosome attachment to the spindle)<br />
extends along the length of the chromosome. The ultrastructure of these long kinetochores is similar to<br />
those of monocentic chromosomes common to other animals. C. <strong>elegans</strong> meiotic chromosomes have no<br />
discernable kinetochores at the ultrastructural level. Investigation of C. <strong>elegans</strong> chromosomes may<br />
identify conserved features of kinetochores essential to chromosome segregation in mitosis and meiosis.<br />
To understand C. <strong>elegans</strong> kinetochores we have characterized him-10, a gene implicated in mitotic<br />
kinetochore function by an allele that increases free duplication loss. Cloning him-10 revealed that the<br />
gene encodes a novel protein. Protein localization and loss-of-function phenotypes are consistent with<br />
HIM-10 playing a direct role in kinetochore function in mitosis and meiosis.<br />
HIM-10 appears to associate with the kinetochore face of mitotic chromosomes from prophase through<br />
anaphase. HIM-10 staining partially overlaps with a conserved centromeric histone variant HPC-3, with<br />
HIM-10 localizing to the kinetochore region, distal to HPC-3.<br />
him-10 RNAi treatment caused the progeny from injected animals to die as embryos. Fluorescent in situ<br />
hybridization (FISH) to these dead embryos revealed severe aneuploidy suggesting that lethality is due to<br />
aberrant embryonic mitosis. Tubulin staining of dsRNAi embryos showed displaced metaphase<br />
chromosomes, unipolar chromosome attachments, and irregular nuclei. FISH and tubulin staining suggest<br />
that loss of him-10 function causes segregation defects consistent with kinetochore failure.<br />
him-10 is also required during meiosis. The hypomorphic mutation, him-10 (e1511ts), causes a sterility<br />
that can be rescued by mating with wild-type males, suggesting a role for the protein in sperm meiosis.<br />
FISH to dead embryos from the ts sterile adults revealed monosomic and trisomic embryos, suggesting<br />
that the mutation causes chromosome loss in meiosis, not embryonic mitosis. HIM-10 encases<br />
spermatocyte and oocyte chromosomes and duplications, suggesting that a kinetochore-like structure<br />
functions in C. <strong>elegans</strong> meiosis.<br />
14
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A C. ELEGANS CHROMOKINESIN REQUIRED FOR<br />
CHROMOSOME SEGREGATION<br />
Jim Powers, Bill Saxton, Susan Strome<br />
Biology Department, Indiana University, Bloomington, IN 47401<br />
A search of the C. <strong>elegans</strong> genome, using the motor domain of kinesin heavy chain (unc-116), which is a<br />
microtubule motor, identified 18 distinct kinesins. Two of these, CeChromoK-A and B, are most closely<br />
related to the vertebrate chromokinesins. Vertebrate chromokinesins bind chromosomes and can bind<br />
microtubules in an ATP-sensitive manner. Therefore, they may function as mitotic motors. However,<br />
strong evidence that chromokinesins actually move along microtubules has not been reported. The<br />
CeChromoKs lack the putative DNA-binding domain that is found in vertebrate chromokinesins.<br />
We have tested the functions of CeChromoKs in oogenesis and early embryogenesis by RNA<br />
interference (RNAi). We detected no phenotypes after RNAi of CeChromoK-A. However, RNAi of<br />
CeChromoK-B causes marked defects in mitosis. Anti-tubulin and anti-histone staining and observation of<br />
GFP-tagged histone in embryos suggest that spindle formation is normal, but that chromosomes<br />
congress poorly to form a loose metaphase plate. During anaphase, chromosomes do not disjoin<br />
accurately, stretching along the pole-to-pole axis to form numerous anaphase bridges. Some<br />
chromosomes fragment, giving rise to multiple micronuclei during telophase.<br />
Antibodies to CeChromoK-B show bright staining of mitotic nuclei in the distal germline and of oocyte<br />
nuclei in the oviduct. After fertilization, CeChromoK-B becomes concentrated between paired meiotic<br />
chromosomes, and is left near the spindle equator as anaphase proceeds. A similar pattern is seen<br />
during mitosis. CeChromoK-B becomes associated with chromosomes during late prophase, remains on<br />
the chromosomes during metaphase and early anaphase, and later becomes concentrated at the spindle<br />
equator, appearing to associate with the overlapping microtubules of the developing telophase bridge.<br />
This concentration persists in the midbody after cytokinesis.<br />
Our results suggest that despite the lack of a recognizable DNA-binding domain, CeChromoK-B<br />
associates with mitotic chromatin and is important for chromosome-microtubule interactions that ensure<br />
an ordered metaphase plate and accurate anaphase separation of chromosomes. We suspect that<br />
CeChromoK-B modulates the interactions of microtubules with chromosome arms to resolve bipolar<br />
chromatid attachment or chromosome catenation.<br />
15
NUCLEAR ENVELOPE DYNAMICS IN CAENORHABDITIS<br />
ELEGANS<br />
Kenneth Lee, Yosef Gruenbaum, Katherine L. Wilson<br />
*No Address*<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Emerin, MAN1 and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share<br />
a 43-residue N-terminal motif, termed the LEM-domain. We found three putative LEM-domain genes in<br />
<strong>Caenorhabditis</strong> <strong>elegans</strong>, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes<br />
Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-Emerin and Ce-MAN1 migrate on SDS-PAGE as 17<br />
and 52 kDa proteins, respectively. Based on their biochemical extraction properties and<br />
immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the<br />
nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to<br />
determine the timing of nuclear envelope breakdown during mitosis in C. <strong>elegans</strong>. The C. <strong>elegans</strong> nuclear<br />
envelope disassembles very late, compared to vertebrates and Drosophila. The nuclear membranes<br />
remained intact everywhere except near spindle poles during metaphase and early anaphase, fully<br />
disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the<br />
lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos,<br />
but exist until anaphase in 2-24 cell embryos. Intranuclear mRNA splicing factors disassembled after<br />
prophase. The timing of nuclear disassembly in C. <strong>elegans</strong> is novel, and may reflect its evolutionary<br />
position between unicellular and more complex eukaryotes.<br />
16
THE FORMIN PROTEIN CYK-1 ACTS IN PARALLEL TO AN<br />
AURORA-LIKE KINASE/MKLP-1 PATHWAY TO EXECUTE<br />
CYTOKINESIS IN EARLY CAENORHABDITIS ELEGANS<br />
EMBRYOS<br />
Aaron F. Severson, Danielle R. Hamill, Bruce Bowerman<br />
University of Oregon, Eugene, Oregon 97403 USA<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The C. <strong>elegans</strong> Formin Homology protein CYK-1 localizes to cleavage furrows in dividing embryonic cells<br />
and, based on an analysis of partial loss-of-function mutations, is required late in cytokinesis (Swan et al.<br />
1998). Analysis of a more severe allele indicates that CYK-1 also is required early in cytokinesis for<br />
contractile ring assembly or function. In addition to CYK-1, embryonic cytokinesis in C. <strong>elegans</strong> requires<br />
the Aurora-like kinase AIR-2 and the mitotic kinesin-like protein ZEN-4. Genetic interactions involving<br />
these loci suggest that an AIR-2/ZEN-4 mitotic spindle pathway functions in parallel to a contractile ring<br />
pathway that includes CYK-1. We have identified temperature-sensitive alleles of both air-2 and zen-4. A<br />
temporal analysis of their function suggests that AIR-2 acts in metaphase or early anaphase, to localize<br />
ZEN-4 to the spindle interzone, while ZEN-4 acts in cytokinesis, during late anaphase or telophase.<br />
Intriguingly, ZEN-4 may also be required well after the apparent completion of cytokinesis, to maintain the<br />
separation of daughter cells. We are currently using the yeast two-hybrid system to determine if AIR-2<br />
and ZEN-4 interact directly. Additionally, we are collaborating with Dr. Jill Schumacher (University of<br />
Texas) to determine if AIR-2 and ZEN-4 associate in stable complexes that can be immunprecipitated.<br />
Our analysis provides genetic evidence that separable, parallel pathways coordinate microfilament and<br />
microtubule functions during cytokinesis in an animal embryo.<br />
17
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE L TYPE CYCLIN SAG-4 IS REQUIRED FOR HEAT-SHOCK<br />
INDUCED PROTEIN EXPRESSION<br />
Wen J. Chen, Yvonne M. Hajdu-Cronin, Paul W. Sternberg<br />
HHMI and Dept. of Biology, Caltech, Pasadena, CA91125, USA<br />
In a screen for suppressors of activated GOA-1 under the control of a heat shock promoter, we identified<br />
four genetic loci that affect heat-shock induction of GOA-1. sag-4 and sag-8 are wild type in appearance,<br />
while sag-3 and sag-5 are egg-laying defective. <strong>West</strong>ern analysis indicated that sag-4 or sag-8 mutations<br />
suppress activated Goa by decreasing heat-shock induced protein expression. Although endogenous<br />
GOA-1 expression is not affected, heat-shock induction of GOA-1 decreased in the suppressor strains.<br />
We cloned sag-4 locus, which encodes a cyclin most similar to cyclin L. The latter is a novel type of cyclin<br />
with unknown function, but also similar to cyclin T, K or C, which was identified as a subunit of TFIIH, part<br />
of RNA polymerase II complex and functions in basal transcription. Only transgenes with hsp16-2<br />
promoter can be affected by sag-4. These results suggest that sag-4 must suppress heat-shock GOA-1<br />
phenotypes by preventing heat- shock mediated transcription in C. <strong>elegans</strong>. We propose that cyclin L is<br />
the type of cyclin acting in TFIIH during heat-shock induced mRNA transcription, which carries function<br />
similar to cyclin T, K or C during basal transcription. sag-3, sag-5 and sag-8 might also be involved in<br />
similar processes.<br />
18
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CDL-1 ENCODES A STEM- LOOP BINDING PROTEIN (SLBP)<br />
HOMOLOG AND MAY BE ESSENTIAL FOR CORE HISTONE<br />
EXPRESSION.<br />
Yuki Kodama 1 , Asako Sugimoto 1 , Joel Rothman 2 , Masayuki<br />
Yamamoto 1<br />
1Dept. of Biophys. & Biochem., Grad. School of Science, Univ. of Tokyo, Tokyo 113, JAPAN<br />
2Neuroscience Research Institute, Univ. of California-Santa Barbara, CA93106<br />
cdl-1 (cell death lethal) mutants show several embryonic defects: 1) delay in appearance of cell corpses<br />
and accumulation of cell corpses in late embryogenesis, 2) defects in elongation, 3) failure in attachment<br />
of the pharynx to the buccal cavity. To understand the cdl-1 function, we cloned the cdl-1 gene. By<br />
transformation assay, we found that a cosmid T19E10 could rescue the cdl-1 phenotypes. RNAi for ORFs<br />
on T19E10 revealed that some of R06F6.1(RNAi) F1s showed cdl-1-like phenotype, although most of<br />
them arrested at the early embryonic stage. We sequenced the corresponding region from cdl-1 mutants<br />
and identified mutations in two alleles, thus concluding that R06F6.1 is the cdl-1 gene.<br />
cdl-1 encodes a member of the stem-loop binding protein(SLBP) family, which binds to the 3’-stem-loop<br />
of core histone mRNAs. It has been described that metazoan core histone mRNAs have a stem-loop<br />
structure instead of a poly-A sequence, and SLBPs have been implicated in post-transcriptional regulation<br />
of core histone mRNAs. In C. <strong>elegans</strong>, 58 of core histone genes contain a conserved stem-loop sequence<br />
in their 3’-UTR sequence. We confirmed the interaction between CDL-1 protein and the stem-loop<br />
structure by yeast three-hybrid system. This result suggests that CDL-1 may also function in the<br />
post-transcriptional regulation of core histones.<br />
To examine the early embryonic phenotypes in cdl-1(RNAi) embryos, we observed them by DAPI staining<br />
and Nomarski optics. In these embryos, chromosomes were less condensed during mitosis, cytokinesis<br />
occurred before completion of the nuclear division, and nuclear fragments existed in some blastmeres.<br />
These observations suggest that the chromatin structure, especially its condensation, might be defective<br />
in cdl-1(RNAi) embryos. We then performed RNAi with core histone genes, the probable targets of<br />
CDL-1. Most RNAi embryos showed early arrest phenotype similar to cdl-1(RNAi) embryos, which<br />
supports the hypothesis that CDL-1 regulates the expression of core histones.<br />
We are currently trying to examine whether the original cdl-1 phenotypes are also caused by the defect of<br />
core histone expression.<br />
19
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UNC-23 IS A MEMBER OF THE BAG FAMILY OF<br />
CHAPERONE REGULATORS<br />
Poupak Rahmani, Donald Moerman<br />
Department of Zoology. University of British Columbia, 6270 University Blvd. Vancouver, BC Canada.<br />
V6T 1Z4<br />
Mutations in the unc-23 gene result in detachment of the anterior body wall musculature and a bent-head<br />
phenotype (Waterston et al, 1980). This phenotype is not observed when animals are grown in liquid<br />
culture (Bullerjahn and Riddle, pers. comm.) Neither muscle cell positioning nor myofilament assembly is<br />
affected in liquid grown unc-23 animals; muscle cell attachment, however, is affected since a small<br />
amount of stress applied results in detachment of the muscle cells from the hypodermis.The result of<br />
immunological staining of unc-23 animals with antibodies to basement membrane and hypodermal<br />
components suggest that the primary defect in unc-23 animals is located within the hypodermis. We have<br />
recently cloned unc-23, and found it to be a protein most similar to a chaperone regulator known as<br />
BAG-2 (BCL2-associated athanogene-2). In humans, the BAG family of chaperone regulators contains a<br />
conserved 45 amino acid region near their C termini (the BAG domain) that binds Hsp70/Hsc70 and<br />
control their chaperone activity (Takayama et al. 1999). Human BAG-2 and UNC-23 share 40% amino<br />
acid identity and 62% similarity over the BAG domain and its upstream region. We are currently<br />
attempting to obtain a full-length unc-23::GFP transgenic line to study the spatial and temporal expression<br />
pattern of this gene<br />
20
MATERNAL UNC-45 PROTEIN CO-LOCALIZES WITH NMY-2,<br />
A NON-MUSCLE MYOSIN AT THE CLEAVAGE FURROW OF<br />
EARLY EMBRYOS<br />
Wanyuan Ao, Dave Pilgrim<br />
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.<br />
unc-45 is an essential gene for normal body wall muscle thick filament development and mutants show a<br />
partial maternal effect. The unc-45 protein product (UNC-45) contains tetratricopeptide (TPR) repeats and<br />
similarity to fungal proteins, but its biochemical function is still unknown. We have previously shown that<br />
UNC-45 is a component of muscle thick filaments and co-localizes with myosin heavy chain B but not<br />
myosin heavy chain A in the body wall muscles of adult worms [1] . Previous genetic evidence also<br />
suggests that UNC-45 may interact with myosin heavy chain isoforms in the muscle cells [2] . We show<br />
here that UNC-45 is also contributed maternally to the embryos and present in all cells of the early<br />
embryo. Zygotic UNC-45 is only detected in the developing muscle cells of the embryo.<br />
Moreover, our yeast two-hybrid screens show that UNC-45 interacts specifically with NMY-2, a<br />
non-muscle myosin. These two proteins are also co-localized at the cleavage furrow of the early embryos.<br />
The localization of UNC-45 at the cleavage furrow is dependent on the presence of NMY-2. NMY-2 has<br />
been previously shown to be required for embryonic polarity and cytokinesis [3,4] . Our results suggest that<br />
the maternal UNC-45 may have a function in the early embryo which is independent of muscle function.<br />
References<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1. Ao, W., and Pilgrim, D. (2000). J. Cell Biol. 148, 375-384.<br />
2. Venolia, L., and R.H. Waterston. (1990). Genetics. 126, 345-354.<br />
3. Guo, S., and Kemphues, K. J. (1996). Nature, 382, 455-458.<br />
4. Shelton, C. A., Carter, J. C., Ellis, G. C., and Bowerman, B. (1999). J. Cell Biol. 146, 439-451.<br />
21
POLYUNSATURATED FATTY ACIDS REQUIREMENTS FOR<br />
PROPER FUNCTIONING OF THE NERVOUS SYSTEM<br />
Jenny Watts, John Browse<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340<br />
Polyunsaturated phospholipids are critical for the function of excitable membranes. Membrane-mediated<br />
information transfers are intimately related to the biochemical events that occur within the neuronal<br />
plasma membrane. Polyunsaturated fatty acid components of phospholipids are necessary to create a<br />
fluid environment as well as to provide precursors of second messenger signaling molecules. C. <strong>elegans</strong><br />
can synthesize a wide range of polyunsaturated fatty acids using only saturated and monounsaturated<br />
fatty acids from E. coli as precursors. In order to study the role of polyunsaturated fatty acids in the<br />
nervous system, we designed a unique biochemical screen which enabled us to isolate a number of<br />
mutant lines exhibiting a range of altered fatty acid compositions. We discovered that many of the<br />
mutations are in known desaturase genes that encode enzymes responsible for inserting double bonds<br />
into a fatty acid chain. The phenotypes of these strains range from no apparent defects in strains lacking<br />
specific classes of 20-carbon polyunsaturated fatty acids to severe locomotion defects and impaired<br />
defecation in strains with more extreme alterations in fatty acid composition. These more extreme strains<br />
also grow slowly and display temperature sensitive embryonic lethality. Providing the worms with dietary<br />
polyunsaturated fatty acids rescues these defects. We are currently performing assays of neurological<br />
function on the whole range of mutants, both unsupplemented and supplemented with various fatty acids.<br />
Comparison of the worm fatty acid composition with the severity of neurological defects will allow us to<br />
determine the polyunsaturated fatty acid requirements for proper movement and behavior.<br />
22
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
TESTING FUNCTIONS OF PHAGOCYTOSIS RECEPTOR<br />
HOMOLOGS IN CELL CORPSE ELIMINATION AND GONADAL<br />
OUTGROWTH<br />
Sambath Chung 1 , Monica Driscoll 2<br />
1UMDNJ-Graduate School of Biomedical Sciences, Piscataway, NJ 08855<br />
2Rutgers University<br />
Cell death can occur as a normal event in development or as a consequence of cell injury. Effective<br />
elimination of cell corpses is essential for maintaining tissue homeostasis, recycling cellular metabolites,<br />
and removing potentially harmful residual cellular contents. Both C. <strong>elegans</strong> programmed cell death<br />
corpses and necrotic-like corpses (such as those generated by mec-4(d), deg-3(d) and other stimuli) are<br />
removed via the action of seven engulfment ced genes--ced-1, ced-2, ced-5, ced-6, ced-7, ced-10, and<br />
ced-12. We have been interested in identifying genes that might specifically be involved in the<br />
recognition/elimination of necrotic cell corpses. Because the receptors that initially mediate recognition of<br />
the necrotic cells might be different from those recognizing the morphologically distinct programmed cell<br />
death corpses, we considered the hypothesis that a subset of nematode genes related to phagocytosis<br />
receptor genes in other organisms might be required for recognition of necrotic cell corpses.<br />
Mammalian CD36 and Drosophila Croquemort are related scavenger receptors that function in cell corpse<br />
removal. We searched the C. <strong>elegans</strong> genomic database and identified six homologs of the<br />
CD36/Croquemort family. We generated a deletion mutation affecting the gene most closely related to<br />
CD36/Croquemort. This allele harbors a deletion of approximately 1kb, starting about 200 bp upstream of<br />
the receptor open reading frame. We named this locus scr-1, for scavenger receptor-like. The scr-1<br />
deletion mutant does not exhibit necrotic or programmed cell death corpse persistence, nor does this<br />
mutation enhance corpse persistence when present in combination with any of the seven engulfment ced<br />
mutations. Interestingly, however, a significant percentage of scr-1 mutants arrest at the L1 larval stage<br />
and appear to have programmed cell death corpses throughout their bodies. scr-1 mutants do exhibit<br />
distinctive defects in distal tip cell migration, similar to that observed in ced-2, ced-5, ced-10, and ced-12<br />
engulfment mutants. This observation suggests that SCR-1 might function as a receptor important in<br />
gonadal outgrowth in the process involving CED-2, -5, -10, -12.<br />
We have also tested for effects of the other five scr homologs using RNAi.<br />
23
REGULATION OF CELL FUSION IN C. ELEGANS<br />
Scott Alper, Cynthia Kenyon<br />
University of California, San Francisco<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Cell fusion is a common process in C. <strong>elegans</strong>. The C. <strong>elegans</strong> hypodermis consists of several<br />
multinucleate syncytia that are generated by the fusion of cells throughout development. The largest<br />
syncytium is hyp7, which spans most of the length of the worm and which contains more than 130 nuclei.<br />
We are carrying out two screens to identify mutations affecting cell fusion. First, we screened for<br />
mutations that prevent fusion of the Pn.p cells with hyp7 on the ventral surface of the worm. Mutations<br />
identified in this screen affect the decision of these cells to fuse. Second, in order to identify mutations in<br />
genes that carry out the cell fusion process, we are screening for mutations that affect fusion of the seam<br />
cells that line the lateral surface of the worm.<br />
We identified several mutations that affect the pattern of Pn.p cell fusion and have been characterizing<br />
two mutations that affect Pn.p cell fusion by altering Hox protein activity. The fusion decision of the 12<br />
Pn.p cells is controlled by two Hox genes, lin-39 and mab-5. lin-39 is expressed in the mid-body [P(3-8).p]<br />
and in hermaphrodites prevents fusion of these cells. mab-5 is expressed more posteriorly [in P(7-11).p]<br />
in both sexes, but is not active in hermaphrodite Pn.ps. In ref-1(mu220) (REgulator of Fusion)<br />
hermaphrodites, P9.p and P10.p fail to fuse with hyp7. This is due, in part, to inappropriate activation of<br />
MAB-5 in ref-1 hermaphrodites. ref-1 encodes a gene with two basic-helix-loop-helix DNA binding<br />
domains of the hairy/E(spl) family.<br />
In males, lin-39 and mab-5 each individually prevent Pn.p cell fusion in P(3-6).p and P(9-11).p,<br />
respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they<br />
somehow neutralize one another’s activities, so that P7.p and P8.p fuse with hyp7. In ref-2(mu218)<br />
males, P7.p and P8.p fail to fuse with hyp7, perhaps because LIN-39 and MAB-5 fail to cancel each<br />
others activities. ref-2 has been mapped to a 40 kb region on the center of the X chromosome and<br />
transformation rescue experiments are in progress.<br />
Descendants of the seam cells fuse with hyp7 at all larval stages and ultimately the seam cells fuse with<br />
each other during L4. We are currently using a membrane localized gfp fusion expressed in the seam<br />
cells to identify mutants in which seam cell fusions fail to occur.<br />
24
ETHANOL SENSITIVITY GENES IN CAENORHABDITIS<br />
ELEGANS<br />
MinGi Hong, JaeYoung Kwon, InYoung Lee, MinSung Choi, Junho Lee<br />
Department of Biology, Yonsei University<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The mechanisms and sites of action of volatile anesthetics and ethanol are not fully understood. In the<br />
hope of understanding the mechanisms of ethanol, we first identified genes that control sensitivity to<br />
ethanol and anesthetics in the invertebrate system <strong>Caenorhabditis</strong> <strong>elegans</strong>. We identified 10 mutations<br />
that confer ethanol resistance either by EMS mutagenesis or transposon insertion mutagenesis. The<br />
genes are being cloned by positional cloning and analyses on the mutations are under way. In the next<br />
experiments, we used the cDNA microarray to identify genes that are either up-regulated or<br />
down-regulated by exposure of the animals to 7 % ethanol for 6 hours. Several gene families including<br />
heat-shock protein family, glutamate receptor family, and gene families with unknown function, were<br />
up-regulated by ethanol. Also, there are gene families down-regulated. We are now examining these<br />
candidate ethanol-affected genes by northen analysis and GFP reporter analysis. To establish an<br />
experimental system by which one can study Fetal Alcohol Syndrome using the nematode, we examined<br />
the effect of ethanol on embryogenesis. After incubating adult hermaphrodites in 7% EtOH for 12 hours,<br />
we observed egg-laying defects and abnormal embryogenesis. Based on this preliminary data, we will<br />
investigate and characterize the ethanol sensitivity genes involved in embryogenesis. In summary, we<br />
identified ethanol resistance genes by EMS or tensposon mutagenesis; we identified genes whose<br />
transcription levels are altered by ethanol in the microarray analysis; and we established an experimental<br />
model system to study Fetal Alcohol Syndrome.<br />
25
STATE-DEPENDENT LEARNING IN C. ELEGANS.<br />
Jill C. Bettinger, Steven L. McIntire<br />
Gallo Center and Program in Biological Sciences, Department of Neurology, UCSF<br />
The execution of learned behaviors may be triggered by contextual information, consisting of<br />
environmental cues or the internal state of the organism. State-dependent learning refers to the ability of<br />
an organism to more effectively execute learned behaviors if the organism experiences internal contextual<br />
influences similar to those experienced when the learning occurred. Such contexts can be<br />
pharmacologically manipulated by treating with cholinergic compounds, opiates, cocaine and<br />
amphetamines, and with neurodepressants such as ethanol and barbituates 1 . <strong>Worm</strong>s become<br />
intoxicated by ethanol in a manner similar to that of most other organisms tested (see abstracts by A.<br />
Davies and H. Kim, this meeting). We have sought to study the mechanisms of ethanol-induced<br />
state-dependent learning in worms.<br />
Olfactory adaptation in C. <strong>elegans</strong> is a decrease in the chemotaxis response to an odorant as a result of<br />
prior exposure to the odorant 2 . We demonstrate a form of state-dependent learning in worms by pairing<br />
olfactory adaptation and ethanol administration. Ethanol does not interfere with olfactory adaptation,<br />
however, worms exposed to an odorant while being treated with ethanol will only show subsequent<br />
adaptation to the odorant if ethanol is again administered during chemotaxis testing. If the odorant is<br />
presented without ethanol during testing, the animals behave as naïve animals and therefore fail to alter<br />
their behavior based on their previous experience or prior exposure to the odorant.<br />
Further, we demonstrate that the state-dependent effects of ethanol require normal dopaminergic<br />
function. The dopamine-defective cat-1 4 and cat-2 5 mutants are able to adapt to volatile odorants,<br />
however, they do not show state-dependency when they are adapted to volatile odorants while<br />
intoxicated by ethanol. These results suggest that C. <strong>elegans</strong> is capable of a form of learning and indicate<br />
a conserved role of dopamine in the modulation of behavioral responses to ethanol.<br />
1 Izquierdo. in Neurobiology of Learning and Memory (eds Lynch, McGaugh, and Wienberger) 333<br />
(Guilford, New York, 1984).<br />
2 Shulz, Sosnik, Ego, Haidarliu and Ahissar (2000) Nature 403, 549, and references therein.<br />
3 Colbert and Bargmann (1995) Neuron 14, 803.<br />
4 Duerr et al. (1999) J. Neuroscience 19, 72.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
5 Lints and Emmons (1999) Development 126, 5819.<br />
26
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE UT236 MUTANT IN C. ELEGANS HAS DEFECTS IN THE<br />
INTERACTION OF TWO SENSORY SIGNALS AND AN<br />
ASSOCIATIVE LEARNING.<br />
Takeshi Ishihara 1 , Yuichi Iino 2 , Isao Katsura 1<br />
1Struct.Biol.Cent., Natl.Inst.Genet., Dept of Genet. Grad.Univ.Adv.Stud.<br />
2Mol. Genet. Res. Lab., Univ. of Tokyo<br />
Among many environmental stimuli, C. <strong>elegans</strong> seems to respond to only a few stimuli simultaneously. To<br />
elucidate how the neural circuit processes many sensory signals to respond properly, we have designed a<br />
new assay system to analyze interaction between two responses: chemotaxis to odorants and avoidance<br />
from Cu 2+ ion. In this assay, the behavior depends on the concentration of both stimuli, which are sensed<br />
by different neurons. Our observation suggests that these two responses repress each other. We can<br />
argue that this mutual interaction takes place in a defined part of the neural circuit, which consists of<br />
about 10 pairs of neurons.<br />
One mutant, ut236, has defects in this interaction. It prefers to avoid Cu 2+ ion rather than to go to<br />
odorants, while its dose responses to the odorants and Cu 2+ ion are indistinguishable from those of the<br />
wild type. Positional cloning revealed that the ut236 gene encodes a novel secretory protein (C36B7.7)<br />
with an LDL receptor ligand binding motif. Expression studies with a functional GFP fusion gene and by<br />
immunostaining show that it is expressed in a few sensory neurons and AIY neurons. By using cell type<br />
specific promoter, we found that the expression of C36B7.7 in AIY or ASE is sufficient for the rescue of<br />
this phenotype. Also, by using a heatshock promoter construct, it is shown that the expression of<br />
C36B7.7 in embryonic stage is not sufficient for the rescue but the expression in the late larval or adult<br />
stage is. These results suggest that C36B7.7 is necessary for the neuronal function of these neurons, but<br />
is not for the development of the nervous system.<br />
The ut236 mutant seems to have another defect in an associative learning by paired presentation of NaCl<br />
and starvation. This phenotype can be also rescued by the C36B7.7 transgene. Therefore, we postulate<br />
that this novel secretory protein is involved in the modification of sensory signals in the neural circuit to<br />
regulate their preference in chemical stimuli.<br />
27
MUTATION IN THE LIM HOMEOBOX GENE LIM-6 DISRUPTS<br />
ASYMMETRIC FUNCTION OF THE ASE CHEMOSENSORY<br />
NEURONS<br />
J.T. Pierce-Shimomura, M.R. Gaston, B.J. Pearson, S.R. Lockery<br />
Institute of Neurosci, Univ of Oregon, Eugene, OR 97405<br />
C. <strong>elegans</strong> detects chemicals with 11 pairs of bilaterally symmetric sensory neurons. All of the right-left<br />
members of these pairs share similarity in lineage, morphology, and synaptic connectivity. However, one<br />
of these pairs (ASE) expresses genes asymmetrically 1 . ASEL expresses two putative chemoreceptor<br />
guanylyl cyclases gcy-6&7 and the homeobox gene lim-6, while ASER expresses a different guanylyl<br />
cyclase gcy-5. The ASE neurons are important for chemotaxis to soluble attractants including salts 3 . We<br />
tested whether the asymmetry in expression pattern correlated with an asymmetry in function by ablating<br />
ASER, ASEL, or both neurons in individual worms and tracked each animal during chemotaxis in<br />
gradients of ammonium chloride, sodium acetate, potassium acetate, and ammonium acetate. To<br />
measure chance performance, we tracked worms (n=74) in the absence of a gradient, but analyzed them<br />
as if they were in a gradient. Most ASER- (n=29) and ASE- (n=27) worms failed to reach the peak of the<br />
NH 4Cl gradient, while most ASEL- worms (n=22) reached the peak similar to sham worms<br />
(anaesthetized and recovered but not ablated; n=35). Likewise, most ASER- (n=17) and ASE- (n=15)<br />
worms failed to reach the peak of the K-acetate gradient, while most ASEL- worms (n=20) reached the<br />
peak similar to sham worms. Surprisingly, most ASEL- (n=26) and ASE- (n=12) worms failed to reach the<br />
peak of the Na-acetate gradient, while most ASER- worms (n=25) reached the peak similar to sham<br />
worms. <strong>Worm</strong>s did not perform better than chance in the ammonium-acetate gradient (n=37). Thus,<br />
ASER controls chemotaxis to Cl - and K + , while ASEL controls chemotaxis to Na + . In lim-6 deletion<br />
mutants gcy-5 is ectopically expressed in ASEL, while gcy-6&7 expression is correctly restricted to<br />
ASEL 2 . To examine if LIM-6 specifies asymmetry in ASE function, we tested whether ASE neurons retain<br />
their asymmetric function in lim-6 mutants. In contrast to wildtype worms, many ASER- lim-6 worms<br />
(n=28) were able to reach the peak of the ammonium chloride gradient. Thus, a homeobox gene is<br />
involved in breaking symmetry in neuronal function.<br />
1. Yu et al (1997) PNAS 95:3384-7<br />
2. Hobert et al (1999) Dev 126:1547-62<br />
3. Bargmann & Horvitz (1991) Neuron 7:729-42<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
28
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
INFORMATION CODING IN THE C. ELEGANS OLFACTORY<br />
SYSTEM<br />
PD Wes, A Sagasti, G Jansen, RHA Plasterk, CI Bargmann<br />
HHMI and Department of Anatomy. University of California, San Francisco, CA 94143-0452<br />
In order to better understand the molecular and cellular basis of behavior, we have begun to genetically<br />
scrutinize odorant discrimination in C. <strong>elegans</strong>. C. <strong>elegans</strong> senses a broad array of attractive odors with<br />
just two pairs of sensory neurons, AWA and AWC. Each member of the pair are thought to be largely<br />
identical, except that the G protein-coupled receptor, STR-2, is stochastically expressed in either the left<br />
or right AWC neuron. A number of odorants activate each neuron. Isoamyl alcohol (iaa),<br />
2,3-pentanedione (pd), butanone (bu) and benzaldehyde (bz) all activate AWC. Each odorant utilizes the<br />
same cGMP signaling cascade, yet animals retain the ability to distinguish amongst some of these<br />
odorants. For instance, when placed in a uniform field of bu, animals will no longer chemotax toward a<br />
point source of bu (defined as saturation), yet will chemotax toward bz. A screen was conducted to<br />
identify mutants that failed to chemotax toward a point source of bz in a field of bu (defined as<br />
cross-saturation). One mutant, ky542, displayed total cross-saturation, as well as defects in pd<br />
chemotaxis and olfactory adaptation. ky542 was cloned and found to be an allele of nsy-1, a "neuronal<br />
symmetry" mutant in which STR-2 is expressed in both AWC neurons. Other nsy mutants, including<br />
nsy-2, nsy-3 and egl-2(gf), also showed cross-saturation phenotypes. One model proposes that odorant<br />
discrimination is achieved by expressing the bu receptor in the STR-2 "ON" cell, the pd receptor in the<br />
STR-2 "OFF", and the bz receptor in both cells. A variety of forward and reverse genetic studies and<br />
ablation experiments support this model. Nevertheless, neuronal asymmetry cannot fully account for<br />
odorant discrimination since the 2 AWC neurons can distinguish at least 4 classes of odorants. For<br />
instance, iaa is distinguished from bu, bz and pd. Therefore, informational processing must also occur<br />
intracellularly. Since at least 6 Ga proteins are expressed in AWC, we reasoned that this protein family<br />
may provide the requisite degree of diversity for signaling specificity. Indeed, gpa-5 is required for<br />
discrimination between iaa and bu in AWC, suggesting that different odorants may employ specific<br />
modulatory signaling pathways within single cells.<br />
29
ROLES OF OSM-9/CAPSAICIN RECEPTOR FAMILY<br />
MEMBERS IN SENSORY BEHAVIORS<br />
D. Tobin 1 , D. Madsen 2 , G. Moulder 3 , R. Barstead 3 , A.V. Maricq 2 , M.<br />
deBono 4 , C. Bargmann 1<br />
1University of California San Francisco, HHMI<br />
2University of Utah<br />
3Oklahoma Medical Research Foundation<br />
4MRC, Cambridge<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The osm-9 gene is required for a wide range of sensory modalities in C. <strong>elegans</strong> including<br />
chemosensation, mechanosensation, osmosensation, and certain forms of olfactory adaptation. osm-9<br />
encodes a putative ion channel with striking homology to the vertebrate capsaicin receptor, a cation<br />
channel expressed in pain-sensing neurons and gated by the active component of chili peppers. In the<br />
amphid neuron AWA, OSM-9 localizes to sensory cilia, suggesting a direct role in sensory transduction.<br />
The vertebrate capsaicin receptor VR1 responds to heat, capsaicin, and low pH. Expression of<br />
mamamlian VR1 under an AWA-specific promoter partially rescued osm-9’s AWA defects. Thus, the<br />
homology between the two proteins may have functional relevance. However, OSM-9 is not sensitive to<br />
capsaicin. We found that expression of mammalian VR1 in C. <strong>elegans</strong> nociceptive neurons created a<br />
robust capsaicin-avoidance behavior. Heterologous expression of VR1 should be a useful tool for specific,<br />
drug-inducible neuronal activation.<br />
Four relatives of osm-9 are each expressed in subsets of osm-9-expressing cells. We call these genes<br />
ocr (osm-9/capsaicin receptor-related)genes and have generated mutations in two of them. ocr-1 is<br />
expressed primarily in AWA while ocr-2 is expressed in AWA, ASH, ADL, and ADF. Based on their<br />
expression patterns and similarity to osm-9 we hypothesized that the OCR channels might coassemble<br />
with OSM-9 to form heteromultimeric complexes. Mutations in ocr-2 recapitulate some of osm-9’s defects:<br />
there are dramatic defects in AWA-mediated chemotaxis and ASH-mediated avoidance behaviors. We<br />
propose that different combinations of subunits may account for the distinct functions of osm-9 in different<br />
sensory neurons.<br />
Interestingly, ocr-2 has a role in C. <strong>elegans</strong> social behavior. npr-1 encodes a putative neuropeptide<br />
receptor that regulates the choice between solitary and social foraging behavior. osm-9 and ocr-2 mutants<br />
suppress the clumping behavior of npr-1 mutants. We have performed single-cell rescue experiments to<br />
identify the neurons in which ocr-2 expression is required for social behavior. Identification of these<br />
neurons should help define the sensory signals and neuronal circuitry that mediate clumping.<br />
30
EXECUTION AND REGULATION OF MALE C. ELEGANS<br />
SPICULE MUSCLE CONTRACTIONS DURING MATING<br />
L. René García, Paul W. Sternberg<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HHMI & California Institute of Technology, Division of Biology, Pasadena, CA 91125 U.S.A.<br />
As the C. <strong>elegans</strong> male attempts to penetrate the hermaphrodite vulva with his spicules, the protractor<br />
muscles that are attached to these copulatory structures contract and relax rapidly such that the spicules<br />
prod the vulva slit at a frequency of 7.2 ± 1.3 Hz. Phasic protractor muscle contractions persist until the<br />
vulva slit is partially penetrated. Once the vulva is breached, the protractor muscles contract tonically and<br />
the spicules extend completely into the hermaphrodite. The spicule muscles stay contracted for 75 ± 20<br />
seconds, sufficient time for sperm to transfer into the hermaphrodite. The male protractor muscles are<br />
innervated by the SPC motor neurons (2), and removal of these cells results in male impotency (1). The<br />
SPC neurons are essential for tonic, but not phasic spicule muscle contractions; the spicules of<br />
SPC-ablated males can not fully extend into the hermaphrodite, but can prod the vulva at a frequency of<br />
5.1 ± 1.1 Hz.<br />
The ACh agonists levamisole, nicotine, arecoline, and oxotremorine can stimulate the protractor muscles<br />
to contract; and the SPC motor neurons support the expression of the unc-17-encoded VAChT. Therefore<br />
ACh most likely regulates the contractile state of the spicule muscles. The ACh agonists differentially<br />
require egl-19 and unc-68-encoded calcium channels to mediate the behavioral output; thus suggesting<br />
that calcium mobilization from these channels might have non-overlapping roles during copulation.<br />
During mating the spicules of egl-19 males can prod the vulva at a frequency of 5.0 ± 1.5 Hz, but they can<br />
not breach the vulva lips. In contrast, the spicules of unc-68 males can insert into the hermaphrodite, but<br />
prior to penetration, the spicules prod the vulva at a frequency of 0.48 ± 1.4 Hz. Therefore, the UNC-68<br />
channel is utilized in phasic muscle contractions, and the EGL-19 channel is required for tonic muscle<br />
contractions.<br />
1. Liu, K.S., and Sternberg, P.W. (1995).Neuron 14, 79-89.<br />
2. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78, 542-576.<br />
31
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GENETIC ANALYSIS OF NICOTINE ADAPTATION IN C.<br />
ELEGANS.<br />
Jinah Kim 1 , Laura E. Waggoner 2 , Kari A. Dickinson 2 , Daniel S.<br />
Poole 2 , William R. Schafer 3<br />
1Department of Neuroscience, University of California, San Diego, La Jolla, CA.<br />
2Department of Biology, University of California, San Diego, La Jolla, CA.<br />
3Departments of Biology and Neuroscience, University of California, San Diego, La Jolla, CA.<br />
In many organisms, prolonged exposure to nicotine causes long-lasting changes in the abundance and<br />
functional activity of nicotinic receptors, processes thought to underlie nicotine addiction in humans. At<br />
present, the molecular basis for nicotine adaptation is poorly understood. We have begun using a genetic<br />
approach to identify molecules required for nicotine adaptation in the egg-laying circuitry of C. <strong>elegans</strong>.<br />
Acute exposure to nicotine or the nicotinic agonist levamisole have dramatic effects on behavior, including<br />
stimulation of egg-laying. These acute effects of nicotine on egg-laying require the activity of a nicotinic<br />
receptor containing subunits unc-29, unc-38, and lev-1, as mutations in these genes confer resistance to<br />
nicotine and levamisole. This receptor appears to function in the vulval muscle, since expression of an<br />
unc-29 transgene under a vulval muscle-specific promoter in unc-29 mutant animals restored egg-laying<br />
sensitivity to levamisole. Upon long-term nicotine treatment, wildtype worms undergo adaptation, and<br />
acquire a long-lasting loss of egg-laying sensitivity. This effect is at least partially mediated at the level of<br />
receptor abundance, as the expression of an UNC-29:GFP chimera was drastically reduced upon<br />
overnight treatment with nicotine.<br />
In order to identify molecules required for the long-term effects of nicotine, we examined known mutants<br />
and screened for new mutants with defects in adaptation. From the known mutants, tpa-1, a PKC<br />
homologue, was shown to be necessary for the control of UNC-29 receptor abundance, since tpa-1<br />
mutants remained sensitive to levamisole and retained high UNC-29 receptor levels in the vulval muscles<br />
even after long-term nicotine treatment. We also identified a new gene, nic-1, in a screen for adaptation<br />
defective mutants. These mutants displayed hypersensitivity to nicotine and a defect in adaptation, as<br />
well as an unusual locomotive phenotype and a deficiency in male mating ability, all of which are<br />
consistent with a defect in cholinergic function in the neuromusculature. Currently, we are in the process<br />
of mapping nic-1 in order to clone it, and hope to discover the identity of the molecule encoded by this<br />
gene.<br />
32
NEURAL CONTROL OF LOCOMOTION IN C. ELEGANS<br />
Saleem Mukhtar 1 , Jane Mendel 2 , Jehoshua (Shuki) Bruck 1 , Paul W.<br />
Sternberg 2<br />
1Mail Code 136-93, Caltech, Pasadena. CA 91125.<br />
2Biology 156-29, Caltech, Pasadena. CA 91125.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Our long-term goal is to understand the functioning of the neural circuit responsible for movement in C.<br />
<strong>elegans</strong>. We hope to accomplish this by perturbing the nervous system and observing what effect this has<br />
on the dynamics of the worm.<br />
We have developed an automated worm tracking system that enables us to record moving worms. We<br />
have also developed an automated worm recognition system that enables us to extract quantitative data<br />
describing the worm’s movement from these videos. Using these tools we have been able to uncover<br />
important constraints on the dynamics of wildtype worms that are imposed by the underlying neural<br />
circuitry. Based on these constraints we have formulated a set of physically relevant metrics that can be<br />
used to quantitatively compare the dynamics of two sets of worms.<br />
By applying these tools to the full spectrum of perturbations - genetic, pharmacological and cell ablations,<br />
we hope to elucidate the functioning of the ventral cord circuit for locomotion.<br />
33
ANALYSIS OF GLUTAMATERGIC NEUROTRANSMISSION BY<br />
KNOCKOUT OF GLUTAMATE TRANSPORTER GENES.<br />
Itzhak Mano, Monica Driscoll<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, NJ.<br />
The amino acid L-Glutamate (Glu) is a neurotransmitter that mediates most of the excitatory<br />
neurotransmission in the human brain and is thus central to development, basic physiology and higher<br />
brain functions. Exaggerated activation of glutamatergic transmission leads to neuronal cell death (in a<br />
process termed excitotoxicity) and is believed to be a cause or a contributing factor to many acute and<br />
chronic neurological disorders, including stroke and ALS. In order to ensure accurate response to rapid<br />
Glu signals and to avoid buildup of toxic levels of this transmitter, synaptic Glu is rigorously pumped out of<br />
the synapse by specialized Glu Transporters (GluTs). The molecular components of Glu synapses, and in<br />
particular GluTs, are highly conserved from nematodes to humans, suggesting that a detailed description<br />
of the molecules and the processes involved in normal and pathological Glu neurotransmission in C.<br />
<strong>elegans</strong> might be used to gain insight to these processes in higher animals.<br />
Since a key feature common to the initial steps of many neurodegenerative disorders is a decline in GluT<br />
efficacy, we decided to create synaptic Glu buildup by systematic knockout of C. <strong>elegans</strong> GluT genes. We<br />
have so far knocked out 3 of the 6 GluT genes in the worm, and we are continuously screening additional<br />
deletion libraries for more GluT mutants. Analysis of the phenotypes of the current GluT deletion mutants<br />
by themselves and in combination with other Glu-related mutants is underway. Initial observations<br />
indicate that GluTs are key regulators of pharyngeal pumping and of the responses to a range of<br />
chemical, osmotic and mechanical stimuli. Together with other studies of Glu neurotransmission in C.<br />
<strong>elegans</strong>, these observations serve as initial steps for a detailed molecular and cellular description of key<br />
processes of synaptic function and behavior. Furthermore, the phenotypes of these GluT knockout<br />
mutants can serve as the bases for genetic screens aimed at the identification of additional components<br />
of the pathways involved in normal and pathological Glu neurotransmission.<br />
34
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ELECTROPHYSIOLOGICAL ANALYSIS OF C. ELEGANS<br />
IONOTROPIC GLUTAMATE RECEPTORS<br />
Jerry E. Mellem, Penelope J. Brockie, David M. Madsen, Andres V.<br />
Maricq<br />
Dept. of Biology University of Utah 257 S. 1400 E. Salt Lake City UT 84112<br />
A fundamental problem in neurobiology is to understand how neuronal circuits function to control<br />
behavior. A simple neuronal circuit that controls movement has been identified in the nematode<br />
<strong>Caenorhabditis</strong> <strong>elegans</strong>. In this circuit, the command interneurons AVA, AVB, AVD, AVE, and PVC are<br />
essential for coordinated movement and escape from tactile stimuli.<br />
Our lab has shown that six putative ionotropic glutamate receptor subunits are expressed in the command<br />
interneurons and that perturbation of the subunits nmr-1 and glr-1 lead to altered locomotion (see abstract<br />
by Brockie et. al.). To better understand how ionotropic glutamate receptors modulate the activity of the<br />
locomotory control circuit, we are undertaking an electrophysiological analysis of the receptors expressed<br />
in the command interneuron AVA.<br />
We have modified the slit worm dissection to isolate neurons of the locomotory control circuit. Using<br />
patch-clamp techniques, we have recorded glutamate-dependent currents from the neuron AVA. The<br />
current can be elicited by glutamate, kainate, and NMDA and can be partially blocked by selective<br />
pharmacological antagonists. The current rapidly desensitizes to glutamate and recovery from<br />
desensitization is slow. The current evoked by NMDA is voltage dependent and exhibits an outward<br />
rectification.<br />
We have also determined the electrophysiological defects underlying the behavioral phenotypes observed<br />
in the glr-1 and nmr-1 deletion mutants. Both mutants display a diminshed response to glutamate. The<br />
nmr-1 mutant, in addition, has an aberrant response to NMDA.<br />
We will describe the dissection used to isolate the command interneuron circuitry, our findings on how<br />
glutamate receptors function in wild type and mutant animals, and our initial insights into how glutamate<br />
receptors modulate the activity of the locomotory control circuit.<br />
35
ELECTROPHYSIOLOGICAL ANALYSIS OF UNC-18<br />
MUTANTS.<br />
J. E. Richmond, R. Weimer, W. S. Davis, E. M. Jorgensen<br />
University of Utah, Salt Lake City, Utah 84112<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UNC-18 is a C. <strong>elegans</strong> neuron-specific protein belonging to a conserved protein family implicated in<br />
vesicle fusion. UNC-18 interacts with the SNARE protein syntaxin, which is believed to mediate fusion of<br />
synaptic vesicles with the plasma membrane. Mutations in unc-18 result in severe locomotory defects and<br />
acetylcholine accumulation, implying a critical role for UNC-18 in synaptic transmission. Three mutants<br />
were examined, unc-18(e81 and e234) which have premature stop codons and unc-18(b403) which<br />
disrupts binding to syntaxin. The mutant phenotype was not caused by developmental abnormalities since<br />
GFP-expression patterns revealed motor neurons had normal morphology and exhibited wild-type<br />
distributions of synapses. We have used electrophysiological techniques to analyze synaptic function in<br />
these unc-18 mutants. Evoked release at the C. <strong>elegans</strong> neuromuscular junction was reduced to 5% of<br />
wild-type amplitudes (from 1800pA to 100pA) in all three mutants. A similar reduction in endogenous<br />
synaptic event frequency (from 44Hz to 5 Hz) was observed. The mutant synaptic event amplitudes were<br />
unaffected demonstrating that the reduction in the evoked response was not due to a defect in vesicle<br />
neurotransmitter filling or in post-synaptic receptor sensitivity. Preliminary EM data showed an<br />
accumulation of vesicles at the neuromuscular synapses of unc-18 mutants consistent with a defect in<br />
exocytosis as opposed to defects in vesicle biogenesis or retrieval. In the absence of external Ca2+, the<br />
rates of spontaneous fusion events were also reduced, suggesting that either the docking or fusion<br />
competence of unc-18 mutants is defective. Given these data, how might UNC-18 function in exocytosis?<br />
UNC-18 forms a heterodimer with syntaxin, which is mutually exclusive of SNARE complex formation.<br />
Several studies suggest that the interaction of UNC-18 and syntaxin is an essential step in regulated<br />
release, and that this interaction may be required to promote a conformational change in syntaxin which<br />
facilitates SNARE complex formation. To test this hypothesis, we have engineered a double mutation in<br />
syntaxin, which, in vitro eliminates UNC-18 binding, and induces the syntaxin open configuration. We are<br />
currently examining the effects of this mutation in wild-type, unc-64 (syntaxin) and unc-18 mutants.<br />
36
ELECTROPHYSIOLOGICAL ANALYSIS OF SEROTONIN<br />
MODULATION OF BODY WALL NEUROMUSCULAR<br />
PHYSIOLOGY.<br />
Jon Madison, Joshua Kaplan<br />
361 LSA, Univ. of Cal., Berkeley, CA 94720<br />
The ability of neurons to alter their synaptic function underlies our ability to control behavior. We are<br />
interested in understanding how neurotransmitters like serotonin (5-HT) cause changes in neuron and<br />
synapse function to effect behavioral changes. In C. <strong>elegans</strong>, 5-HT modulates the rate of locomotion.<br />
Recent analysis has shown that 5-HT acts presynaptically on motor neurons through a G-protein Gao<br />
subunit, GOA-1, to reduce acetylcholine (ACh) release at body wall neuromuscular junctions (NMJs) [1].<br />
Using a recently developed dissection technique and whole cell voltage clamp recordings [2], we have<br />
recorded the effects of 5-HT on adult body wall NMJ physiology.<br />
We have recorded from body wall muscle both miniature excitatory post-synaptic currents (mEPSCs) and<br />
muscle responses to ACh application in the presence of 5-HT. We see two effects of 5-HT: a reduction in<br />
EPSC frequency and a reduction in ACh-activated current amplitude. The amplitude of ACh-activated<br />
current is reduced by 40% in the presence of 5-HT. Previous physiological analysis by Richmond and<br />
Jorgensen has shown there to be two classes of ACh receptors at the body wall NMJ; one ACh receptor<br />
class is sensitive to the cholinergic agonist levamisole and one receptor is insensitive [2]. Recordings of<br />
levamisole-activated current show that 5-HT reduces this current up to 90%. These data suggest that the<br />
levamisole activated ACh receptors may be specifically modulated by 5-HT. To resolve the contribution of<br />
post-synaptic modulation by 5-HT to presynaptic changes in mEPSC frequency and to further understand<br />
the mechanism of the post-synaptic response, we have recorded from mutants that might eliminate the<br />
post-synaptic ACh receptor modulation. Since 5-HT’s downstream effects are mediated by G-protein<br />
coupled pathways, we have tested a number of candidate signal transduction mutants expressed in<br />
muscle for defects in ACh receptor modulation. We have tested the G-protein Ga subunit mutants, gpa-14<br />
and gpa-7, and an adenylate cyclase, acy-1, and all are normal for ACh receptor modulation by 5-HT.<br />
Future physiologic and genetic experiments will help further our understanding of 5-HT’s modulation of<br />
synaptic function and behavior.<br />
1. Nurrish et al., Neuron 24: 231-242 1999.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
2. Richmond and Jorgensen, Nat Neurosci 2: 791-797 1999.<br />
37
SEROTONIN SIGNALING IN THE PHARYNX<br />
Timothy Niacaris 1,2 , Leon Avery 1,3<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1University of Texas Southwestern Medical Center, Department of Molecular Biology, Dallas, TX<br />
75390-9148<br />
2tim@eatworms.swmed.edu<br />
3leon@eatworms.swmed.edu<br />
Serotonin increases the rate of pharyngeal pumping. This effect occurs by at least two different<br />
mechanisms. Serotonin can indirectly stimulate pharyngeal muscle by increasing the activity of the<br />
pharyngeal motorneuron MC. Serotonin can also act independent of the pharyngeal nervous system to<br />
increase pharyngeal pumping rate, presumably by acting directly on pharyngeal muscle. Serotonin has<br />
two additional MC-independent effects on the pharynx. It decreases the duration of pharyngeal<br />
contractions and enhances the effect of the pharyngeal motorneuron M3. To identify the receptor that<br />
mediates these effects, we have searched the genomic sequence for candidate serotonin receptors and<br />
determined their expression patterns. One strong candidate for mediating the effects of serotonin on<br />
pharyngeal muscle is ser-1. ser-1 is expressed predominantly in pharyngeal muscle and has recently<br />
been determined to be responsive to serotonin in a heterologous expression system 1 . We have isolated<br />
a deletion that eliminates the 3’ end and UTR of ser-1. ser-1(ad1675) truncates the receptor and<br />
eliminates a large portion of the C-terminal intracellular tail, a region important for several aspects of<br />
receptor desensitization. ser-1(ad1675) mutants have an exaggerated response to exogenous serotonin,<br />
consistent with an inability to desensitize in the presence of serotonin. We are currently characterizing<br />
worms defective in other components of the G-protein signaling cascade to identify proteins responsible<br />
for mediating the effects of serotonin on pharyngeal muscle.<br />
1 Hamdan et al. (1999) J. Neurochemistry 72(4):1372-83<br />
38
A MUSCARINIC CONTRIBUTION TO THE REGULATION OF<br />
FEEDING<br />
Kate Steger 1,2 , Leon Avery 3<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1Department of Biology, McGill University. 1205 Dr. Penfield Ave. Montreal, QC, Canada<br />
2kate@eatworms.swmed.edu<br />
3Department of Molecular Biology, UT Southwestern Medical Center. 6000 Harry Hines Blvd. Dallas, TX<br />
Acetylcholine (Ach) is essential for worms’ survival and can stimulate feeding through nicotinic receptors<br />
on the pharyngeal muscle. However, we suspect that Ach affects the pharynx through muscarinic<br />
receptors as well. <strong>Worm</strong>s that lack Ach (e.g. cha-1 mutants) have a more severe feeding defect than<br />
worms that lack pharyngeal nicotinic transmission (eat-2; eat-18). Atropine, a muscarinic antagonist,<br />
causes worms to appear starved, although it increases their pumping rate.<br />
Three ORFs in the worm genome closely resemble vertebrate muscarinic receptors: C15B12.5 (acm-1),<br />
F47D12.2 (acm-2) and C53A5.12 (acm-3). We have examined the expression of these three genes: all<br />
three are expressed in pharyngeal tissue (muscle, neurons or both) and in the extra-pharyngeal nerve<br />
ring.<br />
We have obtained mutant alleles of two putative muscarinic receptors: acm-1, and acm-2 (a gift from<br />
Stefan Eimer and Rolf Baumeister). The two mutations have very different effects on worm physiology.<br />
acm-2 worms are hypersensitive to aldicarb (an inhibitor of cholinesterase) and to nicotine. They pump<br />
more rapidly than wild type worms in the absence of drug, and decrease their pumping rate in the<br />
presence of atropine. We suspect that acm-2 mutants are defective in down-regulating nicotinic<br />
transmission. We hypothesize that acm-2, which is expressed in neurons both inside and outside the<br />
pharynx, acts as a negative regulator at nicotinic synapses.<br />
acm-1 mutants, in contrast, are resistant to aldicarb, and are not hypersensitive to nicotine. In an unc-17<br />
background of reduced cholinergic transmission, acm-1 worms are hypersensitive to atropine. We<br />
suspect that acm-1, probably in combination with acm-3, affects pumping efficiency or the adaptation of<br />
feeding behavior to particular circumstances. We are currently designing assays and appropriate genetic<br />
backgrounds to examine these possibilities<br />
39
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
WORMBASE: FROM ACEDB TO A MORE COMPLETE AND<br />
USABLE DATABASE<br />
Paul W. Sternberg, Erich Schwarz, Norma Foltz, <strong>Worm</strong>Base<br />
Consortium<br />
Division of Biology 156-29, Caltech, Pasadena, CA 91125<br />
We briefly describe the current status and plans for <strong>Worm</strong>Base, initially an extension of the existing<br />
ACeDB database with a new user interface. The <strong>Worm</strong>Base consortium includes the team that developed<br />
ACeDB (Richard Durbin and colleagues at the Sanger Centre; Jean Thierry-Mieg and colleagues at<br />
Montpellier); Lincoln Stein and colleagues at Cold Spring Harbor, who developed the current web<br />
interface for <strong>Worm</strong>Base; and John Spieth and colleagues at the Genome Sequencing Center at<br />
Washington University, who along with the Sanger Centre team, continue to annotate the genomic<br />
sequence. The Caltech group will curate new data including cell function in development, behavior and<br />
physiology, gene expression at a cellular level, and gene interactions. Data will be extracted from the<br />
literature, as well as by community submission. We look forward to providing the C. <strong>elegans</strong> and broader<br />
research community easy access to vast quantities of high quality data on C. <strong>elegans</strong>. Also, we welcome<br />
your suggestions and criticism at any time. <strong>Worm</strong>Base can be accessed at www.wormbase.org.<br />
40
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE C. ELEGANS ORFEOME PROJECT<br />
Jerome Reboul 1 , Philippe Vaglio 1 , Cindy Jackson 2 , Troy Moore 2 ,<br />
Jean Thierry-Mieg 3 , Danielle Thierry-Mieg 3 , Jim Hartley 4 , Gary<br />
Temple 4 , Mike Brasch 4 , Nia Tzellas 1 , Marc Vidal 1<br />
1Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, MA, USA<br />
2Research Genetics, Huntsville, AL, USA<br />
3IGM-CNRS, Montpellier, France<br />
4Life Technologies Inc., Rockville, MD, USA<br />
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and<br />
microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein<br />
interaction mapping, protein localization mapping, and biochemical and structural genomics. Most of<br />
protein-based approaches rely upon the availability of near complete set of open reading frames<br />
("ORFeomes") cloned into various expression vectors (i.e., for each ORF: the sequence between the start<br />
and the stop codons, in the absence of 5’ and 3’ untranslated sequences and introns).<br />
To clone the C. <strong>elegans</strong> ORFeome into various expression vectors, we use a Recombination Cloning<br />
technique (RC) referred to as Gateway TM (Walhout et al., 2000, Science, 287, 166-122). RC allows both<br />
the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific<br />
recombination in vitro. In addition, RC is amenable to automation in 96-well (or 384-) plate settings, which<br />
is crucial for large-scale ORFeome cloning. So far we have cloned 2,000 C. <strong>elegans</strong> ORFs. At the current<br />
throughput (~400 ORFs/week), ~70% of the C. <strong>elegans</strong> ORFeome should be cloned by the end of the<br />
year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a<br />
description of the cloning quality, iv) how this resource will be made available to the community, and v)<br />
how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et<br />
al).<br />
41
ANALYSIS OF SPLICING AND REGULATORY ELEMENTS<br />
USING THE INTRONERATOR<br />
W. James Kent, Alan M. Zahler<br />
University of California, Santa Cruz<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The Intronerator (http://www.cse.ucsc.edu/~kent/intronerator/) is a set of web-based tools for exploring<br />
RNA splicing, gene structure, and cross-species alignments in C. <strong>elegans</strong>. It features a display of<br />
mRNA/DNA and cross-species DNA/DNA alignments. It also includes a flexible tool for extracting<br />
sequences, a database of introns, and a catalog of alternatively spliced genes. This catalog of 845<br />
alternatively spliced genes identified through comparisons of ESTs with genomic sequence is currently<br />
the largest database of alternatively spliced genes available for any organism. The use of the Intronerator<br />
will be demonstrated and we will explain in brief the algorithms behind the program, and the use of the<br />
program to explore regulatory regions. We will present observations made during the alignment of 8<br />
million bases of C briggsae genomic DNA with the C. <strong>elegans</strong> genome, including characteristics of introns<br />
present in one species but not the other, the ability to confirm the identification of genes for which no EST<br />
data is available, and the degree of rearrangement that has occurred in the ~50 million years since the<br />
two species diverged.<br />
42
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A GLOBAL PROFILE OF GERM LINE GENE EXPRESSION<br />
USING MICROARRAYS REVEALS GERM LINE-SPECIFIC<br />
REGULATION OF THE X CHROMOSOME IN MALES AND<br />
HERMAPHRODITES<br />
Valerie Reinke 1 , Harold E. Smith 2 , Jeremy Nance 2 , Abby F.<br />
Dernburg 1 , Anne M. Villeneuve 1 , Samuel Ward 2 , Stuart K. Kim 1<br />
1Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305<br />
2Department of Molecular and Cellular Biology, University of Arizona, Tuscon, AZ 85721<br />
We have produced DNA microarrays containing genomic PCR products corresponding to 11,917 C.<br />
<strong>elegans</strong> genes. To identify genes expressed in the germ line, we compared wild-type gene expression<br />
levels to that of glp-4 mutants, in which the germ line precursor cells do not proliferate. We also compared<br />
a mutant strain making only sperm, fem-3(gf), to a mutant strain producing only oocytes, fem-1(lf), to<br />
identify both sperm-enriched and oocyte-enriched genes. Using a statistical criterion for significance,<br />
these experiments together define 1416 germ line-expressed genes that fall into three categories: 650<br />
sperm-enriched, 258 oocyte-enriched, and 508 germ line-intrinsic. We have determined the temporal<br />
expression pattern of each of these genes during development. These germ line genes comprise a<br />
molecular definition of germ line components, and also provide the framework to identify individual genes<br />
involved in specific germ line functions. The sperm-enriched group contains an unusually large number of<br />
protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of<br />
embryonic signaling pathways. The germ line-intrinsic group, defined as genes expressed similarly in<br />
germ lines making only sperm or only oocytes, contains a family of piwi-related genes that may be<br />
important for stem cell proliferation. Surprisingly, we found evidence for germ line-specific regulation of<br />
the X chromosome. Sperm-enriched and germ line-intrinsic genes are nearly absent from the X<br />
chromosome, and X-linked oocyte-enriched genes are expressed at about three fold lower levels than<br />
autosomal genes. Further, a marker for active gene expression (acetylated histone H4) is detectable on<br />
autosomes but staining is strongly reduced on the X chromosomes in germ line nuclei.<br />
43
THE PROMISE AND PERIL OF GENOMICS: SPERM<br />
DEVELOPMENT AS MODEL SYSTEM<br />
Harold Smith, Marci Millhouse, Sam Ward<br />
University of Arizona, Tucson, AZ 85721<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Microarray screening allows the investigator to profile expression patterns on a genome-wide scale.<br />
However, even a successful screen can generate a daunting amount of data. In our effort to identify<br />
genes involved in sperm development, we compared expression levels in fem-3(gf) mutants, which make<br />
only sperm, to fem-1(lf) mutants, which make only oocytes. We identified 650 sperm-enriched genes (as<br />
well as 258 oocyte-enriched genes) out of 11,917 genes screened (see Reinke et al.). Now, how do we<br />
use this information? We have focused our efforts in two areas: 1) obtaining deletion mutations in<br />
potentially interesting genes; and 2) identifying sperm promoter elements and their relevant transcription<br />
factors.<br />
Prior work with the calmodulin inhibitor trifluoperazine (TFP) had implicated Ca++ function in both sperm<br />
activation and motility; therefore, we generated a deletion allele of the sperm-enriched Ca++ channel<br />
gene K01A11.4 (since named spe-39). The spe-39 mutant exhibits reduced fertility and an aberrant<br />
sperm morphology that mimics TFP treatment. We can now examine the role of other spe mutants in<br />
Ca++ sensing, sperm activation, and motility.<br />
We are using an in silico approach to identify potential sperm promoter elements, and molecular and<br />
genetic analyses to confirm functional significance. The algorithm (written by John Anderson, NCBI)<br />
determines which sequences are over-represented in the 5’ upstream sequences of sperm genes<br />
compared to non-sperm genes. One of these sperm-enriched sequences contains two potential binding<br />
sites for the GATA transcription factor elt-1. Prior work has shown that elt-1 is required to specify<br />
hypodermal cell fates in the developing embryo; however, our microarray data also identified elt-1 as a<br />
sperm-enriched gene. Yeast one-hybrid screening demonstrates that elt-1 binds to and activates<br />
transcription from these putative promoter elements. In collaboration with Barbara Page, we are currently<br />
investigating the role of elt-1 in sperm development.<br />
44
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
FUNCTIONAL ANALYSIS OF CHROMOSOME I<br />
Andrew Fraser, Ravi Kamath, Peder Zipperlen, Maruxa<br />
Martinez-Campos, Julie Ahringer<br />
Wellcome-CRC Institute, Tennis Court Road, Cambridge, UK<br />
RNAi is a powerful and specific means of inhibiting gene function. However, the most widely used<br />
technique for RNAi, the injection of dsRNA into adult animals, is too labour-intensive to allow efficient<br />
genome-wide screening for gene function by RNAi. An alternative approach for RNAi is to feed bacteria<br />
expressing dsRNA to worms. We have determined conditions for which RNAi by bacterial feeding is as<br />
potent as RNAi by injection. We have constructed a library of dsRNA-expressing bacteria that can be<br />
used to target 90% of genes on chromosome I by RNAi. This reagent can be used for an unlimited<br />
number of low-cost screens for gene function. We have used this library to screen for all genes on<br />
chromosome I that give a clear phenotype when targeted by RNAi. We will present our results and<br />
discuss their implications for genome-wide functional analysis of C. <strong>elegans</strong> genes.<br />
45
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
OPTIMIZING THE MUTAGENIC PROPERTIES OF THE MOS1<br />
TRANSPOSON IN C. ELEGANS<br />
Daniel C. Williams, Jean-Louis Bessereau, Erik M. Jorgensen<br />
Department of Biology, University of Utah, Salt Lake City, UT, 84112<br />
Forward genetic analysis in C. <strong>elegans</strong> provides an unbiased method to determine the function of<br />
individual genes. However, identifying the physical location and molecular nature of a mutation that<br />
results in a specific phenotype is quite laborious. One way to avoid genetic mapping is to use<br />
transposable elements as mutagens, which provide a tag at the mutation site. The C. <strong>elegans</strong> genome<br />
contains multiple copies of endogenous transposons, which hinder efforts to isolate the insertion of<br />
interest. Recently, we demonstrated that Mos1 (a Tc1/mariner family member from D. mauritiana) can<br />
hop in C. <strong>elegans</strong>. Mos1 insertions in the germ-line result in a unique tag at the precise location of gene<br />
disruption. Unfortunately, these results also demonstrate that Mos1 is an inadequate mutagen for two<br />
reasons: (1) the frequency of transposition is too low for genetic screens and (2) Mos1 insertions may be<br />
phenotypically silent.<br />
We are taking a number of approaches to make Mos1 a more effective mutagen. In order to increase<br />
transposition frequency, we determined whether the local DNA structure around Mos1 substrate<br />
molecules affects hopping. We determined that transposition of Mos1 can occur if substrate molecules<br />
are located in repetitive or complex arrays. Current experiments address whether Mos1 copy number<br />
within an array correlates with transposition frequency<br />
Exonic Mos1 insertions may be phenotypically silent because they are spliced out during mRNA<br />
processing in the same manner as Tc1 elements (Rushforth et al. 1996). We generated recombinant<br />
Mos1 elements that contain a polyadenylation signal in the middle of the transposon that should prevent<br />
splicing of the insertion and result in a truncated transcript. These experiments also allow characterization<br />
of the cis factors present within Mos1 that are necessary for transposition. During these experiments we<br />
isolated an allele of dpy-17. Using inverse PCR the location of this insertion was determined to be 176<br />
base pairs upstream of the putative DPY-17 open reading frame F54D8.1. Determination of the physical<br />
location of this mutation was extremely rapid, suggesting that Mos1 will be a valuable tool for forward<br />
genetic analysis.<br />
Rushforth, A. M. and P. Anderson (1996). Mol Cell Biol 16(1): 422-9.<br />
46
SNAP-25, A PROTEIN IMPLICATED GENETICALLY IN C.<br />
ELEGANS ANESTHETIC MECHANISMS, BINDS THE<br />
GENERAL ANESTHETIC ISOFLURANE<br />
Jason Berilgen 1 , Mike Crowder 1,2<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1Department of Anesthesiology, Washington University School of Medicine<br />
2Department Molecular Biology/Pharmacology, Washington University School of Medicine<br />
Mutations in the neuronal syntaxin gene unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of<br />
C. <strong>elegans</strong>. Two hypomorphic unc-64 alleles confer hypersensitivity to the VAs isoflurane and halothane,<br />
but a third unc-64 hypomorph, md130, which produces a truncated syntaxin product, is VA resistant. The<br />
difference between the isoflurane EC 50s of the hypersensitive and resistant alleles is over 30-fold; this is<br />
by far the biggest allelic variation in VA sensitivity thusfar seen in any animal. Given that these allelic<br />
differences cannot be explained by indirect effects on synaptic transmission and can be rescued and<br />
knocked-in, we hypothesized that syntaxin or a syntaxin-binding protein binds VAs and that the truncated<br />
md130 product somehow interferes with that binding. We have looked for isoflurane binding to syntaxin<br />
and to two syntaxin-binding proteins, VAMP and SNAP-25, which form a ternary complex with syntaxin to<br />
mediate synaptic vesicle fusion. Recombinant expression proteins (C. <strong>elegans</strong> syntaxin and VAMP -<br />
coded for by snb-1, and rat SNAP-25 - plasmids all kindly provided by M. Nonet) without their<br />
transmembrane domains or lipid modifications were used. We used 19 F-NMR to measure binding of<br />
isoflurane to the purified proteins. The transverse relaxation time (T2) of a nucleus is known to decrease<br />
when its Brownian motion decreases; thus, binding of small molecules (eg. isoflurane) to larger ones (eg.,<br />
a protein) shortens T2s. We found that the T2 of the CF 3-moiety of isoflurane was significantly shorter in<br />
solutions containing SNAP-25 than in buffer alone and was protein and isoflurane concentration<br />
dependent, changing over the in vivo relevant isoflurane concentration range. Recombinant syntaxin had<br />
no effect on the T2. We are currently gathering data for VAMP and the ternary complex. Thus, our data<br />
show that SNAP-25 but not syntaxin binds isoflurane at relevant concentrations; SNAP-25 is the first<br />
neuronal protein shown to bind VAs. Given our genetic data in C. <strong>elegans</strong> and electrophysiologic data in<br />
vertebrates showing that neurotransmitter release is reduced by VAs, we think that SNAP-25 may be a<br />
VA target that mediates some aspects of general anesthesia.<br />
47
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HAPPY WORMS: FURTHER CHARACTERIZATION OF<br />
FLUOXETINE (PROZAC) RESISTANT MUTANTS<br />
Robert K.M. Choy 1,2 , James H. Thomas 2,3<br />
1Program in Molecular and Cellular Biology, Box 357360, University of Washington, Seattle, WA<br />
98195-7360<br />
2rchoy@genetics.washington.edu<br />
3Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195-7360<br />
Although over 20 million people have taken fluoxetine for a wide range of mental disorders, its molecular<br />
mechanism of action remains unproven. Fluoxetine is a member of the selective serotonin reuptake<br />
inhibitor (SSRI) class of antidepressants, which inhibit the presynaptic serotonin reuptake transporter.<br />
However, it is still unclear whether this inhibition is responsible for their antidepressant action.<br />
Furthermore, the targets responsible for the various side-effects of SSRIs are poorly characterized.<br />
We previously reported that in C. <strong>elegans</strong>, SSRIs induce contraction of nose and body-wall muscles by<br />
acting on a non-serotonergic target. We also reported the isolation and characterization of several<br />
mutants that are nose resistant to fluoxetine (Nrf). These mutants are cross-resistant to other SSRIs, but<br />
are fully sensitive to several other drugs that also induce nose muscle contraction. Therefore, these Nrf<br />
mutations may identify novel genes relevant to antidepressant action.<br />
Mutations in three of the Nrf genes (nrf-5, nrf-6 and ndg-4) have a common secondary phenotype of<br />
producing pale eggs (Peg). These mutants are defective in yolk transport and accumulate yolk in the<br />
pseudocoelomic space. nrf-6 and ndg-4 both encode homologous transmembrane proteins and define a<br />
novel gene family of several dozen members in C. <strong>elegans</strong> and Drosophila. By a combination of mosaic<br />
analysis and tissue specific promoters, we have found that nrf-6 function is required in the intestine for<br />
both fluoxetine-induced nose contraction and yolk transport. We have also cloned nrf-5 and found that it<br />
is homologous to the mammalian BPI/CETP family of secreted lipid binding proteins. One possibility is<br />
that these genes are involved in a novel aspect of antidepressant transport. Alternatively, they may<br />
mediate some aspect of lipid metabolism that is required for antidepressant-induced nose muscle<br />
contraction.<br />
48
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UNC-43 CA2+/CALMODULIN-DEPENDENT KINASE II<br />
(CAMKII) MUTANT WORMS HAVE CONVULSIONS IN<br />
RESPONSE TO THE SEIZURE-INDUCING DRUG PTZ<br />
Elizabeth M. Newton, James H. Thomas<br />
Dept. of Genetics, University of Washington, Seattle WA 98195<br />
Epilepsy is one of the most common neurological disorders, effecting an estimated 50 million people<br />
worldwide. While most seizures are treatable with present anti-convulsant drugs, approximately 20-30%<br />
of patients remain refractory to drug therapy. Recent work in mouse models has begun to identify genes<br />
involved in seizure susceptibility, but much about the mechanism of seizure susceptibility and the<br />
mechanism of anti-epileptic drugs remains to be elucidated.<br />
Null mutations in C. <strong>elegans</strong> unc-43 CaM Kinase II cause worms to appear nervous and jerky with<br />
occasional spontaneous muscle contractions that resemble convulsions. To test if these contractions<br />
could be related to seizure-like convulsions, we put unc-43 null worms on plates containing<br />
pentylenetetrazole (PTZ), a seizure-inducing drug commonly used in rodent epilepsy models. The<br />
phenotype observed is dramatic: the worms begin to have fast, strong simultaneous contractions of the<br />
anterior and posterior body muscles in an uncontrolled fashion, completely disrupting normal locomotion.<br />
(A videotape of this phenotype will be shown.) Wild-type worms appear mildly hyperactive in response to<br />
PTZ, as expected for a CNS stimulant, and become slightly jerky and less coordinated over time but<br />
never have convulsions, even at doses 10 fold higher than used to induce convulsions in unc-43 mutants.<br />
Other drugs that induce muscle contraction but are not convulsive in mouse models, such as aldicarb and<br />
levamisole, do not induce convulsions in unc-43 mutants but instead induce the expected<br />
hypercontraction/paralysis phenotype. a-CaM Kinase II knock-out mice are epileptic, supporting the idea<br />
that worm convulsions may be related to seizures in rodent models. The unc-43 convulsion phenotype<br />
has a neuronal basis as it is rescued by a transgene expressing an unc-43 cDNA from the aex-3<br />
promoter, which drives expression in all neurons but not muscle.<br />
We tested a number of different mutant strains that might be expected to have hyper-excited neuronal<br />
activity, such as loss of function mutations in potassium channels and mutations in the goa-1 pathway,<br />
but none had convulsions in response to PTZ. We are currently screening for more seizure-susceptible<br />
mutants to determine if this phenotype is specific to unc-43 CaM kinase II. We have also begun screening<br />
for suppressors of the unc-43 PTZ-induced convulsion phenotype, and we have isolated several<br />
independent mutants, indicating that it is likely that there are many genes involved in this response.<br />
Studying suppressors of convulsions may identify new theraputic targets for the future.<br />
49
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
NEUROTOXIN SENSITIVITY OF DOPAMINERGIC NEURONS<br />
IN C. ELEGANS: ROLE OF THE DOPAMINE TRANSPORTER<br />
AND CELL DEATH PATHWAYS<br />
R. Nass 1,2 , J. Duerr 3 , , J. Rand 3 , D. M. Miller 2,4 , R. D. Blakely 1,2<br />
1Dept of Pharmacol, Vanderbilt U. Med. School, Nashville, TN<br />
2Ctr. for Molecular Neuroscience, Vanderbilt U. Med. School, Nashville, TN<br />
3Program in Molecular and Cell Biology, OMRF, Oklahoma City, OK<br />
4Dept of Cell Biology, Vanderbilt U. Med. School, Nashville, TN<br />
The dopamine transporter (DAT) constitutes the primary mechanism for the inactivation of dopamine (DA)<br />
neurotransmission in the brain. DATs are targets for many psychoactive drugs and the cellular gateway<br />
for the accumulation of the neurotoxin 6-hydroxydopamine (6-OHDA) which evokes neuronal death and<br />
Parkinson-like syndrome in animal models. We have previously cloned and tagged the C. <strong>elegans</strong> DAT<br />
(CeDAT), and have shown that it is functionally similar to mammalian DATs and expressed exclusively in<br />
the DA neurons (Jayanthi et al. 1998, Nass et al. 1999). We have also developed WT and DAT knockout<br />
transgenic lines which target a CeDAT promotor-GFP fusion to all 8 DA neurons in the hermaphrodite.<br />
Intense GFP expression is seen throughout the axons and dendrites in the live animals. We now show<br />
that a brief exposure to 6-OHDA results in the blebbing of DA neuronal processes, swelling of DA<br />
neuronal cell bodies, and the loss of CeDAT-GFP expression, while co-exposure with CeDAT substrates<br />
or antagonists significantly reduces the 6-OHDA induced response. We also show that this response is<br />
dependent on the expression of CeDAT, since the CeDAT knockout line is insensitive to the effects of the<br />
neurotoxin. Furthermore, the effect appears to be dependent on a necrotic cell death pathway, since the 6<br />
OHDA induced response occurs in a caspase-deficient ced-3 knockout background. These studies as<br />
well as our progress on toxin-based genetic screens for regulators of CeDAT function, localization, and<br />
the toxin mediated cell death will be presented. Supported by MH19732-07 (RN), GM38679 (JR),<br />
NS26115 (DMM), MH58921 (RDB)<br />
50
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GENE EXPRESSION IN TRANSGENIC C. ELEGANS ANIMALS<br />
EXPRESSING THE HUMAN BETA AMYLOID PEPTIDE.<br />
Chris Link 1 , Carolyn Johnson 1 , Amy Fluet 1 , Kyle K. Duke 2 , Stuart K.<br />
Kim 2<br />
1Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309<br />
2Department of Developmental Biology, Stanford Medical School, Stanford, CA, 94305-5427<br />
We have used Andy Fire’s smg-1 dependent expression vectors to engineer worms with inducible<br />
muscle-specific expression of the human beta amyloid peptide. These animals appear wild type at the<br />
permissive temperature (16 degrees C), but become paralyzed approximately 24 hrs after upshift to the<br />
non-permissive temperature (23 degrees C). As expected, this upshift is accompanied by a large increase<br />
in the synthesis of the beta amyloid peptide. We have examined gene expression profiles in these<br />
animals using DNA microarrays containing probes for all predicted C. <strong>elegans</strong> genes. For our initial<br />
experiments, we compared staged L4 populations from uninduced animals and animals upshifted 22 hr<br />
(harvested before onset of paralysis) or 29 hr (all animals arrested), prepared independently in triplicate.<br />
Approximately 150 genes show an average expression increase > 2X at both induced time points, while<br />
approximately 100 genes show an average expression decrease >2X at both timepoints. We have not yet<br />
independently confirmed these putative gene expression changes, with the exception of HSP-16-2, which<br />
is clearly also upregulated at the protein level, as demonstrated by immunoblots. Perhaps the most<br />
obvious pattern of expression changes is observed in the down-regulated class, where there appears to<br />
be a coordinated decrease in expression of a number of genes involved in energy production and<br />
mitochondrial function (e.g., enolase, GPDH, citrate synthase, succinate dehydrogenase, etc.) (These<br />
changes precede any obvious changes in motility.) These results are consistent with reports of abnormal<br />
glucose metabolism in the brains of Alzheimer patients. to send it in.<br />
51
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A NEMATODE MODEL FOR MITOCHONDRIAL DISEASES<br />
William Y. Tsang, Bernard D. Lemire<br />
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.<br />
The mitochondrial respiratory chain (MRC) is composed of 5 protein complexes capable of generating<br />
ATP for most tissues. Its biogenesis requires the coordinate expression of genes from both the nuclear<br />
and the mitochondrial genomes. Defective MRCs are often associated with myopathies, neuromuscular<br />
and heart diseases. We are developing <strong>Caenorhabditis</strong> <strong>elegans</strong> as our model system to investigate the<br />
biochemical, genetic, and phenotypic consequences of mutations affecting the MRC.<br />
We have identified and cloned 3 MRC mutations. The first two mutations are nuclear deletions in the<br />
nuo-1 (C09H10.3) and in the atp-2 (C34E10.6) genes encoding the active site subunits of Complex I and<br />
V, respectively. Both mutations are homozygous lethal and arrest at the L3 stage. atp2 homozygotes<br />
have a normal life span and cannot enter dauer. Furthermore, development of these animals to L3<br />
requires maternal contribution of atp-2 mRNA.<br />
The third mutation is a mitochondrial DNA (mtDNA) deletion that removes 4 MRC and 7 tRNA genes.<br />
These animals are heteroplasmic and aphenotypic despite the different proportions of mutant mtDNA<br />
(~20-80%). However, exposure of N2 gravid adults to ethidium bromide, an inhibitor of mtDNA replication,<br />
results in L3 arrest progeny with characteristics similar to the nuclear mutants. The arrested animals<br />
exhibit a progressive decrease in the steady-state level of mtDNA with age.<br />
We speculate that the L3 arrest phenotype is due the failure of a common energy-requiring step in<br />
development. In support of this hypothesis, we have determined that the passage from L3 to L4 is<br />
associated with a 3-fold increase in the mtDNA copy numbers, as well as a significant increase in the<br />
levels of ATP-2. In addition, we believe that mitochondrial energy metabolism is linked to the regulation of<br />
dauer formation.<br />
52
BACILLUS TOXIN (BT) SUSCEPTIBILITY AND RESISTANCE<br />
IN C. ELEGANS<br />
Lisa Marroquin 1 , Dino Elyassnia 1 , Joel Griffitts 1 , Johanna O’Dell 1 ,<br />
Jerald Feitelson 2 , Raffi Aroian 1<br />
1U.C. San Diego<br />
2Akkadix Corporation<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in<br />
agriculture and have been expressed in transgenic corn, potato, and cotton to provide organic crop<br />
protection against insect pests. Despite successful and extensive use of these toxins, little is known about<br />
toxicity and resistance pathways in target insects since these organisms are not ideal for molecular<br />
genetic studies. To address this limitation and to investigate the potential use of these toxins to control<br />
parasitic nematodes, we are studying Bt toxin action and resistance in <strong>Caenorhabditis</strong> <strong>elegans</strong>. We<br />
demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. <strong>elegans</strong><br />
hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with<br />
toxin effects in insects. We have screened for and isolated ten recessive mutants that resist the toxin’s<br />
effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more<br />
components are required for Bt toxicity than previously known. We find that a second, unrelated<br />
nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. <strong>elegans</strong> can be<br />
used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold<br />
promise as nematicides. More recently, we have achieved rescue of one of these mutants with a<br />
subclone containing a single predicted open reading frame. Once confirmed by sequencing of mutant<br />
alleles, this would result in the first definitive identification of a gene required for Bt toxin action in any<br />
organism.<br />
53
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
NEW DAUER GENES AND PATHWAYS<br />
Michael Ailion 1 , James H. Thomas 2<br />
1Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195<br />
2Department of Genetics, University of Washington, Seattle, WA 98195<br />
We have isolated and characterized mutants that are strongly Daf-c at 27° but that are not Daf-c at 25°,<br />
a phenotype we call Hid (high temperature-induced dauer formation). Previously identified mutants with<br />
this phenotype include unc-64, unc-31, unc-3, egl-4, daf-3 and the dyf mutants. We screened for new Hid<br />
mutants at 27° and isolated 100 mutants, including twenty-three alleles of known Daf-c genes (many<br />
weak alleles), sixteen alleles of dyf or other Daf-d genes and five alleles of unc-31 or unc-3. We also<br />
isolated alleles of a number of new dauer genes. These include alleles of the genes pdk-1, akt-1, aex-6,<br />
kin-8 and hid-1.<br />
Many of the Hid mutants are fully suppressed by mutations in daf-16, suggesting that these genes act in<br />
the daf-2/age-1 (insulin-receptor /PI3 kinase) pathway. These include unc-64 and unc-31, which encode<br />
neurosecretory proteins that may be involved in insulin secretion, and pdk-1and akt-1 which encode<br />
PI3-dependent protein kinases that act downstream of age-1. The aex-6 and hid-1 genes may also act in<br />
this pathway. In addition to their Hid phenotype, the aex-6 and hid-1 mutants also have defects in<br />
movement and defecation, suggesting that they may function in a common process to regulate multiple<br />
behaviors. We cloned hid-1 and found that it encodes a novel protein with many transmembrane<br />
domains. The HID-1 protein is strongly conserved in Drosophila and humans, with each organism<br />
appearing to carry only one gene of this type.<br />
Reexamining the epistatic interactions of strong Daf-c genes at 27° leads to several new inferences.<br />
Mutations in the TGF-b pathway Daf-c genes and daf-2 are completely suppressed by daf-5 and daf-16,<br />
respectively, at 25° but only partially suppressed at 27°. This suggests that there are additional branches<br />
of the TGF-b and insulin pathways that are not detected at 25°. Furthermore, epistasis results based on<br />
pheromone response at 25° show qualitative differences from epistasis results at 27°, indicating that<br />
gene interactions inferred from epistasis experiments performed under one set of conditions may not be<br />
the same as those under a different set of environmental conditions.<br />
54
TEMPORAL REGULATION OF AGING IN THE NEMATODE C.<br />
ELEGANS<br />
Andrew Dillin, Cynthia Kenyon<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biochemistry and Biophysics, University of California at San Francisco<br />
It has long been assumed that the aging process is stochastic, beginning early in life and eventually<br />
gaining enough activity to cause the eventual decline and death of an organism. In contrast, aging can be<br />
viewed as a developmental process, much like early embryonic development, involving signals, receptive<br />
cues and timely action of these processes during discrete periods of an animal’s life cycle. I have tested<br />
these opposing theories by determining when several genes required for lifespan extension actually<br />
function. Aging in C. <strong>elegans</strong> is regulated by a subset of genes that form a signal transduction pathway.<br />
daf-2 is an insulin-like receptor that acts upstream of a PI3-kinase, age-1. Both genes normally function to<br />
limit life span by down regulating the activity of daf-16, a Forkhead-like transcription factor. daf-16<br />
normally functions to increase life span. I will present data indicating that the aging function of the genes<br />
age-1, daf-2 and daf-16 is exerted during a specific period of the worm’s life cycle. These results suggest<br />
that aging, at least in C. <strong>elegans</strong>, is regulated during specific periods in life. In addition, I have also started<br />
work on a novel screen to identify other genes required for life span regulation. Once these genes are<br />
identified I will test when they function.<br />
55
A LONGITUDINAL ANALYSIS OF ADULT NEURONS IN C.<br />
ELEGANS<br />
Mark I. Snow, Pamela L. Larsen<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Molecular Biology Program and Division of Biogerontology, University of Southern California, Los<br />
Angeles, CA 90089<br />
An intriguing question that remains to be answered is what happens to the cells of the nervous system in<br />
an individual as it ages? Previous studies following the neuron number in rodents and humans have used<br />
a cross sectional design and the correlative conclusions drawn from these experiments are controversial<br />
with regard to whether or not there is a decline in neuron number with advancing age. C. <strong>elegans</strong> show a<br />
progressive decline in neuronally regulated behaviors as they age, so we chose to follow specific neurons<br />
longitudinally in worms by using integrated transgenic GFP arrays.<br />
The analysis of GFP expression for the 26 GABA and the 8 dopaminergic neurons in hermaphrodites was<br />
performed at early adulthood and again just before death. We found that 84% of the old animals with a<br />
wild-type background do not lose GFP expression in their GABA neurons. For the 16% of animals that<br />
express GFP in less than 26 GABA neurons just before dying, the loss of expression appears to be<br />
cell-specific. In animals expressing GFP in the dopaminergic neurons, 94% do not lose GFP expression<br />
with age. The small number that did display changes were in specific neurons.<br />
Two daf-2 alleles, m41 and e1370, were introduced into the strains expressing GFP in either the GABA or<br />
dopaminergic neurons. The GFP expression patterns of the double mutant strains were not altered and<br />
the life spans of the animals were that of the daf-2 alleles. No change in the number of neurons<br />
expressing GFP was observed in any of the animals with the daf-2(m41) or daf-2(e1370) alleles. The<br />
daf-2 mutation appears to prevent loss of GFP expression in older animals.<br />
The experiments described here are the first to follow neurons in a longitudinal manner in individuals as<br />
they age. Assuming that the endogenous gene and the transgene are similarly regulated, absence of<br />
GFP expression may indicate functional loss because the unc-25 and cat-2 genes encode enzymes<br />
necessary for neurotransmitter biosynthesis. From this approach, we conclude that loss of GFP<br />
expression is not widespread and the daf-2 mutations are neural protective.<br />
56
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GERM-LINE CELLS THAT REGULATE AGING IN C. ELEGANS<br />
Nuno Arantes-Oliveira, Javier Apfeld, Cynthia Kenyon<br />
Department of Biochemistry and Biophysics, University of California San Francisco<br />
The lifespan of C. <strong>elegans</strong> is regulated by a hormonal pathway that integrates signals from the nervous<br />
and reproductive systems of the animal. Ablation of the germ-line precursor cells produces a lifespan<br />
extension of approximately 60%. This extension is dependent on the presence of the somatic tissues of<br />
the gonad and thus is not likely to be caused by sterility. We have used a variety of genetic approaches,<br />
RNAi and laser ablations to ask which cells in the germ-line (sperm, oocytes, mitotic precursors) regulate<br />
lifespan. Our findings support the interpretation that mitotically-dividing germ-line cells produce the signals<br />
that regulate lifespan. These signals may act by down-regulating daf-16.<br />
57
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A SCREEN FOR GENES THAT CONTROL PROGRAMMED<br />
CELL DEATH IN THE GERM LINE<br />
S Milstein 1,2,3 , A Gartner 4 , M Hengartner 5<br />
1Program in Genetics, SUNY Stony Brook<br />
2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 USA.<br />
3milstein@cshl.org<br />
4Max Planck Institut for Biochemie, D-82152 Martinsried, Germany<br />
5Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 USA<br />
Although we know a great deal about the apoptotic program in C. <strong>elegans</strong>, we know very little about how<br />
the program is regulated. Since this cell fate is responsible for the demise of about 10% of the somatic<br />
cells, and as many as half of the germ cell, we are interested in defining the genes that may be involved<br />
in the decision of a cell to die.<br />
In order to find such genes, we developed a screen using the vital dye acridine orange, which specifically<br />
stains apoptotic cells in the germ line. Using this screen we have looked at approximately 40,000<br />
genomes and have identified 21 mutants that have increased levels of germ cell death. The ten mutants<br />
that have thus far been mapped represent eight complementation groups, only one of which has multiple<br />
alleles. Of these I am currently cloning two by single nucleotide polymorphism mapping, gla-1(op212) and<br />
gla-3(op234).<br />
In order to determine if these mutants are specifically defective in the regulation apoptosis, or if they are<br />
pathologically compromised, and thus have damage that causes the cells to undergo apoptosis, we made<br />
double mutations with ced-3 and rad-5. Most of the mutations examined are suppressed in the ced-3<br />
background, suggesting that the damage is non pathological (we would have expected necrosis or some<br />
other gross morphological consequence if this were the case). In the rad-5 background, a mutation that<br />
we have shown to render worms insensitive to DNA damage, these mutations are not suppressed. This<br />
again suggests specificity since mutations that somehow result in DNA damage would be suppressed.<br />
58
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
C. ELEGANS P53: REQUIREMENT FOR<br />
RADIATION-INDUCED PROGRAMMED CELL DEATH,<br />
STRESS RESISTANCE, AND NORMAL ADULT LIFESPAN<br />
FOLLOWING DIAPAUSE.<br />
W. Brent Derry, Aaron Putzke, Joel H. Rothman<br />
Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, CA<br />
93106 USA<br />
We have identified the apparent C. <strong>elegans</strong> homologue of the p53 tumor suppressor gene, cep-1, and<br />
have begun to characterize it genetically. CEP-1 appears to be the only p53 family member present in the<br />
completely sequenced C. <strong>elegans</strong> genome and includes all of the signature domains of p53, including 4<br />
of the 5 conserved arginine residues frequently mutated in human cancer. A cep-1::gfp reporter shows<br />
ubiquitous expression throughout embryonic development, which becomes restricted to a subset of<br />
pharyngeal muscle and neuronal cells postembryonically. Ectopic expression of cep-1 or of human p53<br />
fails to induce cell cycle arrest but potently promotes the rapid ced-3-independent necrotic death of all<br />
somatic cells, demonstrating that regulation of cep-1 expression in a critical range is essential for survival.<br />
We isolated a TMP-induced cep-1 mutation that removes the conserved DNA binding domains. Though<br />
cep-1(-) homozygotes show a low level of embryonic lethality, they are generally viable. However, they<br />
are highly resistant to radiation-induced apoptosis of germ cells. Cep-1 deletion mutants also show<br />
enhanced hypoxia-induced lethality, indicating that C. <strong>elegans</strong> p53 is essential for normal physiological<br />
stress response. Furthermore, cep-1 mutants display both reduced viability and reduced lifespan after<br />
release from starvation-induced L1 diapause. These results provide the first evidence linking p53 to<br />
longevity of an animal in response to physiological stress.<br />
59
IDENTIFICATION OF CELL-SPECIFIC REGULATORS OF<br />
PROGRAMMED CELL DEATH IN C. ELEGANS.<br />
Shai Shaham, Cori Bargmann<br />
Dept. of Anatomy, UCSF<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Programmed cell death (PCD) is a common metazoan cell fate. In C. <strong>elegans</strong> 12% of somatic cells and<br />
half of germ cells born undergo PCD. Excess or insufficient PCD can lead to a variety of human diseases.<br />
In C. <strong>elegans</strong> PCD is regulated by egl-1 (a BH3 domain protein), ced-9 (a bcl-2 family member), ced-4<br />
(similar to human Apaf-1), and ced-3 (a caspase protease). Although much is known about the machinery<br />
that executes PCD, only in a few instances have the signal transduction pathways leading to activation of<br />
this machinery been elucidated.<br />
Several lines of evidence suggest that ced-9, ced-4, and ced-3 are expressed in most if not all cells. We<br />
are interested in defining molecular events that trigger PCD activation in specific cells. As a first step, we<br />
identified a novel gene (cip-1) whose protein product interacts with the CED-9 protein. CIP-1 (CED-9<br />
interacting protein) binds to CED-9 in a two-hybrid assay. Precipitation experiments using GST-CED-9<br />
and 35 S-labelled CIP-1 also indicate that these proteins may interact. Reminiscent of C. <strong>elegans</strong> EGL-1,<br />
CIP-1 may contain a BH3 domain (bcl-2 homology domain 3) mediating interaction with CED-9.<br />
To understand the function of CIP-1 we expressed the protein in worms using heat-shock promoters.<br />
Animals expressing HS-CIP-1 are dead. This inviability is rescued by ced-3, ced-4, or ced-9(gf)<br />
mutations. Thus, CIP-1 may act upstream of the global PCD machinery to regulate PCD activation.<br />
Preliminary data suggest that cip-1::GFP is expressed in a small number of cells, suggesting that this<br />
gene may well be a cell-specific PCD activator.<br />
In addition to characterizing cip-1, we are screening for genes controlling PCD of the male-specific CEM<br />
neurons, which normally die in hermaphrodites. Using a pkd-2::GFP transgene specifically expressed in<br />
the CEMs, we have isolated 25 independent mutants that abnormally express pkd-2::GFP in<br />
hermaphrodites. Of these, five are alleles of the PCD activators ced-3 and ced-4, and one is an allele of<br />
the tra-2 sex-determination gene. Seventeen mutations promote CEM survival and do not result in any<br />
other gross abnormalities. These mutations may affect either CEM-specific PCD regulators or CEM cell<br />
fate genes. Two allelic mutations define a novel gene required to suppress CEM-like development of<br />
other head neurons.<br />
60
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
BIOCHEMICAL, STRUCTURAL, AND GENETIC ANALYSES<br />
OF THE ACTIVATION OF PROGRAMMED CELL DEATH<br />
Jay Parrish 1 , Betsy Metters 1 , Lin Chen 2 , Ding Xue 1<br />
1Dept. of MCD Biology, University of Colorado, Boulder, Colorado 80309<br />
2Dept. of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309<br />
In the nematode C. <strong>elegans</strong>, three genes egl-1, ced-3, and ced-4, are required for activation of<br />
programmed cell death whereas one gene, ced-9, prevents programmed cell death. Molecular<br />
characterization of these genes has shown that egl-1 encodes a BH3-only (Bcl-2 homology domain 3) cell<br />
death activator, ced-3 encodes an aspartate-specific cysteine protease (caspase), ced-4 encodes an<br />
Apaf-1-like (apoptotic protease activating factor-1) protein, and ced-9 encodes a Bcl-2 homologue.<br />
Genetic studies have placed these genes in a negative regulatory pathway, with egl-1 antagonizing the<br />
activity of ced-9, which inhibits the activity o f ced-4 to activate CED-3 and eventually cell death. In<br />
combination with in vitro biochemical studies, these genetic findings suggest that a protein interaction<br />
cascade involving EGL-1, CED-9, CED-4, and CED-3 regulates the activation of programmed cell death<br />
in C. <strong>elegans</strong>. We have initiated biochemical, structural, and genetic analyses to study the mechanisms<br />
that activate the death protease CED-3, including the protein interactions involved in this activation<br />
process. Specifically, we are attempting to reconstitute the events necessary for activation of CED-3 in<br />
vitro using purified recombinant proteins. Furthermore, we are using a structural modeling approach to<br />
analyze these protein interactions and guide our efforts to demonstrate the importance of these protein<br />
interactions in vivo. So far, we have been able to recapitulate the early events of cell death activation in<br />
vitro, including the EGL-1 mediated release of CED-4 from CED-4/CED-9 complexes and are in the<br />
process of purifying additional factors that are required for the activation of CED-3. Structural modeling of<br />
the EGL-1/CED-9 complex and corresponding biochemical analyses have revealed important molecular<br />
interactions between EGL-1 and CED-9 and the nature of an unusual gain-of-function mutation in ced-9,<br />
facilitating successful design of its second-site suppressor mutations. Further studies of this kind should<br />
help address the central question of how cell death and the cell death protease CED-3 are activated in<br />
the appropriate cells and at the right time.<br />
61
ANALYSIS OF RNA ASSOCIATED WITH P GRANULES IN<br />
GERM CELLS OF C. ELEGANS ADULTS<br />
Jennifer A. Schisa 1 , Jason N. Pitt 2 , James R. Priess 2<br />
1Fred Hutchinson Cancer Research Center<br />
2Howard Hughes Medical Institute, Seattle, WA 98109<br />
The germ plasm of many species contains germline-specific cytoplasmic granules. These granules (called<br />
P granules in C. <strong>elegans</strong>) have been hypothesized to have some function in germline development; and<br />
several of the proteins that are required for germline specification in the embryo (PIE-1, POS-1, MEX-1)<br />
transiently associate with P granules during the first few cell divisions. Because P granules are associated<br />
with germ cell nuclei during most of development, we have begun a characterization of the<br />
nuclear-associated P granules in adult gonads. These studies have shown that P granules in the gonad<br />
are tightly associated with clusters of nuclear pores 1 .<br />
Several protein components of P granules have been identified and found to have potential RNA-binding<br />
motifs. We find that P granules in the gonad contain RNA using a probe for SL1, a transpliced leader<br />
found on many C. <strong>elegans</strong> RNAs. We have also examined sterile animals lacking glh-1 and glh-2; germ<br />
cells in these gonads do not appear to assemble P granules, as ascertained by immunostaining with<br />
several P granule antibodies 2 . Animals lacking glh-1 and glh-2 show hybridization of SL1 on perinuclear<br />
foci. We examined the germ nuclei in these animals by electron microscopy and found perinuclear foci of<br />
abnormal, electron dense material that appeared to be associated with nuclear pores. Thus, it appears<br />
that the putative RNA-binding proteins GLH-1 and GLH-2 are not essential for RNA to localize outside the<br />
nuclear envelope.<br />
We examined various classes of maternally-expressed RNAs by fluorescence in situ hybridization to<br />
determine which specific mRNAs are in P granules. While ribosomal RNAs and some abundant<br />
housekeeping mRNAs do not appear to be enriched in P granules above the general level in the<br />
cytoplasm, several maternal mRNAs that are translated only in the early embryo appear to accumulate on<br />
germ cell P granules.<br />
1 Pitt et al., 2000. Dev. Biol. 219: 315-333.<br />
2 Gruidl et al., 1996. PNAS 93, 13837-13842.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
62
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE SPLICING SM PROTEINS COLOCALIZE WITH P<br />
GRANULES IN GERM CELLS AND PARTICIPATE IN P<br />
GRANULE LOCALIZATION IN THE EARLY EMBRYO<br />
Scott A. Barbee, Alex L. Lublin, Thomas C. Evans<br />
Department of Cellular and Structural Biology, University of Colorado Health Science Center, Denver,<br />
Colorado, 80262<br />
Early embryonic polarity in C. <strong>elegans</strong> is initiated by asymmetric cell division which leads to the<br />
localization of maternally contributed proteins and mRNAs. In turn, these factors control the temporal and<br />
spatial expression of gene products that determine the developmental fate of cells. Conventional genetic<br />
screening has identified more than 20 genes involved in this process. However, these screens may not<br />
uncover pleiotropic genes that have a necessary function at other stages. With this in mind, we have used<br />
RNA interference (RNAi) to screen a cDNA library for new genes involved in generating asymmetries in<br />
the early embryo.<br />
One gene identified in the RNAi screen is a homolog to human SNRPE (SmE). SmE is one of several Sm<br />
proteins which form a complex required for mRNA splicing and import of snRNAs (U1, U2, U4, and U5)<br />
into the nucleus. RNAi of SmE and other Sm proteins, which together form a subcomplex, resulted in<br />
disruption of P granule segregation in the embryo. P granules are cytoplasmic ribonucleoprotein particles<br />
that segregate to the posterior germline precursor cells. RNAi of several subunits of the Sm complex<br />
caused mislocalization of P granules at various stages. Other aspects of early polarity may also be<br />
disrupted. However, RNAi of other distinct Sm proteins had no effect. RNAi of the core splicing factors<br />
U170K and U2AF65, or of RNA polymerase II (ama-1), also had no effect on P granule localization.<br />
These data suggest that the P granule phenotype is not the result of a general splicing defect. Therefore,<br />
Sm proteins may have a role in generating early embryonic polarity that is independent of snRNA import<br />
and splicing. Interestingly, using a monoclonal antibody against mammalian Sm proteins, we found<br />
colocalization of the Sm’s with PGL-1 in P granules at all stages of development. Sm proteins, therefore,<br />
may be P granule components that participate in P-granule localization and/or other polarity functions.<br />
63
POD-2 DEFINES A NEW CLASS OF MUTANTS REQUIRED<br />
FOR ANTERO-POSTERIOR ASYMMETRY IN THE EARLY<br />
CAENORHABDITIS ELEGANS EMBRYO<br />
Akiko Tagawa, Raffi V. Aroian<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
University of California, San Diego. La Jolla, CA 92093-0349<br />
The generation of asymmetry in the one-cell C. <strong>elegans</strong> embryo is indispensable for a proper segregation<br />
of developmental determinants and thus development of the multicellular organism. We have isolated a<br />
mutation in a previously unidentified gene, pod-2 (for polarity and osmotic defect), through a screen for<br />
cold sensitive embryonic lethals. Many pod-2 mutant embryos resemble par mutant embryos and show a<br />
loss of physical and developmental asymmetry at the one and two-cell stage. More strikingly, pod-2<br />
resembles another mutant discovered in our lab, pod-1, in that mutants embryos are also osmotically<br />
sensitive. Double mutant analysis indicates these two genes work in the same pathway thus defining<br />
pod-1 and pod-2 as a new class of polarity genes and a new pathway important for early embryonic<br />
development. In addition to polarity and osmotic defects, pod-2 mutation also causes abnormal<br />
localization of germline components such that in 25% of mutant embryos, germline components are<br />
located to a single cell where EMS normally would be. By GFP analysis, we show that this defect can be<br />
traced back to abnormal localization of germline components in the one-cell embryo. Our data suggests<br />
that in the mutant, germline components remain bundled but do not move to the proper position in the<br />
one-cell embryo. Thus pod-2 appears to be required for proper positioning of developmental components<br />
and perhaps other asymmetries. Cloning of pod-2 is underway, and we have recently achieved rescue of<br />
the mutant with a single cosmid.<br />
64
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
OOC-5 ENCODES A PUTATIVE ATPASE REQUIRED FOR THE<br />
REESTABLISHMENT OF ASYMMETRIC PAR PROTEIN<br />
LOCALIZATION IN TWO-CELL EMBRYOS<br />
Stephen E. Basham, Lesilee S. Rose<br />
Section of Molecular and Cellular Biology, University of California, Davis, CA 95616 USA<br />
C. <strong>elegans</strong> embryos display two distinct patterns of spindle orientation. Divisions in the AB lineage occur<br />
in an orthogonal pattern while some division in the P lineage repeatedly occur on the same axis, due to a<br />
rotation of the nuclear-centrosome complex. Genetic and molecular analyses suggests that PAR-3 protein<br />
plays an important role in preventing nuclear rotation in the AB cell, while PAR-2 protein functions to allow<br />
nuclear rotation in P1 by restricting PAR-3 localization within this cell.<br />
We have screened maternal-effect lethal mutants for alterations in cleavage pattern. Hermaphrodites<br />
homozygous for mutations in the ooc-5 or ooc-3 gene produce reduced sized embryos that fail to undergo<br />
P1 nuclear rotation. Our phenotypic characterization of ooc-5 and ooc-3 mutants indicates that polarity is<br />
perturbed in the P1 cell of 2-cell mutant embryos. In particular, we have found that in most ooc-5 and<br />
ooc-3 mutant embryos, PAR-3 localization appears normal at the 1-cell stage but is no longer restricted to<br />
the anterior of the P1 cell in 2-cell embryos. PAR-2 is also normal in 1-cell mutant embryos, but is absent<br />
from the periphery of the P1 cell in most 2-cell ooc mutant embryos. Finally, P granules are localized<br />
normally in 1-cell mutant embryos, but often fail to localize normally to the posterior of the P1 cell in 2-cell<br />
mutant embryos. Thus, ooc-5 and ooc-3 may function (directly or indirectly) in polarizing the P1 cell. One<br />
model for OOC-5 and OOC-3 is that they function in P1 to maintain PAR-2 at the posterior of P1, thus<br />
restricting PAR-3 localization in this cell. We have cloned ooc-5 and it is predicted to encode a 350 amino<br />
acid protein containing an amino-terminal signal sequence, a transmembrane spanning region and an<br />
ATP binding domain. A BLAST search indicates that ooc-5 is related to two other predicted genes in the<br />
C. <strong>elegans</strong> genome and is a member of a family of predicted ATPases that have been identified in a<br />
variety of species. A mutation in one of the human orthologs of this gene results in a neuromuscular<br />
disease, but the biological basis for this disease is not well understood. We are currently generating GFP<br />
reporter constructs and anti-OOC-5 antibodies to examine OOC-5 expression and localization.<br />
65
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
RIC-8 (SYNEMBRYN): A NOVEL REGULATOR OF G PROTEIN<br />
SIGNALING<br />
Kenneth G. Miller, Melanie D. Emerson, John R. McManus, James B.<br />
Rand<br />
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK<br />
73104<br />
Recent studies describe a network of signaling proteins centered around GOA-1 (G oa) and EGL-30<br />
(G qa) that regulates neurotransmitter secretion in C. <strong>elegans</strong> by controlling the production and<br />
consumption of diacylglycerol (DAG). We sought other components of the G oa-G qa signaling network by<br />
screening for aldicarb resistant mutants with phenotypes similar to egl-30 (G qa) mutants. In so doing we<br />
identified ric-8, which encodes a novel protein named RIC-8 (synembryn). Through cDNA analysis we<br />
show that RIC-8 is conserved in vertebrates. Through immunostaining we show that RIC-8 is<br />
concentrated in the cytoplasm of neurons. Exogenous application of phorbol esters or loss of DGK-1<br />
(diacylglycerol kinase) rescues ric-8 mutant phenotypes. A genetic analysis suggests that RIC-8 functions<br />
upstream of EGL-30 (G qa), or in a parallel intersecting pathway. Both ric-8 and goa-1 reduction of<br />
function mutants also exhibit partial embryonic lethality. Furthermore, the embryonic lethality of ric-8<br />
mutants is enhanced to 95-100% by a 50% reduction in maternal goa-1 gene dosage. In a separate study<br />
we investigated the roles of RIC-8 and GOA-1 in early embryos (pre 8-cell stage) and found that goa-1<br />
and ric-8 mutant embryos exhibit defects in the movements of centrosomes. Comparing the roles of<br />
RIC-8 and GOA-1 in the nervous system and the embryo reveals potentially informative differences<br />
between the two pathways. First, EGL-30 (G qa) does not appear to play a role in the embryonic pathway.<br />
Second, in the embryonic pathway, reduction of function mutations in goa-1 and ric-8 lead to similar<br />
phenotypes that are enhanced in goa-1; ric-8 double mutants, whereas in the adult neuronal pathway the<br />
same goa-1 and ric-8 mutants have opposite phenotypes and suppress each other. Based on these<br />
findings, we propose that RIC-8 is required, directly or indirectly, for proper activation of G proteins in the<br />
early embryo and in the nervous system.<br />
66
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MED-1 AND -2 ACT AT THE CONVERGENCE POINT OF<br />
SKN-1 AND POS-1 TO SPECIFY MS AND E IDENTITY<br />
Morris F. Maduro, Regina Broitman-Maduro, Joel H. Rothman<br />
Department of MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106<br />
EMS produces daughters with different fates: E produces the endoderm while MS makes mesoderm,<br />
including the posterior pharynx. Depletion of the zygotically expressed MED-1 and -2 GATA transcription<br />
factors by RNAi results in a penetrant MS ’ C transformation and an impenetrant E ’ C transformation,<br />
similar to that seen in maternal skn-1 mutants. However, unlike skn-1 mutants, ABa still makes anterior<br />
pharynx in med-1,2(RNAi) embryos, indicating that the med genes are not required for the<br />
GLP-1-mediated signal that induces ABa pharynx. pos-1 mutants also misspecify MS, yet retain<br />
GLP-1-dependent pharynx (Tabara et al. 1999). These observations suggest that POS-1 is also required<br />
for an activity downstream of SKN-1.<br />
The med genes appear to act at the convergence of SKN-1 and POS-1 function in the EMS lineage.<br />
While expression of a med-1::GFP::MED-1 reporter is detected in the early E and MS lineages, it is<br />
undetectable in both skn-1(RNAi) and pos-1(RNAi) embryos. Ectopic med expression is detected in<br />
embryos expressing SKN-1 ectopically. In addition, expression requires a cluster of upstream conserved<br />
SKN-1 binding sites, which bind bacterially expressed SKN-1 in vitro. We propose that POS-1, which is<br />
primarily cytoplasmic, modulates an activity required for transcriptional activation of the meds by SKN-1.<br />
end-1 and end-3 specify E fate, and are likely downstream targets of med-1,2 in the E lineage. Indeed,<br />
GFP-tagged MED-1 binds in vivo to an extrachromosomal array containing the end-3 promoter. This<br />
interaction also occurs in MS, where end-1 and end-3 are known to be repressed by POP-1. Thus, the<br />
mechanism by which POP-1 represses E fate in MS does not preclude binding of MED-1 to end-3.<br />
Our data indicate that SKN-1 directly activates the med genes through a POS-1-requiring mechanism. As<br />
the meds are required for both E and MS specification, their activity appears to be modulated by POP-1,<br />
the target of the Wnt/MAPK pathways in the E blastomere, perhaps at a step after their binding to target<br />
promoters.<br />
67
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MASS SPECTROMETRIC IDENTIFICATION OF PLP-1 AND ITS<br />
ROLE IN MESENDODERM SPECIFICATION<br />
E. Witze 1 , E. Field 2 , D. Hunt 2 , J.H. Rothman 1<br />
1Department of MCD Biology, University of California, Santa Barbara, CA<br />
2Department of Chemistry, University of Virginia, Charlottesville, VA<br />
end-1 encodes a GATA type transcription factor that is sufficient to specify the endoderm progenitor, E.<br />
end-1 is activated in the E lineage by the combined action of SKN-1 and the Wnt/MAP kinase-activated<br />
form of POP-1 (see abstract by Kasmir et al.). It is repressed in MS, the sister of E, by POP-1 in the<br />
absence of the Wnt/MAPK signal.<br />
We are taking a biochemical approach to characterize additional factors that activate end-1 in the E<br />
lineage and repress it in MS. We developed procedures for isolating factors from early developing C.<br />
<strong>elegans</strong> embryos that bind key regulatory elements in end-1. Mass spectrometry of affinity-purified<br />
proteins identified several end-1 binding factors, one of which, encoded by the plp-1 gene, is an apparent<br />
homologue of a mammalian transcription factor called pur alpha, whose in vivo function is unknown.<br />
We performed RNAi to examine the role of PLP-1 in mesendoderm development. Less than 10% of<br />
plp-1(RNAi) embryos arrest before hatching. A fraction of these contain extra intestinal cells and show a<br />
concomitant loss of MS-derived (posterior) pharynx. This phenotype appears to result from an MS to E<br />
transformation, consistent with our finding that end-1 is sometimes expressed in MS and its descendants<br />
in plp-1(RNAi) embryos. Thus, plp-1 appears to function in part to repress end-1 expression in the MS<br />
lineage. As such, it may act as a corepressor with POP-1.<br />
Paradoxically, we also observe an impenetrant (
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE C. ELEGANS NEUROD HOMOLOG CND-1 FUNCTIONS<br />
IN MULTIPLE ASPECTS OF MOTOR NEURON FATE<br />
SPECIFICATION<br />
Steven Hallam 1 , Emily Singer 1 , David Waring 2 , Yishi Jin 1<br />
1Department of Biology, Sinsheimer Laboratories, University of California Santa Cruz, California 95064<br />
2Fred Hutchinson Cancer Research Center, Seattle, Washington 98109<br />
The basic Helix-Loop-Helix transcription factor NeuroD has been implicated in neuronal fate<br />
determination, differentiation and survival. Here we report the expression and phenotypic analysis of<br />
cnd-1, a C. <strong>elegans</strong> NeuroD homolog*. cnd-1 expression was first detected in neuroblasts of the AB<br />
lineage in 14 cell embryos and maintained in many neuronal descendents of the AB lineage during<br />
embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1<br />
reporter genes were expressed in the precursors of embryonic ventral cord motor neurons including<br />
ABplpp, ABprpp and their progeny. A loss of function mutant, cnd-1(ju29), exhibited multiple defects in<br />
the ventral cord motor neurons. First, the number of motor neurons was reduced, apparently caused by<br />
the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the<br />
fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as<br />
manifested by the variable spatial and temporal expression patterns of motor neuron fate specific<br />
markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including<br />
axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal<br />
type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1<br />
may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their<br />
precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1<br />
appears to have combined the functions of several vertebrate neurogenic bHLH proteins, and may<br />
represent an ancestral form of this protein family.<br />
* Singer E et al., <strong>Worm</strong> Breeder’s Gazette 16(1): 52 (October 1, 1999)<br />
69
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
LEFT-RIGHT ASYMMETRY IN C. ELEGANS INTESTINAL<br />
ORGANOGENESIS INVOLVES A LIN-12/NOTCH SIGNALING<br />
PATHWAY<br />
Greg J. Hermann, Ben Leung, James R. Priess<br />
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and Howard Hughes Medical<br />
Institute<br />
The C. <strong>elegans</strong> intestine is a simple tube consisting of a monolayer of polarized epithelial cells. During<br />
embryogenesis, cells in the anterior of the intestinal primordium undergo reproducible movements that<br />
create an invariant, asymmetrical "twist" in the intestine. We have analyzed the development of twist to<br />
determine how left-right and anterior-posterior asymmetries are generated within the intestinal<br />
primordium. The twist requires the LIN-12/Notch-like signaling pathway of C. <strong>elegans</strong>. All cells within the<br />
intestinal primordium initially express LIN-12, a receptor related to Notch. However, only cells in the left<br />
half of the intestinal primordium contact external, non-intestinal cells that express LAG-2, a ligand related<br />
to Delta. LIN-12 and LAG-2-mediated interactions result in the left primordial cells expressing lower levels<br />
of LIN-12 than the right primordial cells. We propose that this asymmetrical pattern of LIN-12 expression<br />
is the basis for asymmetry in later cell-cell interactions within the intestinal primordium that lead directly to<br />
intestinal twist. Like the interactions that initially establish LIN-12 asymmetry, the later interactions are<br />
mediated by LIN-12. However, the later interactions involve a different Delta-related ligand, called APX-1.<br />
We show that the anterior-posterior asymmetry in intestinal twist involves the kinase LIT-1, which is part<br />
of a signaling pathway in early embryogenesis that generates anterior-posterior differences between<br />
sister cells.<br />
70
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE PHO-1 GENE AND THREE KINDS OF GUT POLARITY<br />
Tetsunari Fukushige, James D. McGhee<br />
Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Dr. NW, Calgary,<br />
Alberta, CANADA T2N 4N1<br />
Many years ago (Beh et al., 1991), we described an acid phosphatase activity (pho-1) that was expressed<br />
along the intestinal brush border in every gut cell except int-1 and int-2, beginning at the three fold stage<br />
of embryogenesis. Thus, compared to early gut differentiation markers such as the ges-1 esterase, pho-1<br />
should allow us to investigate three different polarities within the gut: anterior-posterior, apical-basal, and<br />
temporal. The pho-1 gene was cloned and its expression pattern is now being analyzed. First of all,<br />
fusions of the pho-1 promoter to GFP reporters reflect the endogenous expression pattern and do not<br />
express in int-1 and int-2; in other words, the anterior-posterior patterning is at the level of the pho-1<br />
promoter. We have previously shown that A/P patterned expression of a modified ges-1 reporter depends<br />
on zygotic pop-1 activity (Schroeder and McGhee, 1998). This patterning within the E-lineage is<br />
gut-autonomous and occurs after the Wnt-dependent P2-EMS contact of the four cell stage. We are now<br />
investigating whether pho-1 responds to the same A/P patterning cues but in an opposite manner, i.e. we<br />
would expect that high level of pop-1 within the gut cell nuclei would repress pho-1 expression, whereas<br />
high level of pop-1 in gut nuclei activates the modified ges-1 reporter. An experiment to test this model is<br />
to inject lit-1 doublestranded RNA into a pop-1(zu189) mother. The gut is still formed (zu189 results in low<br />
maternal pop-1 and hence a double E cell) but loss of lit-1 activity should prevent downregulation of<br />
zygotic pop-1 within the gut. Our prediction is that pho-1 should be repressed throughout the gut because<br />
of high pop-1 and (in preliminary experiments) this is indeed what we see. The availability of a<br />
temperature sensitive allele of lit-1 should allow us to investigate the gut patterning events in much finer<br />
detail. To investigate apical-basal asymmetry, we are attempting to find a GFP construct that will reflect<br />
the endogenous pho-1 distribution, in order to allow for genetic screens of polarity defects. At the<br />
moment, all we know is that mutations in lin-2, lin-7 and lin-10 (shown by Stuart Kim’s lab to be involved<br />
in epithelial polarity) have no apparent effect on the normal distribution of pho-1 activity. Finally, we are<br />
investigating why pho-1 is expressed 2-3 E-cell divisions later than ges-1. There are GATA sites in the<br />
promoter, that might be a target for elt-2. However, ectopic expression of either elt-2 or end-1 does not<br />
activate ectopic expression of either endogenous pho-1 gene or pho-1 reporter gene, suggesting other<br />
factors or events may be required. We are analyzing the pho-1 promoter to identify the site of action of<br />
these timing factors or timing events.<br />
71
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A ROLE FOR DISHEVELLED IN ASYMMETRIC CELL<br />
DIVISION.<br />
Nancy Hawkins 1 , Gregory Ellis 2 , Bruce Bowerman 2 , Gian Garriga 1<br />
1Dept. of Mol. and Cell Biology, University of California, Berkeley, CA 94720<br />
2Institute of Mol. Biology, University of Oregon, Eugene, OR 97403<br />
Both intrinsic and extrinsic mechanisms can polarize asymmetrically dividing cells. Asymmetrically<br />
distributed intracellular molecules are known to segregate fate to daughter cells during mitosis. Wnt<br />
signaling also polarizes asymmetrically dividing cells by an unknown mechanism. We propose that in C.<br />
<strong>elegans</strong>, Wnt signaling may segregate intracellular molecules that distribute developmental potential.<br />
In the lineage that generates the HSN and PHB neurons, an HSN/PHB neuroblast divides asymmetrically<br />
to generate an anterior daughter cell that dies and a posterior daughter cell, the HSN/PHB precursor. This<br />
precursor then divides to produce the HSN and PHB neurons. In ham-1 mutants, the HSN/PHB<br />
neuroblast frequently divides symmetrically producing two HSN/PHB precursors. ham-1 encodes a novel<br />
protein that is expressed in a subset of cells during embryogenesis, and in mitotic cells, is often restricted<br />
to one side in a crescent shaped pattern. In the HSN/PHB neuroblast, HAM-1 is asymmetrically localized<br />
and is inherited by the HSN/PHB precursor.<br />
To determine if Wnt signaling is also involved in asymmetric division of the HSN/PHB neuroblast, we<br />
focused on the three C. <strong>elegans</strong> dishevelled (dsh) homologs. Upon Wnt stimulation, Dsh becomes<br />
hyperphosphorylated and recruited to the cell membrane. Several lines of evidence indicate that dsh-2<br />
(C27A2.6) is necessary for asymmetric division of the HSN/PHB neuroblast. First, both RNAi with dsh-2,<br />
as well as a deletion allele, result in a Ham-1 phenotype in the HSN/PHB lineage. Second,<br />
overexpression of dsh-2 produces weak defects in asymmetric cell division. Finally, DSH-2 appears to be<br />
primarily membrane associated and is expressed from approximately the 4-6 cell stage until the 1 1/2 fold<br />
stage. In cells expressing HAM-1, the two proteins co-localize and when HAM-1 is asymmetric, DSH-2<br />
localization also appears to be asymmetric. We are performing co-immunoprecipitation and in vitro<br />
binding experiments to determine if HAM-1 and DSH-2 physically interact.<br />
72
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
RHO-1, A TARGET OF THE EXCHANGE FACTOR UNC-73, IS<br />
REQUIRED FOR CELL MIGRATIONS DURING C. ELEGANS<br />
DEVELOPMENT<br />
Andrew G. Spencer, Christian J. Malone, Satoshi Orita, Min Han<br />
Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology,<br />
University of Colorado, Boulder, CO, 80309-0347<br />
Members of the rho family of small GTPases have been implicated and characterized in a variety of<br />
cytoskeletal regulatory events. However, the spatial and temporal regulation of the activities of such<br />
proteins in vivo is not well understood and represents an important aspect of the cell movements and<br />
morphogenesis required during animal development. By expressing dominant-negative and<br />
dominant-active versions of the C. <strong>elegans</strong> rhoA homologue rho-1 from a tissue specific promoter<br />
(col-10), we establish a role for rho-1 during P cell migration to the ventral cord during the first larval<br />
stage. Expression of rho-1 (dn) causes a strong P cell migration defect. This effect appears to be<br />
specifically the result of rho-1 inhibition since expression of the C3 exoenzyme causes a similar P cell<br />
phenotype. Genetic and biochemical analyses of unc-73, a GDP/GTP exchange factor, indicate that<br />
unc-73 acts as an exchange factor for rho-1 during P cell migration in vivo. Another rho family GTPase,<br />
mig-2, is not required for P cell migrations but may contribute to a common pathway with rho-1 in some<br />
manner. This is based on observations that (a) mig-2 null alleles exacerbate a weak P cell migration<br />
defect in unc-73 mutants (Zipkin, et al., 1997), which is partially rescued by rho-1 (gf) (Zipkin, Kindt, and<br />
Keynon, 1997 1 ), and (b) mig-2 gain-of-function alleles cause a P cell migration defect, presumably by<br />
interfering with members of the rho-1 pathway. In order to identify molecules acting downstream of rho-1<br />
during P cell migration, we have studied multiple alleles of let-502. Putative null alleles of let-502 and<br />
let-502 RNAi result in a weak P cell migration defect in worms surviving to L2. A temperature-sensitive<br />
hypomorphic allele causes a similar P cell phenotype. We propose that unc-73 and rho-1 act together,<br />
partially through let-502, to direct P cell migration in C. <strong>elegans</strong>.<br />
1 Zipkin, Ilan D., Rachel M. Kindt, and Cynthia J. Kenyon Cell 1997 90: 883-894.<br />
73
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
PTP-1, A LAR-LIKE RECEPTOR PROTEIN TYROSINE<br />
PHOSPHATASE, MAY ACT IN PARALLEL WITH C. ELEGANS<br />
EPH SIGNALING TO DIRECT MORPHOGENESIS<br />
Robert J. Harrington 1 , Michael Gutch 2 , Michael Hengartner 2 ,<br />
Nicholas Tonks 2 , Andrew Chisholm 1<br />
1Dept. of Biology, University of California, Santa Cruz CA 95064<br />
2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724<br />
Mutations in the C. <strong>elegans</strong> Eph receptor tyrosine kinase, VAB-1, and the ephrins, VAB-2 and MAB-26,<br />
cause defects in neural and epidermal morphogenesis. These defects are incompletely penetrant,<br />
suggesting that other pathways may function in parallel. To identify components of such parallel pathways<br />
we investigated whether vab-1 mutations displayed synthetic lethality with other morphogenetic mutants.<br />
A mutation in a C. <strong>elegans</strong> receptor tyrosine phosphatase, ptp-1(op147), causes mild morphogenetic<br />
defects similar to those of weak vab-1 alleles. We have found that ptp-1(op147) synergizes with vab-1<br />
mutations, in particular, kinase domain mutations. ptp-1(op147) also synergizes to some degree with<br />
mutations in vab-2 and mab-26, suggesting that PTP-1 may act in parallel with Eph signaling to regulate<br />
morphogenesis. We are testing the specificity of this interaction with other morphogenetic mutants that<br />
are not known Eph signaling components.<br />
PTP-1 is most similar to LAR-like receptor protein tyrosine phosphatases. We have found that the ptp-1<br />
locus encodes two isoforms, PTP-1A and PTP-1B. PTP-1A contains 3 Ig-like domains, 8 fibronectin type<br />
III repeats, and two tandem cytoplasmic phosphatase domains. PTP-1B contains only 5 FNIII repeats and<br />
the two phosphatase domains. Both isoforms are expressed in neurons.<br />
We have analyzed the phenotype of ptp-1 single mutants and vab-1 ptp-1 double mutants at the 4D<br />
microscopy level. We have found that ptp-1 mutants have gastrulation and ventral closure defects similar<br />
to Eph signaling mutants. vab-1 ptp-1 double mutants show the same defects, only at a higher<br />
occurrence. Our data suggests that PTP-1 and Eph signaling may function in parallel in neurons to direct<br />
morphogenesis.<br />
74
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GEX-2 AND GEX-3 DEFINE A CONSERVED PROTEIN<br />
COMPLEX REQUIRED FOR TISSUE MORPHOGENESIS AND<br />
CELL MIGRATIONS IN C. ELEGANS<br />
Martha Soto 1 , Katsuhisa Kasuya 2 , Hiroshi Qadota 2 , Kozo Kaibuchi 2 ,<br />
Craig C. Mello 1<br />
1University of Massachusetts Medical Center, Worcester, MA 01605, USA<br />
2Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma,<br />
Nara, 630-0101, Japan<br />
Morphogenesis during C. <strong>elegans</strong> embryogenesis requires coordinated cell movements. We report the<br />
identification of two genes, gex-2 and gex-3, necessary for tissue morphogenesis and cell migrations<br />
during C. <strong>elegans</strong> embryogenesis and for cell migrations in adult worms. Inactivation of gex-2 or gex-3<br />
with the RNAi technique or a loss of function mutation in gex-3 caused a maternal effect embryonic<br />
lethality with failure of dorsal intercalation and of ventral migrations of the epidermis. The gex-3 mutant<br />
larva showed zygotic phenotypes including an egg laying defect (Egl) and defects in distal tip cell<br />
migration.<br />
gex-2 and gex-3 encode proteins with no known motifs that are conserved from nematodes to humans,<br />
suggesting conserved essential functions for GEX-2 and GEX-3. The mammalian homolog of GEX-2,<br />
Sra-1 was identified as a specific interactor of GTP-bound Rac. GEX-3 has a human homolog;<br />
HEM2/NAP1/NCKAP1 is implicated in Rac signaling and is reported to bind the SH2-SH3 adaptor protein<br />
NCK/DOCK. The GEX-3 fly homolog, HEM2/KETTE affects axonal cell migrations, actin distribution and<br />
interacts genetically with NCK/DOCK.<br />
Immunostaining with anti-GEX-2 and GEX-3 antibodies showed that GEX-2 and GEX-3 colocalize to<br />
cell-cell contact sites of all embryonic cells. GEX-2 interacts with GEX-3 in the two hybrid system, and<br />
GEX-2 and GEX-3 proteins can immunoprecipitate each other, suggesting GEX-2 and GEX-3 form a<br />
complex. Our findings suggest that a novel protein complex including GEX-2 and GEX-3 localizes to cell<br />
boundaries to regulate cell migrations and cell shape changes required for embryonic tissue<br />
morphogenesis and for adult cell migrations. This complex may reorganize the actin cytoskeleton to<br />
control cell shape changes necessary for dorsal intercalation, migration of the hypodermal cells in the<br />
embryo and migration of the distal tip cells post-embryonically.<br />
75
PHARYNGEAL EXTENSION: THE SHORT AND THE LONG OF<br />
IT<br />
MF Portereiko, SE Mango<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Huntsman Cancer Institute Center for Children and Department of Oncological, Sciences, University of<br />
Utah, Salt Lake City, UT 84112<br />
Pharyngeal morphogenesis initiates during mid-embryogenesis, when 78 of the 80 pharyngeal cells have<br />
been born, and the embryo has begun to elongate. At this time, the pharyngeal precursors form a<br />
compact ball that is attached to the nascent midgut and surrounded by a basement membrane that<br />
encloses the entire primordium except for a gap at the anterior. Over the next 80 minutes, the pharyngeal<br />
precursors alter their morphology and position to form a linear tube that is linked to the buccal cavity<br />
anteriorly and the midgut posteriorly. We call this process pharyngeal extension. We have used<br />
time-lapse videomicroscopy and GFP reporter constructs to follow the behavior of the pharyngeal<br />
precursors during pharyngeal extension. Our studies demonstrate that pharyngeal extension can be<br />
loosely divided into three stages i) reorganization of cellular polarity within pharyngeal cells (specifically<br />
the pharyngeal epithelial precursors), ii) formation of an epithelium that mechanically couples the<br />
pharyngeal cells to arcade cells in the nascent buccal cavity, and iii) an apparent contraction that shifts<br />
the buccal cavity posteriorly and the pharynx anteriorly. We are considering a ’purse string’ model to<br />
explain the cellular behaviors we see during contraction. Our findings suggest that pharyngeal extension<br />
is the result of ’pulling’ by anterior pharyngeal cells rather than ’pushing’ by posterior pharyngeal cells. To<br />
test this idea, we eliminated the posterior pharynx via genetic and physical means and demonstrated that<br />
pharyngeal extension still occurs normally. Our model also predicts that tension between cells of the<br />
pharynx and buccal cavity is required for the movement we observe during contraction. We tested this<br />
idea by destroying the arcade cells. As expected, this manipulation blocked the anterior-directed<br />
movement of the pharyngeal cells and reduced the posterior-directed movement of the buccal cavity. Our<br />
current goal is to identify molecules required for pharyngeal extension using forward and reverse genetic<br />
approaches.<br />
76
A VAB-8/UNC-51/UNC-14 COMPLEX MEDIATES AXON<br />
OUTGROWTH<br />
Tina Lai, Gian Garriga<br />
Molecular and Cell Biology, University of California, Berkeley, CA 94720<br />
In C. <strong>elegans</strong> most posteriorly directed cell and growth cone migrations require vab-8, a gene that<br />
encodes at least two protein products known as VAB-8L and VAB-8S. VAB-8L is a 1066 amino acid<br />
protein that contains an N-terminal kinesin-like motor domain and functions in vab-8-dependent growth<br />
cone migrations. VAB-8S is colinear with the C-terminal half of VAB-8L, and lacks the kinesin-like motor<br />
domain. VAB-8S is necessary for certain vab-8-dependent cell migrations.<br />
To identify VAB-8-interacting proteins, we conducted a yeast two-hybrid screen using full length VAB-8L<br />
as bait. One protein identified was UNC-51, a serine/threonine kinase required for proper axon outgrowth<br />
(Ogura et al., 1994). In yeast and in vitro, a region between the kinesin and shared domains of VAB-8L<br />
interacts with the C-terminal half of UNC-51, which lacks the kinase domain.<br />
Ogura et al. (1997) have shown that the C-terminal half of UNC-51 also interacts with UNC-14. Using<br />
yeast two-hybrid, we found that a portion of VAB-8L interacts with UNC-14, but full length VAB-8L does<br />
not. These observations suggest that UNC-14 interaction sites are masked in the full length VAB-8<br />
protein.<br />
Several observations suggest that VAB-8, UNC-14 and UNC-51 also interact in C. <strong>elegans</strong>. First, vab-8,<br />
unc-14 and unc-51 mutants display axon outgrowth defects. Second, all three genes are expressed in<br />
neurons that require vab-8 function. Finally, misexpression of the UNC-51-binding domain of VAB-8L<br />
under control of the ceh-23 promoter results in highly penetrant CAN cell and growth cone migration<br />
defects, presumably by interfering with UNC-51 binding with wildtype VAB-8L. We are in the process of<br />
determining whether this misexpression phenotype could be suppressed by simultaneous misexpression<br />
of unc-51.<br />
Our results and those of Ogura et al. (1997) argue that VAB-8, UNC-14 and UNC-51 form a complex that<br />
functions in the outgrowth of certain axons. We have observed that overexpression of vab-8 can suppress<br />
axon outgrowth defects of an unc-51 mutant, suggesting a positive regulatory relationship between the<br />
two genes. Two possibilities we are testing are whether VAB-8L is an UNC-51 target and whether VAB-8L<br />
can regulate UNC-51 kinase activity.<br />
Ogura et al. (1994) Genes & Dev. 8: 2389-2400.<br />
Ogura et al. (1997) Genes & Dev. 11: 1801-1811.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
77
CYTOSKELETAL SIGNALLING IN RESPONSE TO THE UNC-6<br />
AXONAL ATTRACTANT<br />
Zemer Gitai 1 , Erik Lundquist 2 , Marc Tessier-Lavigne 1 , Cori<br />
Bargmann 1<br />
1HHMI, UCSF, San Francisco, CA 94143-0452<br />
2Univeristy of Kansas, Lawrence, KS 66045<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The netrin UNC-6 is involved in attraction and repulsion of circumferentially migrating cells and axons in<br />
C. <strong>elegans</strong>. Attraction to netrin/UNC-6 is mediated by the UNC-40 receptor, but the signalling events that<br />
occur upon UNC-40 activation remain unknown.<br />
In wild type animals UNC-6 is expressed ventrally, and several axons are directed ventrally via the<br />
UNC-40 receptor. This ventral migration is disrupted in unc-6 and unc-40 mutants. To probe the<br />
mechanism of UNC-40 activation by UNC-6, we deleted the extracellular domain of an unc-40 transgene<br />
and added a myristylation signal to the cytoplasmic domain, generating what we refer to as<br />
MYR::UNC-40.<br />
When expressed under the control of the mec-7 promoter, MYR::UNC-40 produced a variety of novel<br />
phenotypes in all mec-7-expressing touch cells. These phenotypes included exuberant axon outgrowth<br />
and branching, defective axon guidance, and enlarged and deformed cell bodies. The effects are<br />
independent of the endogenous UNC-6 ligand and UNC-40 receptor. These results lead us to believe that<br />
the MYR::UNC-40 represents a ligand-independent, constitutively active form of UNC-40.<br />
The generation of a gain-of-function UNC-40 molecule allowed us to search for downstream components<br />
of the UNC-40 signalling pathway by looking for mutations that suppressed the MYR::UNC-40-induced<br />
phenotype. One such mutation was in unc-115, a gene encoding a molecule with three LIM domains and<br />
an actin-binding villin headpiece. Both unc-40 and unc-115 mutants alone had defects in the ventral<br />
migration of the AVM axon. In the unc-40; unc-115 double mutant, the AVM axon guidance defect was<br />
not enhanced, again suggesting that UNC-115 might function in the UNC-40 pathway. The agreement of<br />
the loss-of-function double mutant phenotype with the MYR::UNC-40 suppression further supports the<br />
idea that MYR::UNC-40 represents a gain-of-function of UNC-40.<br />
Biochemical and cell biological experiments are currently underway to determine whether UNC-40 and<br />
UNC-115 physically associate. Structure/function experiments are also being conducted to determine the<br />
portion of the UNC-40 cytoplasmic domain responsible for signalling to UNC-115. Finally, we are<br />
continuing to search for new components of the UNC-40 signalling pathway by looking for additional<br />
suppressors of MYR::UNC-40.<br />
78
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
IDENTIFYING GENES INVOLVED IN AXONAL BRANCHING IN<br />
C. ELEGANS<br />
Joe C. Hao, Marc Tessier-Lavigne, Cornelia I. Bargmann<br />
Department of Anatomy and HHMI, UCSF, San Francisco, CA 94143-0452, USA<br />
In both the developing and mature nervous system, neurons can innervate multiple targets by sprouting<br />
secondary axon collaterals, or branches, from a primary axon shaft. Although positive and negative<br />
regulators of primary axonal growth cone guidance have been identified, little is known about the<br />
molecular mechanisms mediating axonal branching. In order to elucidate genes that may control axonal<br />
branching, we have performed a screen to identify mutants defective in branch formation by using a<br />
reporter expressed in the chemosensory neuron ADL. Unlike other amphid neurons, whose axons enter<br />
the nerve ring via the amphid commissures, the axon of ADL projects into the nerve ring laterally, where it<br />
then branches into both a dorsal and a ventral process.<br />
As a first step, we have examined the effects of known axon guidance genes on ADL axonal morphology.<br />
Interestingly, the unc-6/unc-40 pathway may mediate ADL ventral branch formation. In unc-6 or unc-40<br />
mutants, the majority of ADL axons lack the ventral axonal process. sax-3 mutants also exhibit defects in<br />
branch formation, ranging from the loss of both branches to the absence of either process.<br />
From our screen, we have isolated 36 mutants defective in ADL axonal branching that represent at least<br />
17 complementation groups. These fall into three classes: i. mutants defective in ventral branching; ii.<br />
mutants defective in dorsal branching; and iii. mutants with multiple branching defects. We have named<br />
these the branching of axon defective, or bad mutants. In addition to known axon outgrowth and guidance<br />
mutants, we have also isolated several novel mutants defective in axon guidance and/or branching.<br />
We are currently attempting to clone and further characterize bad-1, a mutant with identical ADL<br />
branching phenotypes as those observed in unc-6 and unc-40 mutants. These three mutants appear to<br />
act in the same genetic pathway and also affect AVM axon guidance and PLM axon branching. However,<br />
bad-1 animals are not uncoordinated and display normal motor neuron axon guidance, suggesting that<br />
unc-5-dependent repulsion is preserved in these mutants. These results suggest a role for bad-1 in a<br />
subset of unc-6/unc-40-mediated processes. We are also mapping and characterizing several other bad<br />
mutants.<br />
79
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UNC-119 SUPPRESSES SUPERNUMERARY BRANCHING IN<br />
C. ELEGANS<br />
Karla Knobel, Warren Davis, Michael Bastiani, Erik Jorgensen<br />
Biology Dept., University of Utah, Salt Lake City, UT 84112<br />
We previously characterized the behavior of migrating GABA motorneuron growth cones in C. <strong>elegans</strong><br />
larvae using time-lapse confocal microscopy. VD growth cones exhibit specific behaviors that result from<br />
the interaction between growth cones and different cellular substrates encountered during migration . The<br />
most dramatic behavior exhibited by migrating VD motorneuron growth cones in vivo is their collapse at,<br />
and subsequent extension beyond the dorsal body wall muscle. To identify molecules required for specific<br />
growth cone behaviors such as collapse we characterized the motorneuron axon outgrowth phenotype of<br />
existing uncoordinated mutants. One mutant, unc-119(ed3), possessed abnormally branched axons, most<br />
of which were located at the body wall muscle. This suggested that unc-119(ed3) growth cones extend<br />
supernumerary branches between the muscle and epidermis. Analysis of the UNC-119 expression<br />
pattern indicated that UNC-119 is expressed in neurons. Expression of UNC-119 under heterologous<br />
promoters demonstrated that it functions cell-autonomously to suppress axon branching. However,<br />
time-lapse analysis of motor neuron development and axon outgrowth indicated that UNC-119 does not<br />
function during growth cone migration. Comparison of axon outgrowth patterns 1 hour and 48 hours after<br />
the completion of growth cone migration indicated that axon branching occurs after axon outgrowth is<br />
completed in unc-119(ed3) mutants. Time-lapse analysis of established axons demonstrated that<br />
secondary growth cones sprout from motorneuron axons and cell bodies. These secondary growth cones<br />
extend extra branches to the dorsal nerve cord. Concurrently axonal extensions along the dorsal nerve<br />
cord are retracted. As a result, unc-119(ed3) adults have many commissural branches as well as large<br />
gaps in the dorsal nerve cord. Synapse development occurs after outgrowth is completed. To determine if<br />
synaptogenesis is affected we characterized synapses in unc-119(ed3) mutants. Synapses form and are<br />
morphologically normal as determined by electron microscopy. However, synapses are localized to<br />
inappropriate locations of the axon, including branches and dendritic regions. These data suggest that<br />
UNC-119 is a member of a new class of cell intrinsic factors that preserve the architecture of the nervous<br />
system and regulate synapse localization.<br />
80
UNC-119 AND AXON OUTGROWTH: TOWARD A<br />
MECHANISM<br />
Wayne Materi, Dave Pilgrim<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9<br />
UNC-119 is crucial for the correct development of the nematode nervous system. <strong>Worm</strong>s mutant for the<br />
neuronally-expressed unc-119 gene show locomotory and sensory abnormalities that are explained by<br />
structural defects resulting from aberrant nervous system development.<br />
Direct examination of nervous system structure using GFP reporters shows that unc-119 mutants have a<br />
variety of neurite outgrowth defects. The axons of chemosensory amphids exhibit highly penetrant ventral<br />
elongation defects within the nerve ring while the axons of anterior mechanosensory neurons have similar<br />
elongation defects in the dorsal direction. These defects suggest a disruption of normal synaptic patterns<br />
that is sufficient to explain both the inability of unc-119 mutants to form dauer larvae and their inability to<br />
respond to touch. The ventral nerve cord is moderately to severely defasciculated in 50% of mutant<br />
worms, supporting a general role for UNC-119 in axon guidance.<br />
Defects in the anteroposterior position of dorsolateral turns suggests an aberrant response to choice<br />
points. However, there are no circumferential pathfinding defects in motor neurons, similar to those seen<br />
in unc-6 , unc-5 or unc-40 mutants. Indeed, although some cell bodies are misplaced in unc-119 mutants,<br />
their axons are often correctly targeted, implying that turning at choice points is somewhat independent of<br />
other pathfinding mechanisms.<br />
Expression and rescue experiments with functional UNC-119::GFP constructs suggest that it acts cell<br />
autonomously. Although sub-cellular localization of the rat homologue, RRG4, has been demonstrated<br />
within photoreceptor ribbon synapses, antibody studies in the worm are still in progress. Yeast two-hybrid<br />
experiments suggest that UNC-119 is involved in a novel neurite development signaling pathway that<br />
mediates interaction between basement membrane collagen’s and an uncharacterized zinc-finger protein.<br />
We have used PCR-based reverse genetics to isolate strains bearing deletions in the latter of these and<br />
have observed a lack of exploratory behavior but otherwise normal locomotion and response to touch<br />
stimuli.<br />
81
THREE DISTINCT FUNCTIONS OF BETA-SPECTRIN (UNC-70)<br />
Marc Hammarlund, Warren S. Davis, Erik M. Jorgensen<br />
University of Utah, SLC UT 84112<br />
We cloned unc-70 and found that it encodes the C. <strong>elegans</strong> homolog of beta-spectrin, an essential<br />
component of the membrane skeleton.1 The membrane skeleton is a protein mesh that binds to the<br />
plasma membrane and that interacts with a large number of membrane and cytosolic proteins. It has<br />
been proposed that the membrane skeleton functions to support the plasma membrane or to generate<br />
cell polarity. However, we found that null mutations in unc-70 do not result in general membrane or<br />
polarity defects. unc-70 null animals have a severe paralyzed, dumpy phenotype. By analyzing this<br />
phenotype we have identified at least three specific processes that are perturbed by loss of beta-spectrin.<br />
First, we found defects in axonal morphology, suggesting that the membrane skeleton plays an essential<br />
role in neuronal development. We suspect that loss of beta-spectrin may specifically affect growth cone<br />
motility and are conducting time-lapse studies of unc-70 growth cones to test this hypothesis. Second,<br />
we found that loss of beta-spectrin causes defects in the myofilament attachment structures of body wall<br />
muscle (dense bodies and M lines). We are using a panel of antibodies against components of these<br />
structures to understand how the membrane skeleton functions in muscle. Finally, the membrane<br />
skeleton appears to function in synaptic transmission. We found that rare dominant alleles of unc-70 do<br />
not exhibit the null phenotypes described above; rather, they have abnormally high levels of<br />
neurotransmission. Preliminary evidence suggests that synaptic vesicles are mislocalized within the<br />
nerve terminal of these mutants. We are analyzing the synaptic ultrastructure of these mutants, and<br />
using electrophysiology to relate structural defects to possible functional roles for beta-spectrin in nerve<br />
terminals.<br />
1. Hammarlund, M. et al., J. Cell Biol., 149(4), 2000.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
82
RPM-1, A CONSERVED NOVEL PROTEIN, REGULATES<br />
PRESYNAPTIC TERMINAL FORMATION<br />
Xun Huang 1 , Mei Zhen 1 , Bruce Bamber 2 , Yishi Jin 1<br />
1Department of Biology, University of California, Santa Cruz, CA 95064<br />
2Department of Biology, University of Utah, Salt Lake City, UT 84112<br />
Presynaptic terminals contain highly specialized subcellular structures to facilitate neurotransmitter<br />
release. We used the synaptic vesicle tagged GFP marker under the unc-25 promoter to visualize the<br />
presynaptic varicosities of the GABAergic DD and VD motor neurons 1 . In wild-type animals GFP<br />
expression is observed as distinct fluorescent puncta, corresponding to individual neuromuscular<br />
junctions along the dorsal and ventral nerve cords. rpm-1 mutations were isolated in a screen for<br />
abnormal morphology of the GFP puncta.<br />
In rpm-1 mutants the total number of these GFP puncta is reduced to various extents depending on the<br />
strength of the mutation, and regions along the dorsal and ventral nerve cords often contained no GFP<br />
puncta. Moreover, the remaining GFP puncta were variable in size and shape. The overall axonal<br />
morphology of the DD and VD neurons appeared normal. At the ultrastructural level, we observed two<br />
defects at GABAergic NMJs in the rpm-1 mutants: 1) "over-developed" synapses that contained more<br />
than two individual electron-dense presynaptic active zones within the same varicosity, 2)<br />
"under-developed" synapses that had few synaptic vesicles and instead were filled with electron-dense<br />
debris-like material. In cholinergic NMJs, we observed the presynaptic terminals with longer active zones.<br />
rpm-1 encodes a large protein similar to Drosophila Highwire (Hiw) and mammalian Pam. All three<br />
proteins have a putative GEF domain and a highly conserved C-terminal with several zinc finger domains.<br />
RPM-1 is localized to the presynaptic region independently of synaptic vesicles. Mosaic analysis indicated<br />
that RPM-1 functions cell-autonomously. Using a temperature sensitive allele of rpm-1, we found that<br />
RPM-1 is required around the time of the formation of presynaptic terminals. Our results suggest that<br />
RPM-1 may regulate the distribution of presynaptic terminals, or function to prevent the formation of<br />
excessive presynaptic structures.<br />
1. Zhen, M and Jin, Y. Nature 1999<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
83
A C. ELEGANS INOSITOL 5- PHOSPHATASE HOMOLOGUE<br />
INVOLVED IN INOSITOL 1,4,5-TRIPHOSPHATE SIGNALING<br />
AND OVULATION.<br />
Yen Kim Bui, Paul W. Sternberg<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena,<br />
CA, 91125 USA.<br />
Understanding the basis of positive and negative regulation of the inositol signaling pathway may<br />
increase our understanding of the control of diverse biological processes dependent on inositol<br />
1,4,5-triphosphate (IP3). J. McCarter in T. Schedl’s lab demonstrated that mutations in lin-3/EGF or let-23<br />
/Receptor Tyrosine Kinase (RTK) result in a sterile Emo phenotype whereby the oocytes fail to be<br />
ovulated and become trapped in the gonad arm and undergo multiple rounds of DNA synthesis. T.<br />
Clandinin in our lab, showed that either a loss of function mutation in thelfe-2 /IP-3 kinase or gain of<br />
function (gf) in the lfe-1/IP3 Receptor can bypass the requirement for RTK signaling indicating that<br />
ovulation in C. <strong>elegans</strong> is dependent on IP3 signaling. Inositol 5-phosphatase plays a role in<br />
dephosphorylating IP3 to inositol 1,4-phosphate. However the contribution of this step in terminating IP3<br />
signaling in vivo remains unclear. The C. <strong>elegans</strong> genome predicts a putative ortholog of the human Type<br />
I inositol 5-phosphatase (CO9B8.1).<br />
Using a PCR based strategy, we screened for a deletion (D) mutant of the type I C. <strong>elegans</strong> inositol<br />
5-phosphatase. In contrast to the lfe-2 null or lfe-1 (gf), the 5-Ptase D mutant shows a novel ovulation<br />
phenotype whereby two oocytes enter the spermetheca during a single ovulation cylce; thus suggesting a<br />
crucial role for the 5-Ptase in regulating IP3 mediated ovulation. In contrast to the lfe-2 overexpression<br />
phenotype whereby the oocyte remains stuck in the dilated spermetheca, overexpression of the 5-Ptase<br />
has no noticeable affect. We are characterizing the mechanism by which it regulates ovulation by genetic<br />
analysis. Our genetic data support the hypothesis that increased IP3 signaling causes more ovulation,<br />
and that either too much signaling or too little signaling prevents ovulation. Like the lfe-2 null mutant, the<br />
5-Ptase D mutant suppresses the sterile defect of lin-3(n1058). This suggests that lin-3 sterile mutants fail<br />
to generate sufficient IP3 to execute ovulation. Furthermore, the 5-Ptase D mutant synthetically interacts<br />
with others mutants that increase IP3 signaling to produce a sterile Emo phenotype.<br />
84
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MECHANISMS REGULATING THE TIMING AND SPECIFICITY<br />
OF ANCHOR CELL ATTACHMENT TO THE VULVAL<br />
EPITHELIUM<br />
David R. Sherwood, Paul W. Sternberg<br />
Division of Biology and HHMI, California Institute of Technology, Pasadena CA<br />
We are interested in understanding the basic cellular and molecular mechanisms that guide<br />
morphogenetic processes during development. Towards this goal we are examining the initial steps<br />
involved in the connection of the uterus and vulva during C.<strong>elegans</strong> development. The initial contact<br />
between the developing uterus and the vulva is established by the anchor cell (AC), which crosses the<br />
basement membranes separating both tissues and specifically attaches to the progeny of the P6.p cell<br />
during the mid- to late L3 stage. Following attachment, the AC invades between the inner descendants of<br />
the P6.p. cell and becomes positioned at the apex of the developing vulva. Using mutants lacking vulval<br />
induction, we have found that the descendants of the underlying induced vulval precursor cell, P6.p, send<br />
a signal to trigger the attachment behavior in the AC. We have also discovered that the competence of<br />
the AC to respond to this signal is regulated: AC attachment is either absent or delayed in the<br />
heterochronic mutant lin-28, which causes precocious vulval development. To understand the molecular<br />
mechanisms that regulate AC attachment, we have examined many known mutants, as well as initiated a<br />
genetic screen to search for new mutants with defective attachment behavior. Through these studies we<br />
have found that the evl-5 mutant, originally isolated in a screen for sterile p-vul’s (Seydoux et al., (1993)<br />
Dev. Biol. 157(2): 423-36), has a defect in AC attachment to the vulval epithelium. In evl-5 animals vulval<br />
induction and AC positioning over the P6.p cell is normal; however, AC attachment either does not occur<br />
or is severely delayed. Visualization of AC behaviour in this mutant revealed the extension of cellular<br />
processes from the AC toward the vulva, indicating that the AC is still attracted to the developing vulva.<br />
However, in many cases the AC processes appeared to flatten or broaden out at the basement<br />
membrane of the developing gonad, suggesting that the AC may not be able to cross this basement<br />
membrane. We are currently molecularly cloning evl-5.<br />
85
MUTATIONS IN CYCLIN E REVEAL COORDINATION<br />
BETWEEN CELL-CYCLE CONTROL AND VULVAL<br />
DEVELOPMENT.<br />
David S. Fay, Min Han<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Howard Hughes Medical Institute and Department of Molecular, Cellular, and Developmental Biology,<br />
University of Colorado, Boulder CO 80309-0347<br />
In screens for mutations affecting vulval development, we have identified strong loss-of-function<br />
mutations in the C. <strong>elegans</strong> cyclin E gene, cye-1. Mutations in cye-1 lead to the under-proliferation of<br />
many postembryonic blast lineages as well as defects in fertility and gut-cell endoreduplication. In<br />
addition, cye-1 is required maternally, but not zygotically for embryonic development. Our analysis of<br />
vulval development in cye-1 mutants suggests that a timing mechanism may control the onset of vulval<br />
cell terminal differentiation: once induced, these cells appear to differentiate after a set amount of time,<br />
rather than a specific number of division cycles. cye-1 mutants also show an increase in the percentage<br />
of vulval precursor cells (VPCs) that adopt vulval cell fates, indicating that cell-cycle length can play a role<br />
in the proper patterning of vulval cells. By analyzing cul-1 mutants, we further demonstrate that vulval cell<br />
terminal differentiation can be uncoupled from associated changes in vulval cell division planes.<br />
86
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
NOVEL CELL-CELL INTERACTIONS DURING VULVA<br />
DEVELOPMENT IN PRISTIONCHUS PACIFICUS<br />
Benno Jungblut 1,2 , Ralf J Sommer 1,3<br />
1Max-Planck Institut für Entwicklungsbiologie, Abt. Evolutionsbiologie, Spemannstrasse 35, 72076<br />
Tübingen, Germany<br />
2email: benno.jungblut@tuebingen.mpg.de<br />
3email: ralf.sommer@tuebingen.mpg.de<br />
Comparative analysis of vulva development revealed several differences between the two nematode<br />
species <strong>Caenorhabditis</strong> <strong>elegans</strong> and Pristionchus pacificus including changes in cell fates and inductive<br />
processes. For example, seven of 12 ventral epidermal cells in P. pacificus die of apoptosis, whereas<br />
homologous cells in C. <strong>elegans</strong> fuse with the hypodermal syncytium. In addition vulva induction by the<br />
anchor cell is a one-step process in C. <strong>elegans</strong>, but requires an interaction of both anchor cell and the<br />
gonad with the vulval precursor cells in P. pacificus. We have identified novel cell-cell interactions while<br />
carrying out combinatiorial cell ablation studies of the vulval precursor cells. In particular the ventral<br />
epidermal cell P8.p and the mesoblast M contribute to vulval pattern formation. In contrast to the<br />
homologous cell in C. <strong>elegans</strong>, P8.p in P. pacificus is incompetent to respond to inductive signaling from<br />
the gonad. Nonetheless it can be induced to adopt a vulval fate by lateral signaling from a neighboring<br />
vulval precursor cell. Immunofluorescence studies showed that P8.p fuses with the hypodermis a few<br />
hours before the birth of the anchor cell. Using cell ablation studies we show that P8.p provides an<br />
inhibitory signal that limits the developmental competence of P(5, 7).p. This lateral inhibition also requires<br />
the mesoblast M. In Ppa-mab-5 mutants, M is misspecified and provides inductive signaling to the vulval<br />
precursor cells, including P8.p. Therefore P8.p differentiates gonad independently in these mutants.<br />
Taken together, vulva development in P. pacificus displays novel cell-cell interactions involving the<br />
mesoblast M and P8.p. In particular, P8. represents a new ventral epidermal cell fate, which is<br />
characterized by novel interactions and a specific response to gonadal signaling.<br />
87
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CELLULAR AND GENETIC ANALYSIS OF G Q MEDIATED<br />
SIGNALING PATHWAYS IN C. ELEGANS<br />
C. A. Bastiani, S. Gharib, P.W. Sternberg, M.I. Simon<br />
Division of Biology, California Institue of Technology, Pasadena, CA 91125<br />
egl-30 encodes the C. <strong>elegans</strong> homologue of the mammalian heterotrimeric G protein alpha subunit, Gq.<br />
Reduction-of-function and loss-of-function mutations in egl-30 affect diverse behaviors in C. <strong>elegans</strong>.<br />
These include: viability, egg-laying, pharyngeal pumping, movement, and spicule protraction. In a screen<br />
for suppressors of the reduction-of-function mutation, egl-30(md186), two intragenic suppressor mutations<br />
were recovered. One of the mutations lies in a region in close proximity to residues involved in guanine<br />
ring binding. The other mutation is in a region that might affect receptor interaction, and is not expected to<br />
make contacts with the region that contains the original egl-30(md186) mutation. These mutants<br />
phenotypically resemble worms that overexpress egl-30, and have been used as a tool to characterize<br />
genes that function in a pathway with egl-30. From these studies, it is clear that EGL-30 regulates egg<br />
laying via downstream signaling pathways distinct from downstream pathways so far defined that control<br />
movement (1), viability (2), and spicule protraction (3). A rescuing gfp::egl-30 transgene confirms that<br />
egl-30 is coexpressed in some cells with egl-8 and lfe-1/itr-1/dec-4, two genes that encode signaling<br />
molecules defined as downstream of Gq in mammalian systems, and that appear to be downstream of<br />
egl-30 with respect to movement and viability, respectively (1,2). However, egl-30 is also uniquely<br />
localized and expressed in some cells. To determine the basis for the differences in genetic interactions<br />
with respect to different behaviors, we are develooping a system to characterize cell-type-specific<br />
molecular interactions with EGL-30 in vivo .<br />
1. Lackner MR, Nurrish SJ, Kaplan JM; Neuron 24: 335-346 1999<br />
2. Hajdu-Cronin YM and Sternberg PW, personal communication<br />
3. Garcia LR and Sternberg PW, personal communication<br />
88
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II<br />
REGULATES C. ELEGANS LOCOMOTION IN CONCERT WITH<br />
A G-PROTEIN SIGNALING NETWORK<br />
Merrilee Robatzek, James H. Thomas<br />
Department of Genetics, University of Washington, Seattle, WA 98195 USA<br />
Calcium/calmodulin-dependent protein kinase II (CaMKII) is an important regulator of synaptic strength<br />
based on studies in mollusks, Drosophila, and mouse. Although the phosphorylation of many gene<br />
products has been attributed to CaMKII from in vitro assays, the mechanism of CaMKII activity in vivo has<br />
not been fully defined. We are using a genetic approach to study the in vivo function of the C. <strong>elegans</strong><br />
CaMKII homolog unc-43. UNC-43 is 83% identical in the kinase domain to mammalian CaMKII, indicating<br />
that the targets of this kinase are probably conserved.<br />
unc-43 regulates several behaviors, including locomotion. A kinase-activated allele of unc-43, n498,<br />
causes severe lethargy, as well as body-wall muscle hypercontraction, reduced egg laying, and reduced<br />
defecation. Several of these effects are genetically separable, suggesting that unc-43 regulates<br />
defecation, body-wall muscle tone, and locomotion rate through different effectors. To understand how<br />
unc-43 controls locomotion rate in C. <strong>elegans</strong>, we performed a genetic suppressor screen with<br />
unc-43(n498) to identify genes that act with unc-43 to control locomotion rate. From a screen of 28,000<br />
EMS-mutagenized haploid genomes, we recovered 19 recessive extragenic suppressors.<br />
Complementation tests showed that we had recovered multiple alleles of the genes goa-1, dgk-1, and<br />
eat-16, all involved in the goa-1/egl-30 heterotrimeric G-protein network , , , and alleles of a fourth gene,<br />
eat-11, that probably affects this same pathway. In addition to suppressing the unc-43(n498) lethargy,<br />
mutations in these genes also suppress the unc-43(n498) egg-laying defect. Quantitative analysis of the<br />
suppression indicates that UNC-43 may control locomotion rate and egg-laying activity by regulating this<br />
G-protein signaling network.<br />
89
A NOVEL LATERAL SIGNALING PATHWAY DETERMINES<br />
ASYMMETRIC OLFACTORY NEURON FATES<br />
Alvaro Sagasti, Cori Bargmann<br />
UCSF and HHMI<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The AWC olfactory neurons in C. <strong>elegans</strong> are a bilaterally symmetric pair of cells required for sensing<br />
certain attractive volatile odorants. Although bilaterally homologous neurons are usually thought to be<br />
identical, the candidate seven transmembrane olfactory receptor str-2 is expressed asymmetrically in the<br />
AWC neurons. About half of a population of worms expresses str-2 in the left homolog of the AWC pair,<br />
and half expresses it in the right homolog. The AWC cells are able to coordinate their fates by<br />
communicating with each other, probably through their axons which contact each other in the nerve ring.<br />
To study the signaling mechanisms by which this communication is accomplished we conducted a screen<br />
for mutant worms that express a str-2::GFP reporter in both AWC neurons. The screen yielded mutations<br />
in three novel neuronal symmetry genes (nsy-1, nsy-2, and nsy-3) and three known genes that affect<br />
calcium signaling: alleles of the voltage-gated calcium channel subunits unc-2 and unc-36 and an allele of<br />
the calcium/calmodulin-dependent protein kinase II homolog unc-43. This suggests that calcium signals<br />
are an important component of the AWC asymmetry signaling pathway. Genes in a cGMP signaling<br />
pathway necessary for olfaction have the phenotype opposite of the calcium signaling genes. They are<br />
required for any expression of str-2 in AWC, perhaps in an activity-dependent mechanism used for<br />
maintenance of receptor expression. Epistasis analysis is consistent with nsy-1 and nsy-2 acting after<br />
calcium signaling but before cGMP signaling. In contrast, the dominant mutant nsy-3 seems to act after<br />
axon outgrowth but before calcium signaling. We recently cloned the nsy-1 gene and found that it<br />
encodes a protein kinase. We are currently performing experiments to determine where nsy-1 is<br />
expressed, whether it is acting in the AWC cells, and how it is activated by CaMKII. We are also setting<br />
up a system that will permit us to identify easily AWC left/right mosaics, allowing us to determine which<br />
genes in this pathway act permissively and which act instructively<br />
90
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE SEARCH FOR DOSAGE COMPENSATION COMPLEX<br />
BINDING SITES ON X CHROMOSOMES<br />
Raymond C. Chan, Tammy F. Wu, Barbara J. Meyer<br />
HHMI and Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204<br />
Dosage compensation (DC) equalizes the level of X-linked gene products between XX hermaphrodites<br />
and XO males. A protein complex (DCC), with remarkable similarity to the Xenopus mitotic 13S<br />
condensin complex, is directed by the sex-determination and dosage compensation (SDC) proteins to the<br />
hermaphrodite X chromosomes, where it represses gene expression. A mechanistic link between dosage<br />
compensation and mitotic chromosome condensation is further indicated by the dual roles of many DCC<br />
subunits in mitosis or meiosis. This link raises an intriguing question of how the DCC specifically<br />
recognizes X chromosomes to effect global repression of gene expression. To study X recognition, we<br />
developed a chromatin immunoprecipitation (ChIP) protocol to isolate DNA bound by the DCC. We<br />
initially devised an affinity purification method to precipitate DC proteins using peptide antibodies against<br />
the DCC. The IP not only confirmed the association of DCC subunits, it also provided a means to isolate<br />
large quantities of DC and mitotic complexes for identifying novel co-purifying factors (see Hagstrom et<br />
al.). When combined with in vivo formaldehyde crosslinking, we can also recover DNA in the precipitate,<br />
thus enabling us to identify candidate X recognition sequences.<br />
To validate this ChIP assay, we tested the known interaction between the SDC proteins and the her-1<br />
promoter. Aside from DC, the SDC proteins bind and repress the promoter of her-1 to establish the<br />
female fate in young XX embryos. In the sdc-3(y52Tra) mutant strain, SDC proteins fail to bind the her-1<br />
promoter leading to the masculinization of XX animals. With our current protocol, we detected her-1<br />
promoter sequences by PCR in both SDC-2 and SDC-3 ChIP. Moreover, ChIP performed on extracts<br />
from sdc-3(Tra) embryos revealed drastically reduced levels of her-1 DNA consistent with the loss of<br />
her-1 binding in these mutants.<br />
We are now focused on identifying X sequences bound by the SDC and DCC proteins. We have<br />
examined 180 kb around myo-2, a known dosage compensated gene on X, and we found an<br />
approximately 4-kb region enriched in the SDC and DCC ChIP. Experiments are in progress to further<br />
define this region and to confirm SDC and DCC protein binding in vivo and by additional in vitro methods.<br />
91
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
RECOGNITION AND ASSEMBLY OF SDC PROTEIN<br />
COMPLEXES ONTO SPECIFIC DNA TARGET SITES<br />
Diana Chu 1 , Heather Dawes 1 , Jason Lieb 2 , Annie Kuo 1 , Barbara J.<br />
Meyer 1<br />
1HHMI and Department of Molecular and Cell Biology<br />
2HHMI and Department of Biochemistry, Stanford University Medical Center, Stanford, CA 94305<br />
SDC (Sex and Dosage Compensation) proteins trigger hermaphrodite development by activating dosage<br />
compensation and repressing the male sex determination gene her-1. We have biochemically defined an<br />
SDC protein complex including SDC-1, SDC-2, and SDC-3. This complex localizes to the her-1 promoter,<br />
where it represses transcription at least 20-fold. It also associates along the entire length of the<br />
hermaphrodite X chromosomes, where it recruits other dosage compensation proteins (DC) such as<br />
DPY-26, DPY-27, DPY-28 and MIX-1 to repress X-linked gene expression 2-fold. We are interested in<br />
how the SDC complex can recognize and act differentially at these separate sites.<br />
Despite the fact that DC proteins are not required for transcriptional repression of her-1, we have found<br />
that the SDC proteins recruit these DC proteins to the her-1 promoter. We showed this co-localization in<br />
transgenic animals that express a GFP-LacI fusion protein from extrachromosomal arrays that also<br />
contain both lac operator repeats (lacO) and the her-1 promoter. Analysis of SDC and DC protein<br />
localization to the her-1 promoter arrays in SDC and DC mutant backgrounds has revealed sequential<br />
requirements for recognition and assembly of the SDC and DC proteins onto target sequences.<br />
To define how SDC proteins recognize specific target sites, we mapped regions within the her-1 promoter<br />
that are important for SDC protein recognition using the assay above. To test the functional significance<br />
of the three regions we identified, we mutated them in the context of a full length her-1 rescuing construct.<br />
Stable transgenic XX strains transformed with a wild-type her-1 show little masculinization. In contrast,<br />
stable transgenic XX strains transformed with mutated her-1 show varying degrees of transformation to<br />
the male fate, including induction of male tail structures and decreased fertility. We interpret this<br />
masculinization as an indication of her-1 derepression. Interestingly, sequences in the regions important<br />
to support SDC-2 localization are not homologous to sequences on the X chromosome. Thus, recognition<br />
of the X chromosome and the her-1 promoter appears to be specified by distinct mechanisms.<br />
92
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE TBP-LIKE FACTOR CETLF IS REQUIRED TO ACTIVATE<br />
RNA POLYMERASE II TRANSCRIPTION IN C. ELEGANS<br />
EMBRYOS<br />
Linda S. Kaltenbach, Susan E. Mango<br />
Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, Salt Lake City,<br />
UT, 84112<br />
Modulation of transcription is crucial to establish differences between cells during development. While the<br />
importance of activators and repressors for regulating transcription is well known, recent data suggest<br />
coregulators and general transcription factors may provide an additional source of control. TATA-binding<br />
protein (TBP) is a component of the basal transcription machinery that is ubiquitously expressed in<br />
metazoans and essential for all transcription in yeast. Recently, two variants of TBP have been<br />
discovered: 1) TBP-Related Factor, known only in Drosophila, which may act as a cell-type specific TBP<br />
and 2) TBP-Like Factor (TLF), found in most, if not all, metazoans. Given the central role of TBP, what is<br />
the function of the TBP paralogs?<br />
On the basis of biochemical studies, four models have been proposed for the role of TLF in RNA<br />
polymerase II (pol II) transcription: i) TLF and TBP could function redundantly, ii) TLF could antagonize<br />
TBP, iii) TLF could be a tissue-specific TBP or iv) TLF and TBP could have unique activities. To<br />
distinguish between these models, we examined the function of C. <strong>elegans</strong> TLF (CeTLF) in vivo.<br />
We have found that CeTLF has an essential role to activate pol II transcription in early embryos. Loss of<br />
CeTLF activity by RNAi results in embryonic arrest at the 70-200 cell stage with no evidence of<br />
differentiation. This phenotype and global expression of CeTLF demonstrate that CeTLF is not a<br />
tissue-specific TBP. Embryos lacking CeTLF fail to gastrulate, a phenotype that resembles loss of ama-1,<br />
which encodes the large subunit of pol II. These embryos also express a marker of elongating pol II at a<br />
reduced level, suggesting that CeTLF is required for some but not all pol II transcription. To test this idea,<br />
we have examined whether CeTLF is necessary to express individual genes normally transcribed in the<br />
gastrula-stage embryo and find that CeTLF is required to activate two of four surveyed genes. Preliminary<br />
experiments indicate that CeTLF physically associates with at least one of these genes in vivo. These<br />
results suggest that CeTLF performs a unique function to activate pol II transcription at some promoters,<br />
and that this activity is distinct from that of CeTBP.<br />
93
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE INTRACELLULAR DOMAIN OF THE FEMINISING<br />
RECEPTOR TRA-2A INTERACTS DIRECTLY WITH THE<br />
TRANSCRIPTION FACTOR TRA-1A<br />
David H. Lum 1 , P. Kuwabara 2 , D. Zarkower 3 , A.M. Spence 1<br />
1Dept. of Molecular and Medical Genetics, University of Toronto, ON, M5S 1A8, CANADA.<br />
2MRC Laboratory of Molecular Biology, Hills Road, Cambridge , CB2 2QH.<br />
3Institute of Human Genetics, Minneapolis, MN 55455, U.S.A.<br />
In XX animals the tra-2 gene negatively regulates the activity of the three fem genes. The absence of fem<br />
activity in XX animals frees tra-1, the terminal regulator of somatic sex determination, to promote female<br />
development. The tra-2 gene encodes two proteins TRA-2A and TRA-2B. The larger of the two proteins,<br />
TRA-2A, is a predicted transmembrane protein with a large C-terminal intracellular domain. TRA-2B is<br />
expressed in the hermaphrodite germline and is predicted to be a soluble protein consisting of just the<br />
C-terminal domain of TRA-2A.<br />
The intracellular domain of TRA-2A negatively regulates the FEMs through a direct interaction with<br />
FEM-3. Overexpression of the C-terminal domain of TRA-2A (TRA-2Ac) is sufficient for FEM negative<br />
regulation and the transformation of XO animals into females. To investigate the mechanism of TRA-2Ac<br />
feminizing activity we overexpressed various TRA-2Ac fragments. Surprisingly, a C-terminal fragment of<br />
TRA-2Ac (TRA-2AcD 5), incapable of interacting with FEM-3 in the yeast 2-hybrid system, maintained<br />
weak feminizing activity. Both yeast 2-hybrid and biochemical data demonstrate that TRA-2AcD 5<br />
physically interacts with TRA-1A. tra-2(mx) mutations affect tra-2 activity in both the soma and germline.<br />
Three mx mutations all disrupted the TRA-2Ac/TRA-1A interaction and reduce or eliminate the feminising<br />
activity of TRA-2AcD 5 in overexpression assays.<br />
Our surprising results suggest that in the soma TRA-2A plays a role in regulating TRA-1A through a direct<br />
interaction. TRA-1A has been demonstrated to directly regulate transcription of several genes and is<br />
predicted to be nuclear localized. We observe strong nuclear localization of both GFP::TRA-1A and<br />
Myc::TRA-1A in transgenic animals. Consistent with a nuclear role for TRA-2A regulation of TRA-1A, we<br />
observe nuclear localization of a highly active GFP::TRA-2Ac fusion protein. Our data raise the intriguing<br />
possibility that in the soma TRA-2A is cleaved to allow the intracellular domain to enter the nucleus and<br />
interact with TRA-1A.<br />
94
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CHW-1 ENCODES A NOVEL PROTEIN THAT INTERACTS<br />
WITH PHA-4<br />
Michael Horner, Linda Kaltenbach, Susan Mango<br />
Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, Salt Lake City,<br />
Utah 84112<br />
The winged-helix transcription factor PHA-4 specifies organ identity during pharynx and rectal<br />
development. Embryos lacking pha-4 activity produce no pharyngeal or rectal cells while embryos<br />
expressing ectopic pha-4 generate extra pharyngeal and rectal cells at the expense of other cell types.<br />
PHA-4 is also expressed in the gonad, but its function in this organ is unknown.<br />
Homologs of pha-4 are found in most if not all metazoans, where they also regulate gut development.<br />
Proteins belonging to this family are predicted to be transcription factors, with a well-characterized DNA<br />
binding domain. However, little is known about how these proteins regulate transcription to produce such<br />
profound effects on development. To begin to address this issue, we have initiated an analysis of PHA-4<br />
protein and PHA-4 cofactors. Our characterization of five pha-4 alleles demonstrates that the amino<br />
terminus is critical for PHA-4 function. Surprisingly, the carboxyl terminus, which is conserved with<br />
vertebrate HNF-3 proteins and Ce-DISTALLESS, is dispensable.<br />
Since transcription factors often function by recruiting other proteins to target genes, we used a yeast<br />
2-hybrid screen to identify proteins that bind the PHA-4 amino terminus. We isolated six potential<br />
cofactors from a screen of >1x106 clones and have chosen one of these proteins for further study (the<br />
chw genes for Components that Heterodimerize with a Winged helix protein). We have confirmed that<br />
CHW-1 can bind PHA-4 in vitro, suggesting these proteins contact each other directly.<br />
PHA-4 and CHW-1 may function together to regulate expression of a subset of PHA-4 target genes.<br />
CHW-1 is a novel, glutamine-rich protein that is expressed in the intestine, rectum and pm6 pharyngeal<br />
muscles, as is PHA-4. In addition, CHW-1::GFP is expressed in body wall muscles where PHA-4 is not<br />
found. We have used RNAi to show that loss of chw-1 activity affects cells that normally express PHA-4.<br />
In many chw-1(RNAi) animals, the pharyngeal grinder is deformed, larvae appear starved, and gonad<br />
migration is defective. Interestingly, these phenotypes are strongly enhanced when PHA-4 activity is<br />
compromised. We suggest that PHA-4 may bind distinct CHW factors to regulate different target genes in<br />
the pharynx, rectum or gonad.<br />
95
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE UNC-4 HOMEOPROTEIN AND ITS TRANSCRIPTIONAL<br />
CO-REPRESSOR UNC-37/GROUCHO REGULATE<br />
NEUROTRANSMITTER VESICLES IN CHOLINERGIC MOTOR<br />
NEURONS<br />
Kim Lickteig 1 , Janet Duerr 2 , Dennis Frisby 3 , David Hall 4 , Jim Rand 2 ,<br />
David Miller 1<br />
1Vanderbilt University, Nashville, TN<br />
2Oklahoma Medical Research Foundation, Oklahoma City, OK<br />
3Univ. of Central Oklahoma, Edmond, OK<br />
4Albert Einstein College of Medicine, Bronx, NY<br />
The UNC-4 homeoprotein is expressed in VA motor neurons to prevent the adoption of synaptic inputs<br />
normally reserved for their lineal sisters, the VB neurons. Work from this laboratory has shown that<br />
UNC-4 functions as a negative regulator of VB-specific genes and that this activity depends on physical<br />
interaction with the ubiquitously expressed UNC-37/Groucho co-repressor protein. UNC-4 is also<br />
expressed in other motor neurons (DA, SAB, VC) but these cells (e.g. DAs) are not miswired in unc-4<br />
mutants. Here we show that unc-4 and unc-37 mutations result in decreased levels of synaptic vesicle<br />
(SV) proteins in "unc-4-expressing motor neurons" and that this deficit impairs the function of these cells.<br />
Antibody staining reveals that five vesicular proteins (UNC-17, ChAT, synaptotagmin, synaptobrevin,<br />
RAB-3) are substantially reduced in unc-4 and unc-37 mutants whereas other non-vesicular presynaptic<br />
proteins (syntaxin, UNC-18, UNC-11) are not affected. This finding is consistent with ultrastructural<br />
analysis of VA motor neurons in unc-4(e120) which show a 40% reduction in SVs at presynaptic<br />
densities. Because the UNC-4/UNC-37 complex has been shown to mediate trancriptional repression, we<br />
believe that these effects must be executed through an intermediate gene. Furthermore, our results<br />
indicate that this intermediate gene ("Gene-X") functions as a negative regulator of SV biogenesis or<br />
stability. Experiments with a temperature sensitive unc-4 mutant indicate that the adult level of SV<br />
proteins strictly depends on unc-4 function during a critical early period of motor neuron differentiation.<br />
unc-4 activity during this sensitive larval stage (L2 to mid L3) is also required for the creation of proper<br />
synaptic inputs to the VA neurons. The temporal correlation of these events offers the intriguing possibility<br />
that a common regulatory mechanism involving unc-4 may account for the specificity of synaptic input as<br />
well as the strength of synaptic output for the VA motor neurons.<br />
96
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE COMPONENTS OF SENSORY CILIA IN C. ELEGANS<br />
Peter Swoboda, Kerry Bubb, James H. Thomas<br />
Department of Genetics, University of Washington, Seattle, WA 98195<br />
Cilia are an important cellular differentiation of sensory neurons for receiving information from the<br />
environment. 60 of the 302 C. <strong>elegans</strong> neurons have ciliated endings. Mutations in a large number of<br />
genes have been identified that affect the structure of these sensory cilia. Of these, daf-19 mutations are<br />
unique in completely lacking all sensory cilia. Aside from the absence of sensory cilia, the neurons seem<br />
to be morphologically normal. We previously reported that daf-19 encodes an RFX-type transcription<br />
factor that is expressed in ciliated sensory neurons.We also showed that DAF-19 regulates via its target<br />
site in promoter regions, the x-box, a number of effector genes involved in sensory cilium formation.<br />
These effector genes are expressed in essentially all ciliated sensory neurons and, when mutated, cause<br />
morphological defects in sensory cilia (e.g. che-2, osm-1, osm-6). Our results strongly suggest that<br />
DAF-19 regulates the differentiation of sensory cilia by activating the transcription of a battery of genes<br />
whose products form the sensory cilium.<br />
Cilia and flagella are structurally very similar subcellular organelles. Work done on the unicellular,<br />
flagellated green alga Chlamydomonas lead to an estimate of around 300 different protein components<br />
required for structure and function of flagella. We expect a similar number for cilium structure and<br />
function. We aim to initiate the identification of all ciliary components by analyzing genes that have an<br />
appropriately spaced x-box promoter element: so called xbx genes, hypothesized to be regulated by<br />
daf-19. In a first search through the C. <strong>elegans</strong> genome sequence, using an algorithm designed to find<br />
promoter elements, we uncovered more than 200 candidate xbx genes, several of which had C. briggsae<br />
homologues that also had an appropriately positioned x-box promoter element. Initial expression analyses<br />
of a subgroup of these xbx genes showed that, indeed, some of them were specifically expressed in<br />
ciliated sensory neurons in a daf-19 dependent manner. In another approach, using oligo-nucleotide<br />
arrays representing nearly all genes of C. <strong>elegans</strong> (1), we compared the gene expression profile of wild<br />
type and daf-19 mutants. A small group of genes was reproducibly down-regulated in a daf-19<br />
background, among them known cilium-structure genes and several xbx genes. In combination with<br />
previous data about sensory cilia, these approaches have the potential to reveal all the genes required for<br />
sensory cilium function and structure.<br />
(1) in collaboration with Allan Jones (Rosetta Inpharmatics, Kirkland, WA, U.S.A.)<br />
97
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
IN VIVO IMAGING OF HSN OUTGROWTH<br />
Carolyn E. Adler, Cornelia I. Bargmann<br />
Department of Anatomy, UCSF, San Francisco, CA 94143<br />
Axons in developing organisms must migrate and extend long distances in order to synapse onto their<br />
proper targets. Little is known about how extracellular guidance cues elicit the changes in cytoskeletal<br />
dynamics that drive axon outgrowth. In order to understand the factors that control the development of the<br />
HSN motorneuron, we are undertaking an observational approach. We are utilizing two-photon<br />
microscopy to analyze the trajectory of HSN as it grows ventrally in late L2/early L3 stage worms. Using<br />
worms expressing GFP under the unc-86 promoter, we are able to visualize HSN during the course of its<br />
outgrowth.<br />
Preliminary imaging indicates that HSN initially extends filopodia in all directions from its cell body, but<br />
that only those filopodia facing the ventral side persist. Eventually these filopodia develop into a large<br />
lamellopod that extends ventrally and stalls at the ventral muscle barrier. A few processes protrude<br />
through the body wall muscle attachment sites, as seen in VD and DD motorneuron growth through the<br />
dorsal muscle attachment sites.1 Although multiple processes extend toward the ventral nerve cord, only<br />
one is stabilized and develops into the axon.<br />
We plan to characterize HSN outgrowth over the course of its development. With this information, we can<br />
analyze the HSN axon in mutant worms known to have defects in ventral growth. In particular, we will<br />
examine worms containing mutations in the extracellular guidance cues to which HSN responds as well<br />
as in cytoplasmic proteins thought to participate in the regulation of cytoskeletal dynamics. In addition, we<br />
plan to label the actin and microtubule cytoskeletons and visualize their dynamics during HSN outgrowth.<br />
This approach will allow us to elucidate the roles of particular proteins in the process of HSN outgrowth.<br />
1. Knobel KM, Jorgensen EM, Bastiani MJ. Development. 1999 Oct;126(20):4489-98.<br />
98
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
TEMPORAL AND SPATIAL REQUIREMENT OF SENSORY<br />
CILIA IN THE REGULATION OF WORM LIFESPAN<br />
Joy Alcedo, Javier Apfeld, Bella Albinder, Jennifer Dorman, Honor<br />
Hsin, Bernadine Tsung, Cynthia Kenyon<br />
Department of Biochemistry and Biophysics, UCSF, San Francisco, CA 94143<br />
C. <strong>elegans</strong> is an excellent organism in which to study the regulation of lifespan. Genetic analyses have<br />
already shown that an insulin/IGF-like hormonal control system, the DAF-2 pathway, regulates lifespan in<br />
the worm. However, many components of the pathway, whose existence has been inferred from previous<br />
studies (Apfeld and Kenyon, (1999). Nature 402, 804-809; and Hsin and Kenyon, (1999) Nature 399,<br />
362-366) have not been identified. To search for more genes that would help to elucidate mechanisms<br />
that control lifespan in the worm, our group has done an EMS mutagenesis screen for long-lived worms<br />
and found 29 independent long-lived mutants.<br />
At least one of the mutants, mu377(IV), maps to and fails to complement the gene daf-10, which is<br />
required for the normal development of the worm’s sensory cilia. Apfeld and Kenyon (1999) have recently<br />
shown that worms defective in sensory cilia, including daf-10 mutants, live longer than normal worms,<br />
which suggests that the lifespan of C. <strong>elegans</strong> might be regulated by the perception of a signal(s) from<br />
the environment. mu377(IV) has a temperature-sensitive defect in its sensory cilia, which is linked to its<br />
temperature-sensitive lifespan phenotype. At the restrictive temperature, its lifespan is longer than<br />
wild-type and it exhibits a defect in its sensory cilia; whereas at the permissive temperature, its lifespan is<br />
the same as wild-type and it exhibits normal sensory cilia. Since the defect in the sensory cilia of<br />
mu377(IV) is reversible, we determined the temporal requirement for the gene product of mu377(IV) in<br />
regulating the lifespan of the worm. To gain insight into the spatial requirement for sensory perception in<br />
controlling the worm’s lifespan, we are also in the process of determining which sensory neurons are<br />
necessary to regulate its lifespan by expressing wild-type sensory genes in subsets of mutant sensory<br />
neurons.<br />
99
THE TTX-3 LIM HOMEOBOX GENE IS A CENTRAL<br />
REGULATOR OF INTERNEURON CELL FATE<br />
Z. Altun-Gultekin, O. Hobert<br />
Columbia University, College of Physicians & Surgeons, Center for Neurobiology and Behavior, New<br />
York, NY 10032<br />
The LIM homeobox (Lhx) gene ttx-3 is required for correct thermotactic behavior (1,2). In ttx-3 mutants,<br />
the AIY interneuron, a component of the thermoregulatory neural circuit, is structurally and functionally<br />
defective. ttx-3::gfp reporter gene fusions are expressed in AIY. We have set out to test what cellular role<br />
the ttx-3 gene plays and present here our analysis of the execution of the AIY cell fate in ttx-3 mutants.<br />
Previously we have shown that in ttx-3 mutants maintenance of postembryonic ttx-3 expression is lost in<br />
AIY due to autoregulation. Using GFP reporter gene constructs, we have now found that expression of<br />
other AIY cell fate markers including a 7-TM receptor, sra-11, a homeobox gene, ceh-23, and a secreted<br />
protein, C36B7.7 (kindly provided by T. Ishihara) is also downregulated in AIY in ttx-3 mutants. A GFP<br />
fusion to the octopamine/serotonin receptor ser-2 (kindly provided by T. Niacaris), which we identified as<br />
being expressed in AIY as well, is also downregulated. Additionally, AIY loses its cholinergic phenotype in<br />
ttx-3 mutants as detected by VAchT antibody staining (antibodies kindly provided by J.Duerr). These<br />
results suggest that AIY fails to differentiate correctly in ttx-3 mutants. So far it is unclear whether ttx-3<br />
directly controls the expression of the genes described above or functions through intermediary<br />
transcription factors. We are addressing this question by delineating and comparing the ttx-3 -dependent<br />
regulatory elements in the distinct AIY cell fate markers.<br />
Previously it was shown that mutations in the Lhx gene lim-4 lead to a switch of the fate of the AWB<br />
sensory neuron to AWC (3). Although many if not all aspects of the correct fate of AIY are lost in ttx-3<br />
mutants, our preliminary tests for a cell fate switch of AIY into any of its lineal, structural or functional<br />
homologs suggest that AIY has not taken over the identity of AIM, AIZ, ASE or AWC. In contrast to AWB<br />
in lim-4 and AIY in ttx-3 mutants, the DVB motor neuron maintains its correct fate in animals mutant for<br />
the Lhx gene lim-6 (O. H., unpubl.). Hence, in Lhx mutants the identity of a neuron may be maintained,<br />
lost or switched.<br />
References:<br />
(1) Hobert et al., 1997, Neuron 19, 345<br />
(2) Mori and Oshima, 1995, Nature 376, 344<br />
(3) Sagasti et al., 1999, Genes Dev 13, 1794<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
100
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE HETEROCHRONIC GENE PATHWAY: REGULATORY<br />
INTERACTIONS AND REGULATORY OUTPUTS.<br />
Victor Ambros 1 , Marta Hristova 1 , Rosalind Lee 1 , Eric Moss 2<br />
1Department of Biology, Dartmouth College, Hanover, NH 03755<br />
2Fox Chase Cancer Center, Philadelphia PA<br />
The heterochronic gene pathway controls the timing of larval developmental events. Central to the action<br />
of this pathway is the developmental down-regulation of lin-14 and lin-28, whereby high levels specify L1<br />
events, and lower levels specify L2 and L3 events. The decrease in lin-14 and lin-28 results from<br />
translational repression by the small RNA product of lin-4. lin-4 RNA is complementary to sequences in<br />
the 3’ UTR of its target mRNAs, and recent experiments suggest that lin-4 acts by gating access to the<br />
3’UTR by other positive and negative inputs. These other inputs include a mutual positive feedback<br />
between lin-14 and lin-28 and a daf-12-dependent negative regulatory input.<br />
The outputs of the heterochronic gene pathway are the expression of cell-type specific developmental<br />
programs. To seek downstream targets of lin-14 for L2 events, we employed a microarray of 12,000<br />
ORFs prepared by the Kim lab at Stanford University. Probes were prepared from mRNAs of<br />
synchronized mid-L1 populations of N2 and lin-14(n179ts) animals. Among the genes that displayed<br />
lin-14-dependent expression by microarray analysis, we chose four genes for follow-up by Northern and<br />
GFP-fusions. By Northern analysis, three of these four genes show precocious mRNA expression in<br />
lin-14(lf) animals, consistent with their temporal regulation by lin-14 in the wild type. The expression of<br />
GFP promoter fusions suggests that at least one of the genes, ins-33, appears to be developmentally<br />
regulated by lin-14 at the transcriptional level.<br />
For L3 events, the chief effectors downstream of lin-14 and lin-28 appear to be daf-12 and lin-46. lin-46<br />
encodes a member of a conserved family of proteins involved in protein-protein interactions, suggesting a<br />
complex of regulators controling L3 targets. In the vulva precursor cells, L3 events controled by the<br />
heterochronic genes are progress through the G1/S phase of the cell cycle and the acqusition of<br />
competence to express vulval cell fates. VPC G1/S is regulated by the transcriptional activation of a cyclin<br />
kinase inhibitor gene, cki-1. We are identifying regions of the cki-1 promoter which mediate the<br />
VPC-specific control of cki-1 transcription by the heterochronic gene pathway.<br />
101
GENETIC AND PHENOTYPIC CHARACTERIZATION OF<br />
EVL-14 AND EVL-20, GENES INVOLVED IN C. ELEGANS<br />
VULVA DEVELOPMENT<br />
Igor Antoshechkin, Min Han<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of MCD Biology, University of Colorado, Boulder, CO 80309<br />
In an attempt to identify new genes that participate in C. <strong>elegans</strong> vulva development, we carried out a<br />
screen for sterile mutants with protruding vulva. The mutants were then examined using Nomarski optics<br />
for abnormal vulva morphology at the "Christmas tree" stage. Several of isolated mutations were alleles of<br />
evl genes previously identified by Geraldine Seydoux . Here we report genetic and phenotypic<br />
characterization of two such genes, evl-14 and evl-20. Both mutations are completely recessive and result<br />
in a 100 % sterile phenotype. Their vulva structures are variably abnormal and are missing anywhere<br />
from 0 to 10 cells suggesting either under induction or cell cycle/division defect. In order to distinguish<br />
between these possibilities we are making doubles with members of Ras signaling pathway and GFP<br />
reporters for vulval cell fates. We are also performing detailed lineage analysis of the mutants. evl-20 also<br />
displays gonad migration defect not seen in evl-14. evl-14 maps to linkage group III just left of pal-1.<br />
evl-20 is located on chromosome II next to rol-6. We have injected cosmids from these regions and<br />
identified cosmid pools that rescue both evl-14 and evl-20. We are in the process of injecting single<br />
cosmids and making subclones to identify single open reading frames that will rescue the mutants.<br />
102
CAENORHABDITIS ELEGANS T05H10.5, A HOMOLOGUE OF<br />
YEAST UBIQUITIN FUSION DEGRADATION PROTEIN<br />
(UDF-2), IS EXPRESSED THROUGHOUT THE NERVOUS<br />
SYSTEM AND IN THE GUT<br />
Wanyuan Ao, Dave Pilgrim<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada<br />
C. <strong>elegans</strong> UNC-45 is a component of muscle thick filaments and co-localizes with myosin heavy chain B<br />
in body wall muscles. The N-terminal of UNC-45 contains three tetratricopeptide repeats (TPR) and its<br />
C-terminal shows limited similarity to fungal proteins. In our yeast two-hybrid screens using unc-45 cDNA<br />
as a bait, we detected that UNC-45 interacts with T05H10.5 and the TRP domain of UNC-45 is sufficient<br />
for that interaction. The predicted T05H10.5 gene encodes a 113.2 KD protein that is a homologue of<br />
yeast ubiquitin fusion degradation protein (UDF-2). In further characterization of T05H10.5, We found that<br />
the GFP reporter driven by T05H10.5 promoter is expressed throughout the nervous system and also<br />
strongly in the gut of the adult worm. The UDF-2-like proteins are highly conserved from yeast to human<br />
and its putative human homologue is expressed in brain tissues. Our results indicates that T05H10.5 is a<br />
nerve cell and gut-specific gene and may interact with TRP-containing proteins (like UNC-45).<br />
103
GENETIC ANALYSIS OF NEUROENDOCRINE CONTROLS OF<br />
FAT METABOLISM IN C. ELEGANS<br />
Kaveh Ashrafi, Gary Ruvkun<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Molecular Biology, Massachusetts General Hospital and Dept. of Genetics, Harvard Medical<br />
School, Boston, MA 02114<br />
Body fat regulation is a complex process that affects behavior, physiology, and metabolism of an<br />
organism in order to balance energy intake with energy expenditure. C. <strong>elegans</strong> is an attractive choice for<br />
the systematic identification of components of fat homeostasis and cell biology. As in mammals, an insulin<br />
signaling pathway in C. <strong>elegans</strong> couples nutritional status to the tempo and mode of metabolism, while<br />
neuronal outputs, e.g. serotonin, translate food sensation to various motor and endocrine outputs.<br />
We have used the phenoxazine dye Nile Red to visualize fat droplets in the intestinal and hypodermal<br />
cells of living C. <strong>elegans</strong>. The Nile Red staining pattern closely matches the pattern observed with the<br />
traditional methods of fat staining in C. <strong>elegans</strong> (e.g Sudan Black B). Moreover, the known and predicted<br />
patterns of fat accumulation arising from mutations in insulin-like (daf-2 / daf-16) and TGF-b like (daf-7 /<br />
daf-3) signaling pathways are well recapitulated by Nile Red staining. Thus, Nile Red staining of living<br />
animals provides a means for genetic screens based on fat metabolism and homeostasis. Two such<br />
screens are presented:<br />
(i) To identify genes involved in the biogenesis and cell biology of fat storage, we screened<br />
EMS-mutagenized N2 animals for mutant worms with abnormalities in lipid droplet size or number. In a<br />
screen of 15,000 mutagenized F2, we identified four mutants that display abnormally large droplet sizes.<br />
(ii) The C. <strong>elegans</strong> insulin signaling pathway determines whether larvae grow to a fast metabolizing adult<br />
stage or enter a slow metabolizing dauer stage. Reduced signaling through the DAF-2 insulin-like<br />
receptor promotes a shift in metabolism to fat storage and dauer arrest. The forkhead transcription factor<br />
DAF-16, is the key target of DAF-2 signaling pathway and is required for the shift in fat metabolism and<br />
dauer arrest. Mutant adult daf-2 animals have a diffuse pattern of Nile Red staining. In contrast, Nile Red<br />
appears in very discrete droplet spots in daf-16 animals. To identify downstream metabolic targets of<br />
DAF-16, we have begun screening EMS-mutagenized daf-16 worms to find mutants that recapitulate the<br />
daf-2 pattern of staining.<br />
104
ZIG GENES AND THE PVT GUIDEPOST NEURON<br />
Oscar Aurelio, Oliver Hobert<br />
Columbia University,New York, NY 10032<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
We have identified a novel family of putative cell adhesion and/or signaling molecules which are defined<br />
by the presence of a signal sequence and 2 Ig domains; none of these molecules contain a predicted<br />
transmembrane sequence, but several contain consensus sites for GPI anchorage. C.<strong>elegans</strong> contains 9<br />
members of this family, termed the zig gene family. We hypothesize that these genes may play a role in<br />
neural patterning. As part of receptor/ligand complexes, they may be involved in cell recognition or,<br />
alternatively, they may serve as secreted and diffusible signaling molecules.<br />
GFP fusion to the promoter region of these 9 genes reveals that 6 of these genes are expressed almost<br />
exclusively in very specific subdomains of the nervous system, while 2 of them are exclusively expressed<br />
in body wall muscles. One zig gene is expressed ubiquitously in the nervous system and in body wall<br />
muscles.<br />
The most intriguing aspect of zig gene expression in the nervous system relates to the PVT neuron: 5 of<br />
the 9 zig genes are expressed in PVT. Aside from the panneuronally expressed zig-1 reporter gene,<br />
zig-2, zig-4 and zig-9 are expressed in few other cells than PVT, while zig-5 is expressed in about one<br />
dozen additional head neurons. PVT acts during embryonic patterning as a guidepost cell for pioneer<br />
neurons of the ventral nerve cord (Durbin, 1987) and is a source of unc-6 expression (Wadsworth et al.,<br />
1996). Given the embryonic role of PVT, we were surprised to find that at least two of the five<br />
PVT-expressed zig genes, zig-2 and zig-4, are not expressed embryonically; instead their expression is<br />
turned on right after hatching and presists throughout adulthood. Additionally, we found that maintenance,<br />
but not initiation of zig-2 and zig-4 expression requires the lim-6 LIM homeobox gene, which is expressed<br />
in PVT.<br />
The postembryonic expression of zig-2 and zig-4 suggests that PVT subserves a postembryonic role in<br />
axonal patterning. What could this role be? In hermaphrodites relatively few neurons extend their axon<br />
into the ventral nerve cord postembryonically; however, in males, many neurons are born<br />
postembryonically in the tail and presumably require cues to extend their axon along the ventral nerve<br />
cord. We will test whether PVT plays a role in this process by driving expression of cytotoxic agents under<br />
control of the postembryonic zig-2 promoter.<br />
105
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ANALYSIS OF GABA RECEPTOR PLASTICITY IN C.<br />
ELEGANS<br />
Bruce A. Bamber 1 , Janet E. Richmond 2 , Pierrette K. Danieu 1<br />
1 Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, 84112<br />
2 Department of Biology, University of Utah, Salt Lake City, UT, 84112<br />
Neuronal activity depends on the balance of excitatory and inhibitory neurotransmission. One way for a<br />
neuron to regulate its activity level is to alter its sensitivity to inhibitory neurotransmitters. Most inhibitory<br />
neurotransmission in the mammalian brain is mediated by g-aminobutyric acid (GABA), which acts mainly<br />
through GABA A receptors (ligand-gated chloride channels). Neurons regulate their GABA sensitivity by<br />
regulating the density and GABA-responsiveness of synaptic GABA A receptors. This receptor plasticity is<br />
important for regulating neuronal excitability in both normal and diseased nervous systems. The<br />
mechanisms of GABA A receptor plasticity are not well understood, in part because it is difficult to apply<br />
genetic techniques to study mammalian synapse function. The C. <strong>elegans</strong> neuromuscular junction is a<br />
powerful model system to study GABA receptor plasticity. A GABA A receptor homolog, consisting of<br />
UNC-49B and UNC-49C subunits, functions in C. <strong>elegans</strong> muscle cells. This receptor exhibits<br />
mammalian-like plasticity: The GABA receptor agonist muscimol acutely paralyzes C. <strong>elegans</strong>, but<br />
chronic exposure results in adaptation (i.e. the appearance of muscimol resistance, and a GABA<br />
receptor-defective phenotype). Patch-clamp analysis of muscle cells in adapted animals revealed an 85%<br />
decrease in GABA-evoked currents compared to untreated animals, indicating that GABA receptor<br />
downregulation underlies adaptation. Muscimol exposure also caused GFP-tagged UNC-49 receptors to<br />
form clusters in extra-synaptic muscle membranes, which implicates the clathrin-mediated endocytosis<br />
pathway in downregulation. We are pursuing two strategies to study GABA receptor plasticity in C.<br />
<strong>elegans</strong>. First, we are identifying UNC-49 sequences required for synaptic localization and<br />
downregulation. Our results have demonstrated that the UNC-49B subunit is necessary and sufficient for<br />
both processes. Second, we are performing a yeast two-hybrid screen to identify GABA<br />
receptor-associated molecules in C. <strong>elegans</strong>. We have identified several interesting candidates, and we<br />
are using RNA interference to determine which of these proteins are important for GABA receptor<br />
regulation.<br />
106
ISOLATION OF SUPPRESSORS OF A DOMINANT SYNAPSE<br />
DEFECTIVE MUTANT, SYD-5(JU89)<br />
Renee Baran, Yishi Jin<br />
Department of Biology, Sinsheimer Labs, University of California, Santa Cruz, CA 95064<br />
syd-5 (ju89)was identified in a screen for mutations that alter the differentiation of GABAergic synapses in<br />
C. <strong>elegans</strong> using the presynaptic vesicle marker, Punc-25::SNB-1-GFP 1,2 . In adult wild-type worms,<br />
Punc-25-SNB-1-GFP is expressed as puncta of uniform size and spacing along the dorsal and ventral<br />
nerve cords, corresponding to neuromuscular junctions made by the GABAergic DD and VD neurons with<br />
dorsal and ventral body wall muscles, respectively. syd-5(ju89) mutant worms are uncoordinated and<br />
exhibit abnormally large and small SNB-1-GFP puncta dispersed in an irregular pattern along the nerve<br />
cords. This phenotype closely resembles the SNB-1 GFP phenotype of loss-of-function syd-3/rpm-1<br />
mutants, which have overdeveloped synapses with multiple active zones 3 . syd-5 maps to chromosome I<br />
between egl-33 and lin-11. syd-5( ju89) behaves genetically as a weak gain-of-function mutation: Df/+<br />
worms are wild-type, but heterozygous ju89/+ worms exhibit a locomotion and SNB-1 GFP phenotype<br />
that is intermediate between wild-type and homozygous ju89 worms. A suppressor screen was performed<br />
to identify loss-of-function syd-5 alleles and genes that may function in the same pathway. syd-5(ju89)<br />
worms were mutagenized with EMS, and 30,000 F1 progeny were screened for suppression of the<br />
syd-5(ju89) uncoordinated phenotype. Twenty-four mutants were isolated that suppress both the syd-5<br />
locomotion phenotype and the synaptic vesicle marker defect. Four suppressors are linked to ju89 and<br />
may represent loss-of-function syd-5 alleles. Preliminary characterization of the syd-5 suppressors will be<br />
presented, along with progress in the cloning and molecular characterization of syd-5.<br />
1 Zhen & Jin. 1999. Nature<br />
2 Nonet, M. 1999. Neurosci. Methods<br />
3 Zhen et al. 2000. Neuron<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
107
CARGO RECOGNITION BY SYNAPTIC VESICLE KINESIN<br />
Ewa Bednarek, Erik M. Jorgensen<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA<br />
Synaptic vesicles are transported to the synapse by kinesin, an ATP-dependent motor protein that moves<br />
along axonal microtubules. In the cell body the kinesin must specifically bind vesicles destined for the<br />
nerve terminal. We would like to identify the molecular basis for vesicle cargo recognition by UNC-104, a<br />
<strong>Caenorhabditis</strong> <strong>elegans</strong> homolog of the motor protein kinesin. One theory suggests that UNC-104<br />
recognizes specific protein molecules associated with the vesicle membranes. Alternatively, we<br />
hypothesize that UNC-104 recognizes synaptic vesicles by binding specific lipids. This is supported by the<br />
fact that UNC-104 has a pleckstrin homology (PH) domain, which is a lipid binding moiety. If PH<br />
domain-lipid binding determines cargo recognition, the following predictions can be made: the PH domain<br />
should bind lipids specific to synaptic membranes, and the PH domain should be required for cargo<br />
recognition. To test these predictions we are first expressing the PH domain in bacteria and determining<br />
whether it binds phosphoinositides. Second, we are testing whether mutations in the PH domain disrupt<br />
synaptic vesicle trafficking.<br />
108
THE EXP-1 LOCUS MAY ENCODE A SUBUNIT OF AN<br />
EXCITATORY GABA RECEPTOR<br />
Asim A. Beg, Erik M. Jorgensen<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Biology, University of Utah 257 South 1400 East Salt Lake City, UT 84112<br />
Gamma-aminobutyric-acid (GABA) is a major neurotransmitter of inhibitory synapses. In the worm, as in<br />
vertebrates, GABA mediates inhibitory neurotransmission (VD/DD to body muscles). However, in the<br />
enteric muscles, excitatory neurotransmission seems to be mediated by GABA. The GABAergic neurons<br />
AVL and DVB directly synapse on the enteric muscles and are the only neurons required for excitation of<br />
the enteric muscles. We have cloned a potential excitatory GABA receptor subunit, exp-1. Of the six<br />
genes required for GABA neurotransmission, only exp-1 specifically eliminated the excitatory function of<br />
GABA while leaving all other functions intact. We hypothesize that EXP-1 is a subunit of a novel<br />
excitatory GABA receptor expressed in the enteric muscles.<br />
We cloned the exp-1 gene by microinjection rescue. Genetic map data showed that exp-1 is located on<br />
Chromosome II between lin-4 and lin-23. One fosmid in this region, H35N03, contains a gene related to<br />
GABA receptors. Transgenes bearing a subclone of this single open reading frame (ORF) completely<br />
rescued exp-1(sa6) mutants (gift of J. Thomas). In addition, we identified point mutations in this ORF in<br />
three alleles, which confirmed that exp-1 encodes this GABA receptor related protein. We isolated exp-1<br />
cDNAs which show that EXP-1 has a divergent transmembrane 2 (TM2) from all known GABA receptor<br />
subunits. TM2 has been shown to confer ion selectivity to the ligand-gated ion channel superfamily.<br />
Consistent with our hypothesis that EXP-1 is an excitatory GABA receptor subunit, TM2 has numerous<br />
negatively charged residues, suggesting this subunit might form a receptor that passes cations rather<br />
than the anion chloride.<br />
The majority of physiological GABA receptors are heteromultimers. By BLAST search we have identified<br />
a second putative subunit that is very similar to EXP-1. Specifically, TM2 of the predicted subunit is<br />
nearly identical to EXP-1, suggesting this might be the physiological partner of EXP-1. We are currently<br />
performing RT-PCR to isolate cDNAs for this subunit. We will coinject Xenopus laevis oocytes with<br />
cRNAs from both subunits and determine GABA gating and ion permeability of the channel using<br />
electrophysiological analysis.<br />
109
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE LIFE SPAN GENE CLK-2 IS ESSENTIAL FOR<br />
EMBRYONIC DEVELOPMENT<br />
Claire Bènard, Brent McCright, Yue Zhang, Stephanie Felkai, Siegfried<br />
Hekimi<br />
Department of Biology, McGill University, 1205 Dr Penfield Ave., Montreal, Quebec, Canada<br />
Mutations in the C. <strong>elegans</strong> maternal-effect genes clk-1, clk-2, clk-3, and gro-1 are highly pleiotropic<br />
resulting in an average slowing of development, adult rhythmic behaviors, reproduction, as well as in an<br />
extended life span. Here we will present the genetic and molecular characterization of the gene clk-2.<br />
This gene is defined by one recessive mutation, qm37, which was isolated in a screen for viable<br />
maternal-effect mutations (Hekimi et al., 1995). At 20C, the duration of embryonic and post-embryonic<br />
development of clk-2(qm37) mutants is lengthened and adult behaviors such as defecation, pharyngeal<br />
pumping, and egg laying are slower. In addition, they live long. In contrast, at 25C, clk-2(qm37) mutants<br />
die as embryos. Moreover, at 20C, clk-2(qm37) mutants can be fully rescued both zygotically and<br />
maternally, while at 25C there is a strict-maternal effect. We believe that clk-2(qm37) is a<br />
temperature-sensitive mutation that behaves as a hypomorph at 20C and as a null at 25C. As we have<br />
found that clk-2 is not required for the development of the germline per se at 25C, we have explored the<br />
origin of the embryonic lethality at 25C by carrying out temperature shift experiments. We have shown<br />
that clk-2 is required before the two-cell stage of embryogenesis. The temporal and spatial expression<br />
pattern of clk-2 deduced from northern and western analyses, and from the use of clk-2::gfp reporter<br />
fusions, is consistent with a maternal role of clk-2 early in development and throughout the life of the<br />
worm.<br />
110
DOES CEH-20, AN EXD/PBX HOMOLOG IN C. ELEGANS,<br />
PLAY A ROLE IN WORM EMBRYOGENESIS?<br />
Q.F. Boese, W.B. Wood<br />
Dept of MCD Biology, University of Colorado, Boulder, CO 80309<br />
Several studies have shown that the TALE (three-amino-acid-loop-extension) homeodomain (HD)<br />
proteins, Exd and Hth in Drosophila and their mammalian homologs, Pbx1-3 and Meis, complex with Hox<br />
proteins to enhance binding specificity for target promoter sites. Studies have further shown that Hth/Meis<br />
interacts with Exd/Pbx to promote nuclear localization facilitating their roles as co-factors in the nucleus.<br />
Recent work in our lab revealed that loss-of-function (lf) mutations in the C. <strong>elegans</strong> Hth/Meis homolog,<br />
unc-62, result in pleiotropic defects including defects in embryonic development. To investigate whether<br />
the Exd/Pbx homolog, CEH-20 might also be involved in embryonic patterning, we have begun<br />
characterizing the defects of two strong ceh-20(lf) alleles (gift from M. Stern) with respect to their potential<br />
interactions with unc-62 and other embryonically required genes (Hox or non-Hox). Both ceh-20 alleles<br />
result in low-penetrance larval lethality and no apparent embryonic defects suggesting that ceh-20 is not<br />
normally required for embryogenesis. Injection of double-stranded ceh-20 RNA into wild-type<br />
hermaphrodites [ceh-20 (RNAi)] results in an Unc Egl phenotype but no embryonic defects.<br />
To test for an interaction with the embryonically required posterior Hox paralog gene nob-1 (1), ceh-20<br />
(RNAi) was performed in the background of a weak allele, nob-1(ct230). No enhancement of the mutant<br />
phenotype was observed. Whether this reflects compensatory functions of two other Exd/Pbx homologs,<br />
ceh-40 and F22A3.x, in the absence of ceh-20, is under investigation.<br />
To test for interactions with unc-62, ceh-20 RNAi was performed in the background of the weak allele<br />
unc-62 (e644). The low penetrance e644 lethal phenotype was enhanced over the uninjected control,<br />
consistent with an interaction between these genes during embryogenesis. To test for interactions with<br />
other non-Hox HD proteins important for embryogenesis, ceh-20 RNAi was performed in the background<br />
of a weak pal-1 allele (e2091). The e2091 mutant phenotype was also enhanced suggesting a previously<br />
undescribed interaction between Exd/Pbx and Caudal/CDX homologs. Further tests for ceh-20<br />
interactions with other embryonically required genes are in progress.<br />
(1) Van Auken, K. et al., 2000, PNAS 91: 4499-503<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
111
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A-DOMAIN-CONTAINING PROTEIN FAMILY IN C. ELEGANS.<br />
Michael Brannan, Joaquin Muriel, Kathryn Taylor, Gordon Lithgow,<br />
Danny Tuckwell<br />
School of Biological Sciences, University of Manchester, Stopford Building, Manchester M13 9PT, UK.<br />
The extracellular matrix (ECM) plays an important structural and functional role in metazoans and its<br />
major components include collagen, laminin, and proteoglycan. The cuticle and basement membrane are<br />
the major forms of ECM in C. <strong>elegans</strong>: the cuticle is an exoskeleton important for shape and motility, and<br />
it is also a barrier between the worm and its environment. The basement membrane provides structural<br />
support and tissue separation. Very little is known about the molecules that organise the ECM or the cell<br />
receptors for ECM molecules in C. <strong>elegans</strong>.<br />
A-domains are 200-amino acids modules identified in a range of mammalian ECM proteins. We have<br />
previously studied the A-domains of the integrin a subunits. Integrins a 1b 1 and a 2b 1 are the two major<br />
receptors in the human body for collagens. We have showed that it is the A-domains found within the a<br />
subunits that are responsible for collagen binding, and also for the interaction of these integrins with other<br />
ECM proteins such as laminin. Studies of a number of other A-domain-containing proteins indicate a<br />
collagen or ECM-binding function.<br />
Using bioinformatics approaches we have identified 8 novel A-domain-containing proteins in the C.<br />
<strong>elegans</strong> genome. For some of the novel proteins, regions flanking the A-domains were found to resemble<br />
mucin core protein-like sequence and cuticulin domains. Since mucins and cuticulins are ECM<br />
components, this suggests that a number of the novel proteins are likely to be ECM molecules. Genetical<br />
and biochemical characterisation of these molecules will give insights into the function of these proteins in<br />
ECM organisation and cell-ECM interactions.<br />
112
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
DISTRIBUTION AND REGULATION OF GLUTAMATE<br />
RECEPTORS IN THE LOCOMOTORY CONTROL CIRCUIT OF<br />
C. ELEGANS.<br />
Penelope J. Brockie, David M. Madsen, Yi Zheng, Jerry E. Mellem,<br />
Andres V. Maricq<br />
Department of Biology, University of Utah, Salt Lake City, UT 84112<br />
Ionotropic glutamate receptors are ligand gated ion channels that mediate rapid excitatory synaptic<br />
communication in most nervous systems. Ten putative ionotropic glutamate receptor subunits (glr-1 -<br />
glr-8, nmr-1 and nmr-2) have been identified in C. <strong>elegans</strong>. Analysis of the predicted amino acid<br />
sequence suggests that GLR-1 - GLR-8 are members of the non-NMDA class of glutamate receptors,<br />
where NMR-1 and NMR-2 are most similar to members of the NMDA class. Using GFP fusions we have<br />
shown that the subunits are differentially expressed in the nervous system with some found in only a<br />
single neuron.<br />
The NMDA subunits and four of the non-NMDA subunits (GLR-1 - GLR-4) are expressed in at least one<br />
of the five pairs of command interneurons that regulate worm locomotion (AVA, AVB, AVD, AVE and<br />
PVC). This provides insight into the possible combinations of receptor subunits that form ion channels in<br />
vivo, and suggests that a diversity of both NMDA and non-NMDA type glutamate receptors function in the<br />
locomotory control circuit. What regulates expression of the glutamate receptors in this circuit? It has<br />
recently been shown that the homeodomain protein UNC-42 regulates glr-1 expression in a number of<br />
neurons, including AVA, AVD and AVE (1). We show that unc-42 is also required for glr-3 and glr-4<br />
expression in these neurons, but not for the expression of glr-2, nmr-1 or nmr-2. This indicates that<br />
expression of glutamate receptors in the command interneurons is differentially regulated.<br />
Like glr-1 mutants, unc-42 mutants are nose touch defective and show additional mechanosensory and<br />
locomotory defects. Using the nmr-1::gfp fusion, we show that unc-42 mutants also have dramatic defects<br />
in axon outgrowth in the command interneurons. In wild-type worms, these neurons send their axons<br />
along the ventral cord. In unc-42 mutants, axons are dorsally, laterally and anteriorally misplaced. Using<br />
the nmr-1 promoter to express the unc-42 cDNA does not rescue the axon defects. This suggests that<br />
expressing UNC-42 in the command interneurons alone is not sufficient to direct proper axon outgrowth in<br />
these cells.<br />
1. Baran, et al., Development 126, 2241-2251 (1999)<br />
113
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MUTATIONS THAT AFFECT SYNAPTIC LOCALIZATION OF<br />
GLR-1<br />
Michelle Burbea, Joshua M. Kaplan<br />
Department of Molecular and Cellular Biology, 361 Life Sciences Addition, UC Berkeley, Berkeley, CA<br />
94720-3200<br />
The orderly flow of information in the nervous system requires that proteins are specifically targeted to<br />
pre- and post-synaptic specializations. We are interested in identifying the molecules that target and<br />
maintain proteins to synapses. We have used the GLR-1 AMPA type glutamate receptor as a marker for<br />
post-synaptic localization. A wild type animal expressing a GLR-1::GFP fusion exhibits punctate spots<br />
along the ventral nerve chord representing clustering of the glutamate receptor at specific synaptic<br />
terminii. To identify genes involved in synaptogenesis, we are screening mutagenized GLR-1::GFP<br />
worms for defects in GLR-1 localization.<br />
We predict classes of mutants to represent trafficking, targeting, localization and stabilization molecules<br />
necessary to initiate and maintain synaptic architecture. Approximately 7000 genomes have been clonally<br />
screened so that sterile or inviable mutants may be recovered as heterozygously. Nine complementation<br />
groups have been isolated which define loci important for the proper localization of GLR-1 in the nervous<br />
system. Several of the loci are in genes whose identity is already known. These include mutations in<br />
unc-11, an AP180 homologue, and unc-101, a medium chain subunit of the AP-1 clathrin adaptin<br />
complex. The implication of these mutants on receptor trafficking and maintanance at the synapse will be<br />
discussed.<br />
114
REGULATION OF C. ELEGANS DAUER FORMATION BY AN<br />
RNA QUALITY CONTROL PATHWAY COMPONENT<br />
J Burgess 1 , JC Labbe 2 , S Hekimi 1<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1Department of Biology, Mc Gill University, 1205 Dr. Penfield Ave, Montreal, Quebec, Canada H3A1B1<br />
2Department of Biology, University of North Carolina at Chapel Hill, 3280 Coker Hall, Chapel Hill, North<br />
Carolina. 27599-3280, USA<br />
Under conditions that do not favor developmental growth and reproduction, worms can enter an alternate<br />
third-larval stage termed the dauer stage. Dauer larvae are developmentally arrested and are adapted for<br />
long term survival and dispersal. In order to make this decision, animals monitor a variety of<br />
chemosensory cues including the concentration of a constitutively secreted pheromone, as well as food<br />
and temperature. It is currently unknown whether internal states, such as the amount of molecular<br />
damage accumulated, is also taken into account when making the decision to enter the dauer stage. The<br />
gene rop-1 encodes the worm homologue of the human Ro ribonucleoprotein 60-kDa constituent, which<br />
has previously been shown to participate in the quality control of 5S ribosomal RNA. Here, we present<br />
biochemical evidence that rop-1 is cleaved at the L2/L3 molt concommittant with entry into L3 but is not<br />
cleaved when animals enter the dauer stage. In addition, rop-1 is not cleaved in daf-2 mutants suggesting<br />
cleavage of rop-1 may require functional insulin-like signaling. Furthermore, we provide evidence that<br />
rop-1 genetically interacts with the insulin receptor-like kinase daf-2 in an allele-specific manner as well as<br />
with the TGF-b transforming growth factor daf-7 in a temperature dependent manner. Finally, we show<br />
that the enhancement of dauer formation by rop-1 in daf-2(e1370) is daf-16 dependent. However, the<br />
processing of rop-1 appears to be daf-16 independent. We suggest that an RNA quality control<br />
checkpoint may exist for dauer formation.<br />
115
SYNAPTIC VESICLE LOCALIZATION IS MISREGULATED IN<br />
UNC-16 MUTANTS<br />
DT Byrd, Y Jin<br />
University of California, Santa Cruz, Dept. Biol., Sinsheimer Labs, CA 95064<br />
The DD motoneurons provide a model in which to study synaptic development and remodeling. The DDs<br />
are born embryonically, innervate ventral body wall muscles in L1 animals, and remodel to innervate<br />
dorsal body wall muscles in L2 through adult animals. We visualize DD synapses and the process of DD<br />
synaptic remodeling in vivo with a synaptic vesicle marker expressed by the DDs and other GABAergic<br />
neurons1. In wild type L1s, the synaptic vesicle marker is strictly localized to the ventral processes of the<br />
DDs until just before the molt to L22. After the L1 molt, the synaptic vesicle marker is localized along the<br />
dorsal processes of the DDs, indicating that the DDs have remodeled.<br />
We isolated a partial loss-of-function mutation in unc-16 in a screen for dorsal localization of the synaptic<br />
vesicle marker in the DDs of early L1 animals. unc-16 mutant L1 animals exhibit full ventral localization of<br />
the synaptic vesicle marker as well as varying amounts of dorsal localization in the DDs. Seven alleles of<br />
unc-16 form an allelic series in which n730, e109, ju79, and s1072 are partial loss-of-function alleles of<br />
increasing severity, respectively, and s453, s529, and s222 are genetic null alleles. unc-16 encodes a<br />
pan-neurally expressed novel protein with interesting motifs that may interact with a number of signaling<br />
pathways. We are currently determining the molecular lesions in the unc-16 mutants.<br />
We hypothesize that the vesicle localization defect may be caused by disruptions in synaptic vesicle<br />
cargo selection, transport, or docking. Supporting this hypothesis, we found that weak unc-16 mutations<br />
partially suppress partial loss-of-function mutations in unc-104, a gene encoding a kinesin. This suggests<br />
that in the DDs, unc-16 may play a role in regulating synaptic vesicle transport.<br />
1. Nonet ML. 1999. J Neurosci Methods 89(1):33-40.<br />
2. Hallam and Jin. 1998. Nature 395:78-82.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
116
THE EGL-21 GENE ENCODES A CARBOXYPEPTIDASE E,<br />
WHICH IS REQUIRED FOR PRO-NEUROPEPTIDE<br />
PROCESSING<br />
Tija Carey, Joshua M. Kaplan<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UC Berkeley, MCB Dept , 361 LSA, Berkeley CA 94720<br />
We are interested in understanding the mechanisms for neurotransmitter and neuropeptide modulation of<br />
C. <strong>elegans</strong> behavior, including locomotion, defecation, and egg laying. We previously reported that the<br />
egl-3 gene encodes a prohormone convertase involved in proteolytic processing of pro-neuropeptides<br />
(Kass and Kaplan IWM99). We will present evidence that the egl-21 gene encodes a carboxypeptidase<br />
E(CPE), which mediates the next enzymatic step in peptide processing, removal of C-terminal basic<br />
residues.<br />
Mutations in egl-21 were first isolated in a screen for egg laying defective mutants, but also showed<br />
defective defecation and uncoordinated locomotion (Trent, Genetics 104: 619-647 1983). The various<br />
defects in egl-21 mutants show differing drug sensitivity: the egg laying defect is resistant to serotonin and<br />
imipramine, while the locomotion phenotype is sensitive to both.<br />
We found that egl-21 mutations map very close to mec-3. The genome sequence of this region indicated<br />
that a gene very similar to vertebrate CPE mapped in this region. We found that a cosmid containing this<br />
CPE rescued the egl-21 defecation defects. Next, we showed that two egl-21 alleles correspond to<br />
mutations in the CPE gene. The n576 allele alters an invariant G in a splice donor sequence whereas the<br />
n476 allele corresponds to a 122 bp in-frame deletion. Since egl-21 encodes the only apparent CPE in<br />
the worm genome, we would expect that the phenotype of egl-21 mutants corresponds to a neuropeptide<br />
null phenotype. We are currently studying the role of egl-21 CPE in modulation of several behaviors.<br />
117
HOW ARE ANTERIOR CELL MIGRATIONS GUIDED BY<br />
MIG-13?<br />
QueeLim Ch’ng, Cynthia Kenyon<br />
Department of Biochemistry, UC San Francisco, CA 94143-0448<br />
mig-13 was previously identified as a guidance factor specifically required for the anterior migrations of<br />
the QR descendants. mig-13 acts by promoting cell migrations in the anterior direction (Sym et. al., 1999).<br />
We are taking several approaches to understand further how the anterior migrations of the QR<br />
descendants are guided by mig-13. mig-13 is predicted to encode a novel transmembrane protein<br />
containing a CUB domain and LDL-receptor repeat in the extracellular region as well as a proline-rich<br />
domain in the intracellular region (Sym et. al., 1999). Our structure/function studies suggest that the<br />
extracellular domain of MIG-13 alone can confer partial function in guiding the QR descendants to the<br />
anterior.<br />
A rescuing mig-13::GFP fusion was previously found to be expressed in the anterior and mid-body ventral<br />
cord motor neurons, which cross the migratory track of the QR descendants. Consistent with this<br />
expression pattern, mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations<br />
of the QR lineage (Sym et. al., 1999). To determine where mig-13 expression is sufficient to guide the<br />
migrating cells, we are expressing mig-13 in broad sets of tissues, as well as in specific subsets of cells<br />
that express mig-13::GFP. We also intend to refine previous mosaic analysis to pinpoint the cells in which<br />
mig-13 acts.<br />
Reference<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Sym M, Robinson N and Kenyon C. Cell, 1999 Jul 9, 98(1):25-36.<br />
118
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
NEW SCREENS FOR NEGATIVE REGULATORS OF LET-23<br />
Monica Chan, Marie Tiongsen, Romel C. Castro, Vanessa Lee, Gregg<br />
Jongeward<br />
University of the Pacific, Department of Biological Sciences, 3601 Pacific Avenue, Stockton, Cal 95211<br />
We are trying to identify additional genes that act to negatively regulate the Epidermal Growth Factor<br />
Receptor (EGF-receptor) during vulval induction in Caenorabditis <strong>elegans</strong>. The gene let-23 encodes the<br />
C. <strong>elegans</strong> homolog of the EGF-receptor. Mutations in let-23 generally result in a number of defects,<br />
including larval arrest, sterility, and defects in the vulva and the male tail. At 20 o C, animals homozygous<br />
for the allele let-23(n1045) display excess vulval differentiation. A similar phenotype is observed in<br />
animals homozygous for lin-2(n768). lin-2 activity is required to properly localize EGF-receptor on the<br />
vulval precursor cell. For as yet unknown reasons, animals homozygous for both let-23(n1045)and<br />
lin-2(n768) display a highly penetrant vulval defect unlike that seen in either homozygote. These doubly<br />
mutant animals fail to form any vulval tissue. We have begun to screen for new alleles that suppress the<br />
egg-laying defect associated with the inability to form vulval tissue in animals of this genotype. Thus far,<br />
we have recovered four new alleles. At least one of these alleles appears to be a new lin-1 mutation, as<br />
animals homozygous for the suppressor display excessive vulval differentiation even if there are no other<br />
mutations present. A second (independent) suppressor causes a similar phenotype. The other two alleles<br />
restore approximately normal vulval differentiation. Animals of the genotype let-23(n1045); lin-2(n768);<br />
new suppressor are generally able to lay eggs but lack prominent pseudovulvae. We are currently<br />
characterizing these new alleles to determine if they are mutations in previously identified genes.<br />
119
C. ELEGANS MRE-11 IS REQUIRED FOR MEIOTIC<br />
RECOMBINATION AND DNA REPAIR BUT NOT FOR THE<br />
MEIOTIC G2 DNA DAMAGE CHECKPOINT<br />
Gregory Chin, Anne Villeneuve<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Developmental Biology, Stanford University School of Medicine Stanford, CA 94305<br />
We have isolated and analyzed mutants defective in the nematode ortholog of yeast MRE11, a<br />
multifunctional protein with roles in diverse cellular processes required to maintain genome integrity,<br />
including meiotic recombination, DNA repair, and telomere length maintenance. While MRE11 is highly<br />
conserved among several species and has been linked to genetic disease in humans, exploration of its in<br />
vivo roles in metazoan systems has been hampered by the fact that vertebrate cells that lack MRE11 are<br />
inviable. We have found that worms homozygous for an mre-11 null mutation are viable, allowing us to<br />
demonstrate an in vivo requirement for MRE-11 in meiotic recombination and DNA repair. In mre-11<br />
mutants, crossovers are not detected and chromosomes lack chiasmata at diakinesis but appear<br />
otherwise intact. Irradiation of mre-11 mutants not only fails to induce chiasmata but also eliminates<br />
progeny survivorship and leads to cytologically visible chromosomal abnormalities including<br />
fragmentation. These results indicate a defect in the ability of mre-11 mutant germ cells to repair<br />
radiation-induced damage. While they are repair-deficient, we show that mre-11 mutant germ cells retain<br />
function of the meiotic G2 DNA damage checkpoint.<br />
Although mre-11 homozyogtes derived from heterozygous parents are fully viable and produce a normal<br />
number of embryos, there is a marked drop both in the number and in the survivorship of embryos<br />
produced by succeeding generations. As a result, the strain cannot be propagated as a homozygous<br />
stock. This progressive loss of fecundity and viability sets mre-11 mutants apart from many other meiotic<br />
recombination-defective mutants, and indicates that MRE-11 performs an additional essential function in<br />
maintaining reproductive capacity in the species.<br />
120
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SUPPRESSOR ANALYSIS OF EPH/EPHRIN DEFECTIVE<br />
SIGNALING IN C. ELEGANS<br />
Ian Chin-Sang, Julie McCleery, Andrew Chisholm<br />
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA<br />
Mutations in the genes vab-1 and vab-2 cause similar defects in neural and epidermal morphogenesis.<br />
The phenotypes range from embryonic lethality, due to epidermal enclosure defects, and a ventral<br />
notched head in larvae and adults. vab-1 encodes an Eph receptor tyrosine kinase that is expressed in<br />
the developing nervous system, and is required in neurons for proper epidermal morphogenesis (George<br />
et al., Cell 92:633). vab-2/efn-1 encodes a GPI anchored ephrin that specifically interacts with VAB-1 in<br />
neurons to regulate neuronal and epidermal morphogenesis (Chin-Sang et al., Cell 99:781). What are the<br />
molecular consequences of the VAB-1/VAB-2 interaction? We have taken a genetic approach to identify<br />
genes that might act in the VAB-1/2 Eph/Ephrin signaling pathway or in parallel pathways. The incomplete<br />
penetrance and variability of phenotypes displayed by vab-1 and vab-2 mutants, made it difficult in the<br />
past to carry out genetic modifier screens. Therefore we sought to create strains that would make vab-1<br />
and vab-2 mutants more suitable for genetic modifier screens. A mutation in a LAR like receptor tyrosine<br />
phosphatase, ptp-1, results in a synthetic lethality with both vab-1and vab-2, suggesting that PTP-1 may<br />
function redundantly with Eph signaling (see abstract by Harrington et al.). The ptp-1(op147);vab-2(ju1)<br />
double mutant is temperature sensitive- viable at 15 0 C and 20 0 C, however, at 25 0 C almost completely<br />
inviable. We screened for suppressors of the ptp-1(op147);vab-2(ju1) lethality at 25 0 C and isolated at<br />
least 19 suppressors from a screen of over 67,000 F1 animals. The suppressors are of two classes: those<br />
that reduce the lethality and those that suppress both the lethality and the notched head phenotype of<br />
vab-2. Five suppressors of the second class map to LGIV and are dominant suppressors of vab-2.<br />
Intriguingly, these suppressors display a low penetrance notched head phenotype (about 2-5% of the<br />
animals), however, unlike vab-1 and vab-2 the notch is always on the dorsal side. We are currently<br />
determining the specificity of the suppression effect. We will present results from the characterization of<br />
these suppressors.<br />
121
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
RNAI SCREEN FOR COMPONENTS OF THE C. ELEGANS<br />
MEIOTIC MACHINERY<br />
Mónica Colaiácovo, Gillian Stanfield, Kirthi Reddy, Anne Villeneuve<br />
Dept. of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305<br />
Meiosis is the specialized cell division process by which diploid organisms generate haploid gametes. In<br />
preparation for the meiosis I division, homologous chromosomes condense, recognize each other,<br />
synapse, and undergo recombination. During this process a proteinaceous structure known as the<br />
synaptonemal complex (SC) forms between homologs maintaining them in a side-by-side alignment.<br />
Crossing over occurs in the context of the SC, leading to formation of chiasmata that connect the<br />
homologs and allow them to orient toward opposite poles of the meiosis I spindle.<br />
We are using a functional genomics strategy to identify genes required for these key meiotic prophase<br />
events, taking advantage of a genome-wide survey of germline gene expression conducted by V. Reinke<br />
and collaborators in the lab of S. Kim. We defined a subset of 206 germline-enriched genes whose<br />
expression profiles most closely match those of known meiosis genes, and designed a screen to identify<br />
those genes for which RNAi elicits defects in meiotic prophase. Defects in pairing, synapsis or<br />
recombination that lead to chromosome non-disjunction are initially identified by the production of inviable<br />
embryos and an increased frequency of male progeny. Defects that lead to arrest in meiotic progression<br />
are initially identified by a sterile phenotype. Candidates fitting either of these profiles are then subjected<br />
to cytological analysis to investigate possible meiotic defects. The efficacy of our screening procedure has<br />
been validated using genes encoding known meiotic recombination and synapsis components as positive<br />
controls.<br />
Our initial screening has already identified several distinct phenotypic classes with defects in<br />
chromosome behavior and/or morphology. These define genes with putative roles in meiotic chromosome<br />
synapsis (see accompanying poster by Reddy et al.), as well as genes that may have both mitotic and<br />
meiotic roles. A summary of the results of this ongoing screen will be presented.<br />
122
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
EXPLORING THE ROLE OF PINCH/UNC-97 IN MUSCLE<br />
DEVELOPMENT AND FOCAL ADHESION ASSEMBLY IN<br />
CAENORHABDITIS ELEGANS AND MAMMALIAN TISSUE<br />
CULTURE CELL LINES<br />
Shaun Cordes 1 , May Dang-Lawson 1 , Poupak Rahmani 1 , Linda<br />
Matsuuchi 1 , Donald G. Moerman 1,2<br />
1Department of Zoology, U.B.C., Vancouver, B.C., Canada<br />
2Biotechnology Laboratory, U.B.C., Vancouver, B.C., Canada<br />
PINCH/UNC-97 is a phylogenetically conserved adaptor protein consisting entirely of 5 LIM domains<br />
which has been implicated in the assembly of dense bodies, focal adhesion (FA)-like structures in worm<br />
muscle. An examination of the subcellular localization of UNC-97 in body wall muscles using a full length<br />
UNC-97::GFP reporter construct reveals that the protein co-localizes with integrin-containing attachment<br />
structures and that it is also in the nucleus. Studies of partial and complete loss of function mutations in<br />
unc-97 demonstrate that the protein is necessary for the integrity of FA-like attachment structures found in<br />
nematode body wall muscles (JCB 144: 45-57). We are using a combination of genetics in C. <strong>elegans</strong><br />
and expression studies in mammalian tissue culture cells to further investigate the role of PINCH in<br />
muscle development and FA assembly. Moreover, we are investigating the functional importance of<br />
nuclear-localized UNC-97 in the regulation of these processes.<br />
To identify the regions of PINCH responsible for its localization, we have designed altered UNC-97::GFP<br />
reporter constructs which delete one or multiple UNC-97 LIM domains. Preliminary results in worms<br />
suggest that LIM2 and/or LIM3 are involved in nuclear localization. When a full length UNC-97::GFP<br />
reporter construct is transiently transfected into COS fibroblasts, the pattern of expression in the<br />
cytoplasm is similar to that of the FA associated protein alpha-actinin. Preliminary results in COS cells<br />
suggest that UNC-97 requires LIM1 for cytoplasmic localization to punctate cytoplasmic structures. Our<br />
results suggest that the interaction between PINCH and its LIM1 binding partners (such as the<br />
integrin-linked kinase; Mol. Cell Biol. 19: 2425-34), is important for the recruitment of UNC-97 to specific<br />
locations in the cytosol. Future experiments will explore the localization of our reporter constructs in the<br />
mouse myoblast cell line C2C12.<br />
123
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE SAD-1 KINASE REGULATES PRESYNAPTIC VESICLE<br />
CLUSTERING IN C. ELEGANS<br />
Justin Gage Crump 1 , Mei Zhen 2 , Kang Shen 1 , Yishi Jin 2 , Cornelia I.<br />
Bargmann 1<br />
1Howard Hughes Medical Institute and Department of Anatomy, University of California, San Francisco,<br />
CA 94143-0452 USA<br />
2Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA<br />
Synaptic development is a multistep process that includes the presynaptic clustering of vesicles and<br />
active zone proteins, the postsynaptic clustering of neurotransmitter receptors and regulatory proteins,<br />
and the termination of axon outgrowth as neurons recognize their targets. We report here the<br />
identification of a novel serine/threonine kinase, SAD-1, that regulates several aspects of presynaptic<br />
differentiation in C. <strong>elegans</strong>. In sad-1 mutant animals presynaptic vesicle clusters in sensory neurons and<br />
motor neurons are disorganized and more diffuse. Sensory axons fail to terminate in sad-1 mutants,<br />
whereas overexpression of SAD-1 causes axons to terminate prematurely. SAD-1 is related to PAR-1, a<br />
kinase that regulates cell polarity during asymmetric cell division. SAD-1 is expressed in the nervous<br />
system at the time of synaptogenesis and localizes to synapse-rich regions of the axons, where it can<br />
function cell-autonomously in presynaptic neurons. Strikingly, overexpression of SAD-1 can induce the<br />
formation of well-organized, evenly spaced vesicle clusters in dendrites, which are normally devoid of<br />
synaptic vesicles. These results reveal a potent function for sad-1 in specifying synaptic regions in<br />
polarized neurons. We have also started to study the molecular events that coordinate the presynaptic<br />
and postsynaptic specialization.<br />
124
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MUTANTS WITH ALTERED SENSITIVITY TO THE EFFECTS<br />
OF ETHANOL ON LOCOMOTION<br />
Andrew G. Davies, Tod R. Thiele, Catharine Eastman, Steven L.<br />
McIntire<br />
Gallo Center and Program in Biological Sciences, Dept. of Neurology, UCSF<br />
Ethanol is reported to have multiple targets in the nervous system, a factor that has complicated efforts to<br />
understand the actions of ethanol that bring about behavioral effects in mammalian systems. We are<br />
using C. <strong>elegans</strong> to confirm suspected targets of ethanol and identify new targets and pathways that can<br />
be affected by ethanol to produce behavioral changes. We have concentrated on the effects of ethanol on<br />
locomotion. With increasing doses of ethanol in a plate assay, wild-type animals display a decrease in<br />
activity, velocity and amplitude of the body waveform. Non-saturating screens for mutants with altered<br />
sensitivity to ethanol for locomotion have produced eight mutants with increased resistance to ethanol<br />
and four mutants with increased sensitivity to ethanol. We have characterized the locomotion, egg laying<br />
and pharyngeal pumping behaviors of these mutants in the presence and absence of ethanol and<br />
examined nervous system function by pharmacological analysis.<br />
Mapping and cloning of the genes mutated in these strains is underway using positioned Tc1<br />
polymorphisms or single nucleotide polymorphisms that result in alterations to restriction enzyme sites<br />
(thanks to Stephen Wicks for unpublished data). We have identified one of the resistance genes as the<br />
calcium-activated potassium channel gene slo-1. Potassium channels of the slo class are thought to<br />
regulate the excitability of neurons. There is in vitro evidence for a direct activating interaction of ethanol<br />
on channels of this class derived from mammalian systems (reviewed by Dopico et al. 1999, Neurochem.<br />
Int. 35:103-106) raising the possibility that we may have identified a conserved direct target of ethanol in<br />
the nervous system of the worm.<br />
125
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A SCREEN FOR DD/DV AXONAL MORPHOLOGY DEFECTS<br />
M. Wayne Davis, Erik M. Jorgensen<br />
Department of Biology, University of Utah, 257 South 1400 East Salt Lake City, UT 84112-0840<br />
Growth cones undergo a series of morphological changes as they migrate from the neuronal cell body to<br />
their target. VD motor neuron growth cones slow down and change their shape when they encounter the<br />
lateral nerve cord and body wall muscle (Knobel et al. Development 1999). In order to pass through the<br />
narrow spaces between the body wall muscles and the hypodermis, the growth cone stops and projects<br />
filopodia through these spaces. When the forward tip of one filopodium reaches the other side of the<br />
muscle the growth cone behind the muscle collapses and re-forms at the tip of this projection. The axon<br />
then branches at the dorsal nerve cord and synapses onto the dorsal muscles.<br />
Using an integrated unc-47::GFP construct that specifically labels the DD and VD axons, I screened for<br />
mutations that disrupt DD/VD axon morphology. In this screen, I identified two main classes of mutations.<br />
The mutations in the first class cause wandering of the axons; mutations in the second class cause<br />
premature termination and branching, either at the lateral nerve cord or at the dorsal muscle boundary.<br />
Most of the wandering axon mutations cause an Unc phenotype, and many of these are likely to<br />
represent alleles of previously known genes. However, the premature branching mutants have no other<br />
obvious phenotype. These mutations may identify new genes necessary for migrating growth cones to<br />
deal with obstacles or changes in substrate.<br />
I have done this screen in the CB4856 Hawaiian background. This should allow the use of<br />
single-nucleotide polymorphisms between this strain and N2 to facilitate mapping of the mutations with<br />
subtle phenotypes.<br />
126
SPN-2 AND SPN-3 FUNCTION TO ORIENT THE SPINDLE<br />
DURING EARLY CLEAVAGES<br />
Leah R. DeBella, Lesilee S. Rose<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Section of Molecular and Cellular Biology, University of California, Davis CA 95616 USA<br />
Orientation of cell division is critical for partitioning cytoplasmic factors and setting up cellular interactions<br />
necessary for proper development. In C. <strong>elegans</strong>, the first cleavage bisects the long axis of the embryo,<br />
generating the AB and P1 cells. Subsequent cleavages in the AB lineage are perpendicular to the<br />
previous axis of division. Cleavages in the P1 lineage occur repeatedly on the same axis, due to a nuclear<br />
rotation event. Previous studies have shown actin, microtubules, dynein, and components of the dynactin<br />
complex to be required for proper spindle orientation in some cells. However, the precise mechanism by<br />
which spindles align is not understood.<br />
We are studying two genes, spn-2 and spn-3, which are required for proper spindle orientation in<br />
embryos. spn-2 embryos show defects in spindle orientation at the 2 and 4-cell stage in both AB and P1<br />
lineages, while spn-2/sDf130 worms show defects in the generation of fertilized embryos. Together these<br />
phenotypes suggest a continuing role for spn-2 in spindle orientation during embryogenesis, as well as a<br />
role prior to embryonic division. Some spn-3 embryos fail to rotate at the 2-cell stage while others have<br />
ectopic rotation at the 4-cell stage. However, spn-3/sDf127 embryos are defective in positioning the first<br />
mitotic spindle. This data suggests spn-3 plays a role in spindle orientation during the first three divisions<br />
after fertilization.<br />
An examination of polarity markers in homozygous spn mutants indicates these markers are localized<br />
normally prior to abnormal spindle alignment. In addition, cell divisions are asymmetric and cell cycle<br />
timing is normal, suggesting that overall polarity is not affected in these embryos.<br />
Our working hypothesis is that the spn genes play a role specifically in spindle orientation by regulating<br />
interactions between the microtubules and actin cytoskeleton. As a first step towards testing our model of<br />
spn gene function, we are isolating these genes using a positional cloning approach. spn-2 has been<br />
placed within 220 kb and spn-3 within 11 kb. Examination of the spn genes, combined with the analysis of<br />
other genes in our lab, will provide a basis for understanding the mechanism behind spindle orientation in<br />
all blastomeres of the early embryo.<br />
127
MOLECULES ACTING IN PARALLEL WITH UNC-34 TO<br />
CONTROL CELL MIGRATION<br />
Megan Dell 1 , N Chugh 1 , N Hawkins 1 , E Kong 1 , J Hardin 2 , G Garriga 1<br />
1MCB, UC Berkeley<br />
2Dept Zoology, U Wisconsin-Madison<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Guidance cues and their receptors direct cell and growth cone migrations by regulating cytoskeletal<br />
dynamics. Several signaling molecules that function downstream of guidance receptors to regulate actin<br />
have been described. We have found that three signaling molecules, UNC-34, the C. <strong>elegans</strong> homolog of<br />
Enabled, WAS-1, a homolog of the Wiskott-Aldrich Syndrome Protein (WASP), and DAB-1, a homolog of<br />
Disabled, act in parallel to regulate cell migrations in C. <strong>elegans</strong>.<br />
Drosophila Enabled (Ena) and its mouse homolog Mena function in axon outgrowth and fasciculation. The<br />
phenotypes of unc-34 mutants show that this gene functions not only in these processes but also in cell<br />
migrations. unc-34 null mutants are viable and display only partially penetrant defects in cell and growth<br />
cone migrations, suggesting the existance of parallel pathways in C. <strong>elegans</strong> that can compensate for the<br />
absence of UNC-34. By conducting RNAi experiments in an unc-34 background, we have found that<br />
WAS-1 and DAB-1 act in parallel to UNC-34.<br />
Because both WASP and Ena have an EVH-1 domain at their N-termini and regulate actin assembly, we<br />
injected was-1 RNA into wild-type and unc-34 mutant animals. While was-1(RNAi) animals appeared<br />
normal, was-1(RNAi); unc-34(null) animals died as embryos due to ventral enclosure defects. Injection of<br />
was-1 RNA into a temperature-sensitive allele of unc-34 resulted in temperature-dependent lethality.<br />
Injection into worms raised at 25° , the restrictive temperature, resulted in embryonic lethality, while<br />
injected worms raised at 20° showed synthetic cell migration defects and partial lethality. These results<br />
show that UNC-34 and WAS-1 act in parallel pathways to control ventral enclosure and cell migration. A<br />
screen for mutations that result in synthetic lethal and migration defects in the unc-34(ts) background has<br />
yielded several potential mutants.<br />
Disabled is a negative regulator of Ena in flies. dab-1(RNAi) animals show defects in embryonic neuronal<br />
migrations, and injection of dab-1 RNA into unc-34(null) mutants results in enhanced migration defects.<br />
This result is different from the genetic evidence in Drosophila in which Disabled mutations suppress the<br />
Ena phenotype and suggests that unc-34 and dab-1 act in parallel pathways to regulate neuronal<br />
migrations in C. <strong>elegans</strong>.<br />
128
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
INSIGHTS INTO THE ROLE OF C. ELEGANS PROTEIN<br />
UNC-119 IN AXONOGENESIS<br />
Chantal Denholm 1 , Wayne Materi 1 , Daniel Gietz 2 , David Pilgrim 1<br />
1Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9<br />
2Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba,<br />
Canada, R3E 0W3<br />
UNC-119 has widespread effects on the development and maintenance of the C. <strong>elegans</strong> nervous<br />
system. Unc-119 mutants have several movement, sensory, and behavioral abnormalities due to<br />
structural and functional defects in the nervous system. Homologues have also been identified in humans,<br />
rat, Drosophila, and zebrafish, and their functional conservation suggests that this protein plays a<br />
fundamental role in neurogenesis.<br />
Human protein HRG4 has 57% identity with UNC-119 and is a novel photoreceptor enriched protein<br />
which begins to be expressed at the time of photoreceptor differentiation in the ribbon synapse. It is<br />
thought to be involved in visual neural signal transmission, however it is also expressed at lower levels in<br />
other tisues including fetal brain.<br />
Although the phenotype of unc-119 mutants has been well described, the biological role of UNC-119 and<br />
HRG4 remains to be determined because their sequences give no clues to their function(s). To gain<br />
insights about how this protein exerts its effects in vivo, yeast-two hybrid screens have been conducted<br />
using UNC-119 and its functional homologue HRG4 to determine with what proteins they interact. Both<br />
screens have yielded good candidate interactions which are consistent with these proteins’ involvement in<br />
neurite outgrowth, and these results, along with possible models of UNC-119/HRG4 function will be<br />
discussed.<br />
129
THE DEFECATION GENE AEX-1 MAY REGULATE A<br />
RETROGRADE SIGNALING PATHWAY AT<br />
NEUROMUSCLULAR JUNCTIONS.<br />
Motomichi Doi, Kouichi Iwasaki<br />
National Institute of Bioscience, Japan<br />
Communication between pre- and postsynaptic cells is crucial for synaptic transmission regulation. In one<br />
regulatory mechanism, retrograde signaling, signals from postsynaptic cells control synaptic connectivity<br />
and transmission of presynaptic cells. Although there is evidence that retrograde signaling occurs at<br />
neuromuscular junctions in C. <strong>elegans</strong> 1) , its mechanism is unknown. Here we report that the defecation<br />
gene aex-1 may be a regulatory component of C. <strong>elegans</strong> retrograde signaling.<br />
aex-1 mutants show phenotypes similar to those in aex-3 mutants, which were consistent with<br />
presynaptic defects. These phenotypes include defecation defects, reduced male mating, and resistance<br />
to the acetylcholinesterase inhibitor aldicarb. Based on these observations we initially hypothesized that<br />
aex-1 encodes a component which functions at presynaptic terminals.<br />
To determine the molecular function of AEX-1, we cloned and characterized aex-1. aex-1 encodes a<br />
novel protein of 1027 amino acids whose C-terminus is similar to the second C2 domain of the mouse<br />
protein Munc-13-4 and the human protein BAP-3. Using GFP fusion constructs we unexpectedly found<br />
that aex-1 was expressed primarily in body wall muscles. Furthermore, aex-1 expression driven by the<br />
muscle-specific unc-54 promoter conferred hypersensitivity to aldicarb but did not change sensitivity to<br />
the acetylcholine agonist levamisole. This suggests that AEX-1 affects presynaptic activities at<br />
neuromuscular junctions.<br />
To investigate whether AEX-1 regulates retrograde signaling, we generated egl-30(gf);aex-1 double<br />
mutants and tested whether or not hyperexcitation of ventral cord motor neurons suppresses the<br />
aldicarb-resistance phenotype of aex-1 mutants. egl-30(gf) expression in ventral cord motor neurons<br />
confers hypersentivity to aldicarb 2) . Our prediction was that if aex-1 functions downstream of ventral cord<br />
motor neurons then the egl-30(gf) mutation would not suppress the aex-1 phenotype. However, the<br />
egl-30(gf) mutation could suppress the aldicarb-resistance phenotype of aex-1 mutants, suggesting that<br />
aex-1 acts upstream of the egl-30(gf) mutation. This observation is consistent with the hypothesis that<br />
AEX-1 regulates retrograde signaling at neuromuscular junctions.<br />
1) Zhao, H. and Nonet, M. L. Development 127 1253-1266 (2000)<br />
2) Lackner, M.R. et al., Neuron 24 335-346 (1999)<br />
@<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
130
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
COSUPPRESSION IN THE GERMLINE: SILENCING IS<br />
GOLDEN<br />
Abby F. Dernburg 1 , Mónica P. Colaiácovo 1 , Jonathan Zalevsky 2 ,<br />
Anne M. Villeneuve 1<br />
1 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA<br />
94305-5329<br />
2 Current address: Department of Biochemistry and Biophysics, University of California, San Francisco,<br />
San Francisco, CA 94143-0448<br />
When genes required in the nematode germline are introduced as trangenes, they show notoriously poor<br />
expression. Paradoxically, the presence of such an array in a wild-type worm can have a bizarre<br />
outcome: it can produce specific phenocopy of loss-of-function mutations in the homologous gene. Similar<br />
"cosuppression" or "quelling" phenomena, in which transgenes induce functional silencing of endogenous<br />
genes, have been reported in diverse organisms, from plants to flies to fungi. We have demonstrated the<br />
generality of this phenomenon and characterized its rules. We show that functional repression is not a<br />
consequence of persistent physical association between transgenes and endogenous genes, or of<br />
mutations in affected genes. Constructing transgenes with different regulatory and coding information, we<br />
obtained evidence that cosuppression is likely to require transcription from the array to generate an RNA<br />
mediator that dictates its target specificity. Because this potential RNA involvement is reminiscent of<br />
RNAi, we asked whether mutations that abrogate RNAi have any effect on cosuppression. This analysis<br />
demonstrated that the genetic requirements for these two silencing phenomena do overlap but show key<br />
differences. Specifically, we find that both rde-2 and mut-7 are essential for cosuppression, but that the<br />
function of rde-1 is dispensable.<br />
Transgene-mediated cosuppression provides a valuable alternative to RNAi as a technique for probing<br />
gene function. Targeting interesting candidate genes, we have elicited diverse germline phenotypes,<br />
including defective chromosome synapsis, failures in spindle assembly, or altered meiotic progression.<br />
Because the effects of cosuppression appear to be largely restricted to the germline, this approach can<br />
reveal germline-specific functions for essential genes for which mutation or RNAi would result in lethality.<br />
131
SUR-9 A SUPPRESSOR OF ACTIVATED LET-60(N1046) IN<br />
THE C.ELEGANS VULVA.<br />
Dennis Eastburn, Min Han<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HHMI, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder<br />
The adult C. <strong>elegans</strong> vulva is induced by an EGF like signal which activates the Ras/MAPK pathway.<br />
Constitutively active alleles of ras result in a multivulva (Muv) phenotype where numerous pseudovulvae<br />
are formed from vulval precursor cells (VPCs) that are normally not induced in a wild-type background. By<br />
initiating suppressor screens of activated let-60 ras, many previously unknown components of this<br />
pathway have been identified. One gene, sur-9, has been isolated from such a screen and is currently<br />
being cloned and characterized. sur-9(ku258) has been mapped to chromosome III and is currently being<br />
fine mapped. The ku258 allele can suppress the Muv phenotype of let-60(n1046) from 80% to<br />
approximately 1%. In addition, ku258 is dominant with respect to its suppression. Cloning and elucidation<br />
of sur-9’s molecular identity could contribute to our knowledge of a highly conserved and important<br />
biological pathway.<br />
132
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
KNOCKOUTS IN C. ELEGANS: MADNESS AND<br />
METHODOLOGY<br />
Mark Edgley 1 , Erin Gilchrist 1 , Greg Mullen 1 , Bin Shen 1 , Margaret<br />
Kotarska 1 , Don Moerman 1,2 , Steven Jones 3 , Anil Dsouza 4 , Gary<br />
Moulder 4 , Malini Viswanathan 4 , Martin Lansdale 4 , Robert Barstead 4<br />
1Biotechnology Laboratory,U.B.C., Vancoouver, B.C., Canada<br />
2Department of Zoology, U.B.C., Vancouver, B.C., Canada<br />
3Genome Sequence Centre, Vancouver, B.C., Canada.<br />
4Department of Molecular and Cell Biology, OMRF, Oklahoma City, Oklahoma<br />
As the first multicellular metazoan to have its genome sequenced the nematode C. <strong>elegans</strong> offers an<br />
unprecedented opportunity to investigate larger scale developmental problems including tissue formation<br />
and organogenesis. The C. <strong>elegans</strong> genome contains approximately 19,000 ORF’s, of which only a<br />
fraction have been characterized through genetic mutational analysis. Functional analysis of the complete<br />
genome may be feasible using reverse genetic methodology. Random chemical mutagenesis with<br />
trimethylpsoralen and UV irradiation, coupled to PCR amplification using primers to a specific gene, can<br />
allow for targeted gene disruption. Potential deletions are detected using agarose gel electrophoresis.<br />
The approach is sensitive enough to detect deletions in single animals in complex populations. Through a<br />
process of "sib-selection" one can derive a clone of animals bearing a specific deletion. The mutations are<br />
then sequenced and mutations conferring lethality are balanced using (where possible) a GFP-balancer<br />
chromosome.<br />
Our laboratories are part of the C. <strong>elegans</strong> Gene Knockout Consortium, an international group of labs that<br />
do gene knockouts by request from the worm community [see web site:<br />
http://www.cigenomics.bc.ca/<strong>elegans</strong>]. In the past year the consortium has received 662 requests for<br />
targeted gene disruptions. From this request list consortium labs have eliminated the function of<br />
approximately 80 genes using the chemical mutagenesis approach [as of January, 2000]. All mutants and<br />
data provided by consortium laboratories are in the public domain and freely available to all researchers.<br />
133
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
USING DNA MICROARRAYS TO IDENTIFY TARGETS OF<br />
HOMEOBOX GENES IN C. ELEGANS<br />
Andreas Eizinger 1 , Tibor Vellai 2 , Fritz Müller 2 , Stuart K. Kim 1<br />
1Stanford University Medical Center, Developmental Biology, 279 Campus Drive B369, Stanford, CA<br />
94305, USA<br />
2University of Fribourg, Institute of Zoology, Perolles, Fribourg CH-1700, Switzerland<br />
Homeobox genes are necessary to specify anterioposterior identity in metazoa. Despite their importance,<br />
targets of homeobox genes are poorly understood. Using our DNA microarrays, which contain nearly<br />
every gene of C. <strong>elegans</strong>, we wish to identify a comprehensive list of homeobox target genes in C.<br />
<strong>elegans</strong>. Currently we are focusing on the four major HOX genes ceh-13, lin-39, mab-5 and egl-5.<br />
Preliminary data showed that heat shock expression of homeobox genes in embryos cause strong<br />
lethality and malformation of the hatched larvae. This shows that embryonic cells are responsive to<br />
excessive or ectopic HOX gene expression. We will use our DNA microarrays to identify the earliest gene<br />
expression changes following induction of HOX gene expression by heat shock. By comparing the gene<br />
expression profiles from heat shock treatment of HOX transgenic embryos to control strains, we will be<br />
able to separate the heat shock and HOX responses. We are also interested in the specificity of HOX<br />
genes. Elucidation of the targets of each of the four major HOX genes will show which targets are<br />
common between all HOX genes and which are specific to individual HOX genes.<br />
134
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
DED GENES DISRUPT CELL DIVISION TIMING AND<br />
PATTERNING IN C. ELEGANS EMBRYOS<br />
Sandra Encalada, Paula Martin, Jennifer Phillips, Rebecca Lyzcak,<br />
Danielle Hamill, Kathryn Swan, Bruce Bowerman<br />
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403<br />
In early C. <strong>elegans</strong> embryos, asymmetric cell divisions produce descendants with characteristic cell cycle<br />
times. To understand how these differences in cell cycle timing arise, and to investigate their role in<br />
pattern formation, we are characterizing ded (delayed division) mutants, in which embryonic cell divisions<br />
are delayed and cell fate patterning is abnormal.<br />
In genetic screens for both non-conditional and temperature-sensitive embryonic lethal mutations, we<br />
identified 25 ded alleles. Twelve mutations define nine genes (ded-1 through ded-9), based on genetic<br />
mapping and complementation tests. A common phenotype of these mutants is a prolonged three cell<br />
stage due to a delay in the division of P1. The terminal phenotype of some ded mutants resembles skn-1<br />
mutants: they produce differentiated cells but lack pharynx and gut and instead make extra hypodermis.<br />
The skn-1 gene encodes a transcription factor that specifies mesoderm and endoderm fate in the EMS<br />
blastomere in a wild-type 4-cell embryo. The protein PIE-1 normally blocks SKN-1 function in the<br />
germiline precursor P2. Analysis of a ded-1;pie-1::GFP strain shows that PIE-1 accumulates to<br />
abnormally high levels in EMS and C in ded mutant embryos. These observations suggest that PIE-1 is<br />
blocking SKN-1 function in both P2 and EMS. Consistent with this conclusion, ded-1;pie-1 double mutants<br />
resemble pie-1 mutants, producing extra phrynx and intestine. Finally, the delayed cell divisions of ded-1<br />
mutants also result in a loss of asymmetry in the size of the daughters of P1, and in the mislocalization of<br />
P-granules.<br />
We have cloned ded-1, and it encodes the B subunit of the DNA polymerase-primase complex. Based on<br />
RNA interference experiments with other DNA replication genes, the other loci we have identified likely<br />
define a set of DNA replication genes. Based on these results, we conclude that (1) interference with DNA<br />
replication delays the onset of mitosis, suggesting that a checkpoint monitors the completion of DNA<br />
replication, and (2) proper control of cell division timing is important for multiple aspects of asymmetric cell<br />
division in early C. <strong>elegans</strong> embryos.<br />
135
VOLTAGE-DEPENDENT CURRENTS IN HOMOLOGOUS<br />
CHEMOSENSORY NEURONS WITH DIFFERENT FUNCTIONS<br />
IN C. ELEGANS<br />
S Faumont, S.R. Lockery<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Institute of Neurosci, Univ of Oregon, Eugene, OR 97405<br />
In C. <strong>elegans</strong>, chemotaxis to the attractant NaCl is largely controlled by a pair of left-right homologous<br />
chemosensory neurons ASER and ASEL. Although the two neurons are bilaterally symetric in their<br />
morphology and pattern of connectivity , recent work argues for functional asymetry between these<br />
neurons in chemotaxis (Pierce-Shinomura et al., abstr. WCWM). Laser ablation of ASER greatly reduces<br />
chemotaxis to Cl- but not Na+; conversely, ablation of ASEL greatly reduces chemotaxis to Na+ but not<br />
Cl-. To determine if this functional asymetry reflects a difference in the ionic currents expressed in the two<br />
neurons, we made whole-cell voltage clamp recordings fron ASEL (n=20) for comparison with previous<br />
recording from ASER (Goodman et al., Neuron 20: 763-72, 1998).<br />
We found that ASEL is similar to ASER in three main respects. First, ASEL exhibits an outward current<br />
activated by depolarisation (-30 to 100 mV) and an inward current activated by hyperpolarisation below<br />
-70 mV. Little or no current is activated in the region between -70 and -30 mV. Second, the outward<br />
current comprises inactivating and sustained components which are probably carried by K+ ions, because<br />
both components are eliminated by substitution of N-methyl-glutamine (NMG) for K+ in the recording<br />
pipette. Third, outward current inactivation is voltage-dependent, because prepulses more positive than<br />
-70mV decreases peak amplitude of the outward current. These results suggest that the functional<br />
asymetry reflect differences in the response to chemical stimuli rather than differences in voltage<br />
dependent ionic current. Support: NIMH 51383 and NSF IBN-9458102.<br />
136
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
VAV IS REQUIRED FOR PHARYNGEAL MUSCLE<br />
CONTRACTION IN C. ELEGANS<br />
R.T. Fazzio, J.E. Mellem, M.C. Beckerle, A.V. Maricq<br />
Dept. of Biology and Huntsman Cancer Institute, Univ. of Utah, SLC, UT 84112<br />
In vertebrates, VAV, VAV-2 and VAV-3 are guanine nucleotide exchange factors for Rho-family GTPases.<br />
Nucleotide exchange is a key mechanism by which Rho GTPases are regulated during cytoskeletal<br />
remodeling. Disruption of this regulation by specific mutations in VAV results in the constituative activation<br />
of Rho-family members and oncogenesis. To better understand the role of VAV in cytoskeletal control, we<br />
have undertaken a genetic and molecular analysis of VAV in C. <strong>elegans</strong>.<br />
We have cloned the C. <strong>elegans</strong> homolog of vav, vav-1, from first-strand cDNA. vav-1 encodes a 975<br />
amino acid protein that shares 34% overall identity with human VAV and contains the predicted exchange<br />
factor domain and other important functional motifs. We have previously reported vav-1 expression in<br />
pharyngeal tissue, body wall muscle, vulval tissue and somatic gonad. In the pharynx, VAV-1 is found in<br />
muscle, in some neuronal cells and in the g1 gland cells. Subcellular localization of VAV-1 will be studied<br />
using a mouse monoclonal antibody generated to this protein.<br />
We have engineered a deletion mutation in vav-1 that removes 85% of the mature protein product. This<br />
disruption yields a pharyngeal contraction defect that ultimately results in early larval lethality. Mutant<br />
animals display asynchronous pharyngeal muscle contraction or, in some cases, complete pharyngeal<br />
paralysis. Electrophysiological analysis of mutant pharyngeal muscle confirms the importance of VAV-1<br />
for synchronous contraction. We can rescue this defect by the transgenic expression of a genomic DNA<br />
fragment that contains the vav-1 open reading frame.<br />
We are currently pursuing experiments designed to provide a better understanding of the vav-1 null<br />
phenotype: Through tissue-specific expression of vav-1 in the null background, and possibly mosaic<br />
analysis, we aim to identify the pharyngeal cells (and other cells) that require VAV-1. In addition, we aim<br />
to complete a mutational analysis of the vav-1 gene to determine the domains/residues of VAV-1 that are<br />
important for function. The generation of multiple alleles in vav-1 will provide the reagents necessary for a<br />
genetic screen designed to identify proteins that function in the VAV signal transduction pathway.<br />
137
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
REGULATION OF C. ELEGANS BODY SIZE BY SENSORY<br />
CUES<br />
Manabi Fujiwara, Hoan Phan, Steven L. McIntire<br />
Gallo Center and Program in Biological Sciences, Dept. of Neurology, UCSF, San Francisco, CA94608<br />
In C. <strong>elegans</strong>, body size may be regulated by the nervous system. Lewis and Hodgkin initially reported<br />
that cilium-defective mutants such as che-2 and che-3 are smaller than wild-type animals. A smaller body<br />
size is also observed in other cilium-defective mutants and in the tax-4 mutant (tax-4 encodes a<br />
cGMP-gated channel that is necessary for chemosensation). Furthermore, DBL-1/CET-1 TGF-b, which is<br />
involved in body size regulation, is expressed in neurons. These observations suggest that if an animal<br />
cannot sense an environmental cue such as food, body size may be reduced through altered neural<br />
activity. Such regulation may be useful if a smaller body is economical. Recently Apfeld and Kenyon<br />
reported that sensory cues also regulate aging of C. <strong>elegans</strong>.<br />
In order to analyze the putative neural regulation of body size, we isolated suppressor mutants of the<br />
che-2 small body size phenotype (chb). 15,000 haploid genomes were screened, yielding 28 candidates.<br />
These suppressor mutants do not suppress the dye-filling defect of che-2, but suppress the small body<br />
size of che-2. Some of these suppressors show the same body size with or without the che-2 mutation in<br />
the background. Such mutants may result from defects downstream of sensory signals. Nine strains of<br />
this category are being mapped using the snipSNP method (Wicks and Plasterk). One of these, chb-1,<br />
maps to the left arm of chromosome IV and may be allelic with odr-9/egl-4. odr-9/egl-4 maps to the same<br />
region. chb-1 has the chemotaxis defect to diacetyl that is found in odr-9/egl-4. chb-1 and odr-9/egl-4 both<br />
have a slightly larger body than wild type. We have mapped chb-1 to a reigion that is less than 100kb and<br />
are attempting to rescue with cosmids covering this region.<br />
138
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
REGULATION OF INTRACELLULAR DYNAMICS OF<br />
MAPKAPK2 IN LIVING C.ELEGANS<br />
Makoto Fukuda, Yves Bobinnec, Eisuke Nishida<br />
Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University,<br />
Sakyo-ku, Kyoto 606-8502, Japan<br />
MAPKAPK2, a target of p38 MAPK, is shown to be exported from the nucleus to the cytoplasm upon its<br />
activation in mammalian cells. This nuclear export is believed to be dependent on its nuclear export signal<br />
(NES). Here, we have examined intracellular dynamics of C. <strong>elegans</strong> MAPKAPK2 (C44C8.6) during<br />
whole processes of development. To visualize subcellular localization of MAPKAPK2 in living C. <strong>elegans</strong>,<br />
we constructed a fusion between GFP and MAPKAPK2 under the control of its own promoter. This<br />
construct was expressed in late embryo, all larval stages and adults. In adults, MAPKAPK2-GFP was<br />
expressed mainly in neuron, neuroblast and vulva. MAPKAPK2-GFP was present mainly in the nucleus of<br />
those cells. However, in response to extracellular stresses that induce activation of P38 MAPK and<br />
MAPKAPK2, MAPKAPK2-GFP was completely exported from the nucleus to the cytoplasm. An<br />
NES-disrupted form of MAPKAPK2 was not exported from the nucleus under any conditions. In addition,<br />
RNAi with C. <strong>elegans</strong> CRM1, which is known as an NES receptor, resulted in the inhibition of nuclear<br />
export of MAPKAPK2. These results define the requirement of the NES of MAPKAPK2 for its nuclear<br />
export. Moreover, we found that MAPKAPK2 was exclusively localized in the cytoplasm in some neuronal<br />
cells under normal condition, suggesting that MAPKAPK2 is activated in these cells. It is possible that<br />
nuclear export (= cytoplasmic localization) of MAPKAPK2 is tightly associated with activation of p38<br />
MAPK and MAPKAPK2. We are currently examining when and where the p38 MAPK/MAPKAPK2<br />
pathway is functioning in living C. <strong>elegans</strong> by this method.<br />
139
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ROLE OF CKI-1 IN TERMINAL EMBRYONIC<br />
DIFFERENTIATION AND CELL-CYCLE ARREST<br />
Masamitsu Fukuyama, W. Brent Derry, Joel H. Rothman<br />
Department of MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106<br />
The CIP/KIP family of Cyclin-dependent Kinase Inhibitors (CKIs) function to arrest the cell cycle at the<br />
appropriate time during metazoan development. In C. <strong>elegans</strong>, two apparent CIP/KIP CKIs are encoded<br />
by the adjacent cki-1 and cki-2 genes (1). We previously reported that deficiencies that remove both cki<br />
genes cause extra embryonic cell divisions in multiple tissues and accumulation of excess cell corpses,<br />
and that a cosmid containing these genes can rescue these deficiency phenotypes (2). We recently found<br />
that the cki-1 transgene alone can rescue the hyperplastic phenotype of homozygous deficiency embryos,<br />
while the cki-2 transgene shows only very weak rescuing activity, suggesting that cki-1 is essential for<br />
embryonic cell cycle arrest. In support of this hypothesis, cki-1(RNAi) but not cki-2(RNAi) leads to<br />
embryonic hyperplasia in most tissues and the appearance of many cell corpses. Interestingly, embryonic<br />
germ cells arrest the cell cycle in a CKI-1-independent manner.<br />
The CIP/KIP CKIs in vertebrates have been implicated in terminal differentiation as well as exit from the<br />
cell cycle. We previously demonstrated that cki-1(RNAi) causes loss of a differentiation marker in the<br />
adult somatic gonad (3). We recently found that cki-1(RNAi) can eliminate expression of two late<br />
differentiation markers during embryogenesis, one in seam cells and one in the gut, suggesting that cki-1<br />
is required to promote terminal stages of differentiation. We are currently attempting to determine (1)<br />
which stage in differentiation is blocked by loss of cki-1 activity, (2) whether terminal differentiation in the<br />
other lineages requires cki-1 activity, and (3) whether CKI-1 promotes differentiation through a<br />
cyclin-dependent kinase activity.<br />
1. Hong, Y et al. (1998). Development 125, 3585-3597. Feng, H et al. (1999). Nature Cell. Biol. 1,<br />
486-492.<br />
2. Gendreau, SB and Rothman, JH. (1997). 11th International <strong>Worm</strong> <strong>Meeting</strong>.<br />
3. Fukuyama, M et al (1998). 1998 <strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong>.<br />
140
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SAX-1 AND SAX-2 ACT IN PARALLEL WITH UNC-34 TO<br />
MAINTAIN NEURON POLARITY.<br />
Maria E. Gallegos 1 , Jennifer A. Zallen 2 , Cori Bargmann 1<br />
1HHMI, Dept. of Anatomy, UCSF, San Francisco, CA 94143<br />
2Dept of Molecular Biology, Princeton University, Princeton, New Jersey, 08544<br />
During development each neuron in C. <strong>elegans</strong> extends a precise number of processes from its cell body.<br />
This polarity is established normally in sax-1 and sax-2 mutants, but later in development ectopic neurites<br />
often emerge from the soma of select neurons. In addition, sax-1 and sax-2 mutants have neurons with<br />
enlarged, irregularly shaped cell bodies (Zallen et al. 1999). Whereas similar phenotypes have been seen<br />
in a variety of mutants known to disrupt neuron function, neurons in sax-1 and sax-2 mutants function<br />
normally suggesting that sax-1 and sax-2 may act more directly with the actin cytoskeleton to maintain<br />
neuron polarity.<br />
To ask if the late-emerging neurites in sax-1 and sax-2 mutants result from a deregulated axon guidance<br />
pathway, I made double mutants of sax-1 and sax-2 with various axon guidance mutants including<br />
unc-34, a homolog of Enabled (M. Dell and G. Garriga, pers. comm.). unc-34 lf mutants have axon<br />
guidance and early axon termination defects suggesting that unc-34 functions to direct axon guidance<br />
and promote axon outgrowth during development. Surprisingly, unc-34;sax-1 and unc-34;sax-2 double<br />
mutants are enhanced for the ectopic neurite outgrowth defect. Furthermore, these ectopic neurites<br />
emerge after initial polarity has been established. These results suggest that sax-1 and sax-2 act in<br />
parallel with unc-34 to inhibit ectopic neurite outgrowth. Since the early axon termination defects of<br />
unc-34 suggest that it functions to promote axon outgrowth one interesting possibility is that unc-34 is<br />
bifunctional. These results also demonstrate that unc-34 plays a role during the establishment and<br />
maintenance of neuron polarity.<br />
sax-1 encodes a S/T kinase related to the Ndr protein kinase in humans (62% id.) and flies (60 % id.)<br />
(Zallen and Bargmann submitted). While the function of the Ndr kinases is unknown, other closely related<br />
kinases have been shown to play a role in cell shape and polarity in nonneuronal cells. Since double<br />
mutant analysis places sax-1 and sax-2 in the same genetic pathway, I am in the process of cloning sax-2<br />
in order to identify other components of this kinase pathway. Progress in cloning and additional<br />
phenotypic characterization will be presented.<br />
141
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
IDENTIFYING PHARYNGEAL TARGETS OF PHA-4 USING<br />
DNA MICROARRAYS<br />
Jeb Gaudet 1 , Michael Horner 1 , Stuart Kim 2 , Susan E. Mango 1<br />
1Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, Salt Lake City,<br />
UT 84112<br />
2Department of Developmental Biology, Stanford University Medical School, Stanford, CA 94305-5427<br />
Development of the C. <strong>elegans</strong> pharynx requires the activity of the gene pha-4. Animals that lack pha-4<br />
activity fail to make a pharynx, while ectopic expression of pha-4 leads to the ectopic production of<br />
pharyngeal markers at the expense of other cell types. These data indicate that pha-4 specifies<br />
pharyngeal organ identity.<br />
The PHA-4 product is a member of the family of winged helix transcription factor and is expressed in all<br />
pharyngeal cells. Several genes are known to be specifically expressed in the pharynx, and at least two of<br />
these (ceh-22 and myo-2) require PHA-4 for their expression. Therefore, pha-4 probably functions by<br />
activating pharynx-specific gene expression.<br />
For pha-4 to direct development of the entire pharynx, it must act either directly or indirectly on all<br />
pharyngeal genes. One possibility is that pha-4 directly activates a small subset of pharyngeal genes,<br />
which then activate other downstream targets. This model proposes a regulatory hierarchy with PHA-4<br />
directly activating those genes immediately downstream of it. Another hypothesis is that pha-4 directly<br />
regulates all pharyngeal genes, whether they act early or late in pharyngeal development. A third<br />
possibility is that pha-4 directly activates a subset of genes at multiple levels in the hierarchy and<br />
indirectly activates others.<br />
To understand how pha-4 directs organogenesis we have identified potential targets of PHA-4 using<br />
microarray experiments. Probes were made from RNA from skn-1 embryos, which produce no pharyngeal<br />
cells, and from par-1 embryos, which produce excess pharyngeal cells.<br />
The results of these experiments have identified ~500 genes whose transcripts are more abundant in<br />
par-1 versus skn-1 embryos. Among these genes are known targets of PHA-4 (such as myo-2) as well as<br />
other genes that are involved in pharyngeal development, including pha-4 itself and ceh-22. We are<br />
currently analyzing the other positives to determine whether they are expressed in the pharynx, and<br />
whether they are direct or indirect targets of PHA-4.<br />
142
AN OVERVIEW OF PREDICTED CYTOCHROME P450 GENES<br />
IN C. ELEGANS<br />
Erin Gilchrist<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
C. <strong>elegans</strong> Reverse Genetics Core Facility, Biotechnology Laboratory, U.B.C., Vancouver, B.C., Canada<br />
P450 heme-thiolate genes (more commonly known as cytochromes P450) are found in all eukaryotes and<br />
most bacteria and Archaea. They encode a family of heme proteins that are responsible for metabolizing<br />
a wide range of endogenous and exogenous compounds. These enzymes have been implicated in<br />
carcinogenesis, and in the bioactivation or detoxification of many drugs and other xenobiotics.<br />
The <strong>Worm</strong>pep database has identified 75 predicted proteins that encode P450 enzymes in C. <strong>elegans</strong>.<br />
These can be sorted into 16 different classes based on their sequence similarity to each other and to<br />
P450s in other systems. The genes in different classes are likely to be involved in different metabolic<br />
pathways, therefore, I have compared the sequences of the C. <strong>elegans</strong> P450 genes with the known<br />
substrate-binding domains of P450s in other systems. This indicates which processes the different<br />
classes of C. <strong>elegans</strong> proteins may be involved in. Future biochemical and genetic analysis of the C.<br />
<strong>elegans</strong> loci will be used to resolve which amino acids in the substrate-binding domain are essential for<br />
the binding of that substrate.<br />
Several classes of P450 proteins in the worm are believed to be specific to C. <strong>elegans</strong>, or at least to<br />
nematodes, while others have orthologues in vertebrate or other invertebrate systems. Some of the<br />
worm’s P450 genes are more than 90% identical to each other, and several of them are clustered,<br />
suggesting that they evolved by gene duplication of some ancestral heme-thiolate locus. In addition, the<br />
polycistronic nature of some of these clusters indicates that their expression is likely to be coordinated<br />
and that their function may be redundant. I am endeavoring to generate a knockout of one of these<br />
clusters of P450-encoding genes: the C. <strong>elegans</strong>-specific CYP13 cluster on cosmid T10B9. I am also<br />
comparing the ORFs and flanking DNA of the C. <strong>elegans</strong> P450 sequences with sequences from C.<br />
briggsae to predict sequences that are important for regulating the expression of the genes in this family.<br />
143
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CLUES TOWARD UNDERSTANDING EGF/ WNT SIGNAL<br />
INTEGRATION IN THE SPECIFICATION OF P12 FATE:<br />
ANALYSIS OF THE EGL-5 PROMOTER<br />
Lisa Girard 1 , Henrique B. Ferreira 2 , Scott Emmons 2 , Paul Sternberg 1<br />
1Howard Hughes Medical Institute and California Institute of Technology Pasadena, CA 91125<br />
2Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461<br />
The C. <strong>elegans</strong> Hox gene egl-5 is most similar, based on sequence analysis, to Abdominal-B. Consistent<br />
with its assignment into this paralog group, egl-5 is expressed in the posterior region of the worm.<br />
Immuno-staining results have shown that in the hermaphrodite, egl-5 is expressed in the hermaphrodite<br />
specific neuron, body wall muscle, posterior lateral microtubule neuron, PVC interneuron, M, V6, the<br />
rectal epithelial cells K, F, B, U and the P12 neuroectoblast cell.<br />
The two most posterior P cells are P11 and P12. The anterior products of their first division are both<br />
neuroblasts. P11.p fuses with the epidermal syncytium and P12.p divides again during late L1. The<br />
anterior division products, the epidermal cells P12.pa and P11.p are distinguishable by their distinct<br />
nuclear morphologies and positions.<br />
Earlier ablation experiments have shown that before interdigitating at the ventral midline during early L1,<br />
either of the two most posterior P cells can adopt the P12 fate. Previous genetic analysis indicates that<br />
P12 fate specification requires the synergistic action of the EGF and the Wnt signaling pathways.<br />
Reduction - or loss of function mutations in components of the EGF or the Wnt pathway result in partially<br />
penetrant P12 to P11 or P11 to P12 transformations. Double mutants of EGF and Wnt pathway<br />
components significantly enhance the frequency of transformation. P12 is not specified in an egl-5(lf)<br />
mutant and overexpression of egl-5 can rescue the loss of P12 specification phenotype of let-23 mutants.<br />
In order to understand how information from the EGF and Wnt pathway are integrated at a cis-regulatory<br />
level, we have undertaken an analysis of the egl-5 promoter to determine elements required for egl-5<br />
expression in P12. Do the two pathways converge on the egl-5 promoter or upstream of it? We have<br />
identified an approximately 1.3 kb. fragment of egl-5 promoter sufficient to drive expression of a<br />
heterologous promoter in the cells K, F, B, U, body wall muscle and P12. We are further dissecting this<br />
region in an attempt to identify P12 specific elements. Additionally, we hope to utilize this P12 enhancer<br />
as a tool to identify additional players involved in egl-5 activation in P12.<br />
144
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SPN-4: A GENE REQUIRED FOR MITOTIC SPINDLE<br />
ORIENTATION IN THE 2-CELL STAGE C. ELEGANS EMBRYO<br />
José E. Gomes, Kathryn A. Swan, Christopher A. Shelton, Bruce<br />
Bowerman<br />
Institute of Molecular Biology, University of Oregon, 1390 Franklin Blvd, Eugene OR 97403<br />
Mitotic spindle orientation is crucial during development for proper segregation of cytoplasmic factors<br />
upon asymmetric cell division. Nevertheless the mechanism underlying the specification of spindle<br />
orientation is not well understood. In the C. <strong>elegans</strong> embryo the P1 blastomere divides asymmetrically<br />
with the mitotic spindle oriented along the anterior-posterior (a-p) axis. The centrosomes, after duplication<br />
and migration, are initially positioned transversely to a-p axis and rotate subsequently, setting up the<br />
spindle longitudinally. The remnant site, a structure derived from the completion of cytokinesis, has been<br />
proposed to be necessary for this centrosome-nucleus complex rotation, by capturing the astral<br />
microtubules, in a process involving the actin cytoskeleton. In addition mutations in the par genes exhibit<br />
defects on spindle orientation in the 2-cell stage suggesting that these genes also play a role in P1<br />
spindle orientation. In genetic screens for non-conditional and for temperature sensitive embryonic lethal<br />
mutations we found three mutant alleles (or25, or80 and or191ts) of a gene, spn-4, exhibiting defects on<br />
mitotic spindle orientation in the P1 blastomere. In spn-4 mutant embryos the P1 spindle fails to set up<br />
along the a-p axis due to a deficiency in centrosome-nucleus complex rotation. In contrast to par mutants,<br />
other aspects of polarity in the 1-cell stage are not affected. Like in wild type embryos the spindle in the<br />
first division is oriented along the a-p axis, AB and P1 blastomeres are unequal in size and divide<br />
asynchronously. Thus spn-4 may be more specifically required for the process of mitotic spindle<br />
alignment in P1. spn-4 maps to linkage group V, position +0.1 map units. We are using a combination of<br />
RNA mediated interference and cosmid rescue to determine its molecular identity. Using the or191ts<br />
allele we have identified set of 8 cosmids that is able to rescue the mutant phenotype.<br />
145
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CA 2+ -SIGNALLING VIA THE NEURON-SPECIFIC CA 2+<br />
SENSOR NCS-1 IS ESSENTIAL FOR THERMOTAXIS, A FORM<br />
OF ASSOCIATIVE LEARNING AND MEMORY IN C. ELEGANS<br />
Marie Gomez 1 , Edouard De Castro 2 , Ernesto Guarin 1 , Patrick Nef 1<br />
1Department of Central Nervous System, F. Hoffmann-La Roche, Basel, Switzerland<br />
2Department of Institute for Behavioral Genetics, University of Colorado at Boulder, USA<br />
A form of associative memory in <strong>Caenorhabditis</strong> <strong>elegans</strong> is the pairing of the temperature of growth with<br />
food. After conditioning, worms seek food at the temperature of growth in a very precise circular fashion<br />
on a radial gradient of temperature. This phenotype is called the isothermal behavior. Here we show that<br />
NCS-1, a neuron-specific calcium sensor highly conserved throughout evolution, is essential for proper<br />
isothermal behavior. ncs-1 knockout animals are unable to perform isothermal track behavior, although<br />
their loco motor and thermal avoidance behaviors are normal. The knockout phenotype is rescued by<br />
re-introducing wild-type NCS-1, but is not rescued by a loss-of-function form of NCS-1 unable to bind<br />
calcium. Calcium signaling via NCS-1 is therefore essential for proper thermotaxis, a pavlovian form of<br />
associative learning and memory in C. <strong>elegans</strong>.<br />
146
CHARACTERIZING THE NEURAL CIRCUITRY OF<br />
CHEMOTAXIS TO VOLATILE ODORANTS<br />
Jesse Gray, Maria Gallegos, Tim Yu, Cori Bargmann<br />
UCSF and HHMI, San Francisco, CA 94143<br />
The C. <strong>elegans</strong> chemotaxis circuit offers the possibility of applying genetics to understand a functionally<br />
complex neural circuit. Further characterization of this circuit, as well as an in vivo physiological assay,<br />
would help relate new results to knowledge of circuitry in other systems. Anatomically, the chemotaxis<br />
circuit can be defined as having three main layers: sensory neurons, interneurons, and motorneurons.<br />
The sensory input to the chemotaxis circuit is relatively well-understood. As a first step in characterizing<br />
the remainder of the circuit, we are using laser ablations to identify functionally important interneurons,<br />
motorneurons, and muscles.<br />
We are also developing new behavioral assays to more specifically control chemotaxis circuit input and<br />
more directly assay circuit output. By flowing odorants over an airtight plate, we can create a temporal<br />
gradient of volatile odorant. It has recently been shown that ASE-mediated (water-soluble) chemotaxis is<br />
accomplished by modulating the frequency of pirouettes (i.e., omega bends and reversals) (3). Using a<br />
video camera to record worm movement during odorant application and subsequently counting omega<br />
bends and reversals, we have begun to characterize the kinetics of the pirouette response to volatile<br />
odorants.<br />
Finally, we will apply imaging techniques to assay physiology. We have constructed a myo3::cameleon<br />
(1) that expresses in body wall muscles. We will use myo3::cameleon worms to identify muscles<br />
important for omega bends and will follow up by ablating the corresponding muscles and motorneurons.<br />
We hope to extend this analysis to neurons in order to characterize circuit activity during olfactory<br />
stimulation and adaptation. We are also exploring other imaging technologies.<br />
1. Miyawaki et al. (1997) Nature. 388, 882-887.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
2. Pierce-Shimomura et al. (1999) J Neurosci. 19(21), 9557-69.<br />
147
STUDIES ON THE NEMATICIDAL BACILLUS THURINGIENSIS<br />
TOXINS<br />
Joel S. Griffitts, Raffi V. Aroian<br />
UC San Diego, Department of Biology<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The insecticidal crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis have been<br />
used for decades to control herbivorous pests. The ability to produce these toxins in transgenic crop<br />
plants has given rise to more extensive application in recent years. Little has been accomplished in<br />
elucidating the mode of action of Cry toxins, and even less in determining how pests generate resistance<br />
to them. One reason for this is that agricultural pests are generally not amenable to molecular genetic<br />
analysis. That C. <strong>elegans</strong> is susceptible to a subset of the toxins in this family makes it a promising<br />
system in which to approach these questions genetically. We present findings based on two toxins, Cry5B<br />
and Cry21. These toxins are similar in their amino acid sequence (42% identical) and in their destructive<br />
effects on the worm gut. Previously, our laboratory has characterized the susceptibility of C. <strong>elegans</strong> to<br />
Cry5B, and isolated mutations in five complementation groups that confer resistance (see abstract by<br />
Marroquin et al). Here, we discuss the cloning of one of these resistance genes, bre-5. Mutations in the<br />
bre-5 gene result in strong resistance to Cry5B toxin. We have mapped the bre-5 gene to a small interval<br />
on chromosome IV. Based on homology, we have identified a candidate gene in this region. We are<br />
currently working to determine whether this gene, or some other nearby gene, is the bre-5 gene. We have<br />
started to characterize the toxicity of Cry21. It appears to be a more potent nematicide than Cry5B,<br />
inducing remarkable destruction of the gut. Additionally, we have not found animals in large mutagenized<br />
populations which escape the toxicity of Cry21, even over a series of dilutions. The results outlined incite<br />
fundamental questions about what molecules in the worm mediate the toxicity response and the<br />
generation of resistance, and about what element or elements in the two toxins under study account for<br />
the aforementioned differences in spite of overall high sequence similarity. The experimental approach we<br />
have developed will undoubtedly shed light on these questions, and perhaps on issues of normal gut<br />
development and maintenance in C. <strong>elegans</strong>.<br />
148
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SYNAPTIC LOCALIZATION OF THE GLUTAMATE-GATED<br />
CHLORIDE CHANNEL GBR-2<br />
Maria E. Grunwald, Joshua M. Kaplan<br />
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720<br />
In C. <strong>elegans</strong> and other invertebrates, glutamate receptors are likely to play a dual role at excitatory, as<br />
well as at inhibitory synapses. Recently, an AMPA-type glutamate receptor (GLR-1) has been shown to<br />
be localized to excitatory synapses (Rongo et al., 1998), and glutamate-gated chloride channels (GluCls)<br />
have been implicated in the formation of inhibitory synapses (Lee et al., 1999). We are interested whether<br />
these different types of glutamate receptors are in fact localized to functionally diverse synapses and if so,<br />
how this differential targeting is achieved.<br />
We are comparing the synaptic targeting of GLR-1 receptors and the glutamate-gated chloride channel<br />
GBR-2, encoded by the avr-14 gene (Dent and Avery, WCWM 1998). Using a GFP-tagged version of<br />
GBR-2, we found that these receptors are expressed in interneurons and localized to distinct synapses<br />
along the ventral cord. In addition, GBR-2 is expressed in motorneurons and in the sensory neurons ALM<br />
and PVD. Thus, compared to GLR-1, which is mainly expressed in interneurons, GBR-2 is more broadly<br />
expressed. We have also compared the subcellular localization of GBR-2 to that of GLR-1. Interestingly,<br />
GBR-2 and GLR-1 receptors are targeted to different synapses. GBR-2 does not co-localize with GLR-1<br />
and only a minor fraction of GBR-2 was found at touch neuron-interneuron synapses, where GLR-1 is<br />
found. However, a significant fraction of GBR-2 was identified at interneuron-interneuron synapses in the<br />
ventral cord. We speculate that these GBR-2-containing synapses correspond to the reciprocal synapses<br />
between forward and backward command interneurons, and that these putatively inhibitory synapses<br />
coordinate transitions between forward and reverse locomotion. We are presently investigating this<br />
hypothesis. Furthermore, we are interested in what causes the differential targeting of GBR-2 molecularly.<br />
Recently, we were able to show that the PDZ protein LIN-10 is responsible for GLR-1 localization (Rongo<br />
et al., 1998). GBR-2 is likely to use alternative pathways for its localization. We are presently searching<br />
for proteins that are responsible for its clustering using EMS mutagenesis, yeast-two-hybrid screening<br />
and co-localization assays with candidate proteins.<br />
149
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
REGULATION AND FUNCTION OF LIN-11 IN C. ELEGANS<br />
VULVAL DEVELOPMENT<br />
Bhagwati P Gupta, Paul W. Sternberg<br />
HHMI and Division of Biology, California Institute of Technology, Pasadena, California, USA<br />
The C. <strong>elegans</strong> hermaphrodite vulva has been a well-established model system to address the<br />
mechanisms of cell fate specification. The highly invariant lineage of the vulval precursor cells (VPCs)<br />
provides an opportunity to study the molecular interactions of genes that control cell types so precisely.<br />
We are interested in understanding the mechanism of the nuclear factors that function to specify the<br />
terminal fate of the vulval cells. Towards this we have been analyzing the role of lin-11 that encodes a<br />
LIM Homeobox family of transcription factor. Mutations in lin-11 gene cause egg-laying defective (Egl)<br />
phenotype. Cell lineage analysis and reporter gene expression experiments suggest its possible function<br />
in the specification of a subset of 2 o vulval cells. In addition, the gene is also required for the normal<br />
development of uterine p precursors that ultimately form vulva-uterus connection, and a subset of<br />
neurons. In order to understand the function and regulation of lin-11, we have characterized its regulatory<br />
region and identified distinct cis-elements for the vulva and uterine - specific function. We have also<br />
re-examined its expression pattern using multiple reporter constructs and the gene function in egg-laying<br />
behavior. Our results show that lin-11 is likely to function in many more cells and at multiple times than<br />
previously reported. In an effort to further understand its function, we have searched the C. <strong>elegans</strong><br />
genome database to identify a putative binding partner of LIN-11 - LBP-1 (LIM Binding Protein-1) - and<br />
currently investigating its function in vulval morphogenesis. We plan to carry out genetic screens using<br />
different genetic backgrounds to identify the upstream and downstream genes of the lin-11 pathway.<br />
These experiments are likely to provide a better understanding of the function of LIM Homeobox family<br />
members that act to specify diverse cell types in multiple organisms.<br />
150
SUR-7, A GENE THAT SUPPRESSES ACTIVATED RAS<br />
Eric Hague 1 , Min Han 2<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1Department of MCD Biology, University of Colorado at Boulder<br />
2HHMI, Department of MCD Biology, University of Colorado at Boulder<br />
Suppressors of let-60 (n1046) ras have proven to be important in the regulation of the ras signal<br />
transduction pathway. The sur (suppressor of activated ras) genes include sur-1/mpk-1, sur-2<br />
(downstream of mpk-1), sur-3/ksr-1, sur-6 (a PP2A regulatory subunit) and sur-8 (a leucine rich repeat<br />
containing protein). ksr-1, sur-6 and sur-8 appear to act positively between ras and raf.<br />
I am attempting to clone sur-7using single nucleotide polymorphisms (SNP’s). sur-7 has no phenotype<br />
alone, but suppresses n1046 better than 99%. sur-7 maps between sdc-1 and sup-10 on LGX.<br />
151
CHARACTERIZATION AND SUPPRESSION OF EAT-16;<br />
SAG-1/DGK-1 LETHALITY<br />
Yvonne M. Hajdu-Cronin, Wen J. Chen, Paul W. Sternberg<br />
HHMI and Division of Biology, Cal Tech, Pasadena CA 91125<br />
goa-1 (Ga o) modulates many behaviors in C. <strong>elegans</strong>, including locomotion, egg-laying, and feeding.<br />
Reducing function in goa-1 causes hyperactive locomotion and constitutive egg laying (1, 2); in contrast,<br />
overexpressing the constitutively activated Q205L mutation causes lethargy and cessation of egg laying<br />
(1). Previously, we screened for suppressors of the paralysis of syIs17, an integrated transgene<br />
containing goa-1(Q205L) under control of a heat shock promoter. Hyperactive mutants were isolated in<br />
two genes, eat-16 and sag-1/dgk-1. We found that in addition to suppressing the phenotype of activated<br />
Ga o, eat-16 and sag-1/dgk-1 mutations also partially suppressed the phenotype of several reduction of<br />
function mutations in egl-30 ( C. <strong>elegans</strong> Ga q). eat-16 encodes a regulator of G protein signaling, which<br />
we believe functions as a GAP for EGL-30, and sag-1/dgk-1 encodes a diacyl glycerol kinase (3) which<br />
likely functions to reduce the levels of diacyl glycerol (DAG), a second messenger produced upon<br />
stimulation of PLCb by activated EGL-30.<br />
eat-16(sy438) and sag-1/dgk-1(sy428) double mutants arrest during larval development. This lethal<br />
phenotype is highly penetrant (>99%). We hypothesized that the lethality of eat-16; sag-1/dgk-1 is caused<br />
by excessive levels of second messengers produced downstream of Ga q, such as DAG. In support of<br />
this hypothesis, the triple mutant egl-30(md186) eat-16(sy438); sag-1/dgk-1(sy428) is viable to adulthood<br />
and fertile. To further understand the cause of death and identify more components in the pathway, we<br />
are seeking other suppressors of eat-16; sag/dgk-1 lethality besides egl-30, both by testing mutations in<br />
genes that have already been described, and by screening for new suppressors. To date we have<br />
screened about 7000 genomes and have backcrossed 7 suppressor mutants. We are beginning to<br />
characterize the lethal phenotype by Nomarski optics and plan to determine the site of action.<br />
References:<br />
1. Mendel et al., 1995. Science 267: 1652-1655.<br />
2. Segalat et al., 1995. Science 267: 1648-1651.<br />
3. Nurrish et al., 1999. Neuron 24: 231-242<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
152
IMPROVED TISSUE PRESERVATION USING METAL MIRROR<br />
FREEZING OR HIGH PRESSURE FREEZING FOR TEM<br />
David H. Hall 1 , Frank Macaluso 2 , Gloria Stepheney 1 , Marie-Christine<br />
Paupard 1<br />
1Center for C. <strong>elegans</strong> Anatomy, Albert Einstein College of Medicine, Bronx NY 10461<br />
2Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx NY 10461<br />
Several recent reports (see below) have demonstrated that C. <strong>elegans</strong> tissues can be very well preserved<br />
for electron microscopy by high pressure freezing (HPF) followed by freeze substitution, perhaps<br />
substantially better than by standard chemical immersion fixation. HPF shows the potential to capture a<br />
more "life-like" view of the worm’s ultrastructure. We have been testing both HPF and a related technique,<br />
rapid freezing on a metal mirror (MMF) followed by freeze substitution. Both methods obtain similar high<br />
quality fixation, although there are some freezing artifacts using the metal mirror device that are<br />
eliminated in HPF. For MMF, live animals are concentrated on a small piece of filter paper and plunged<br />
against a metal mirror at liquid nitrogen temperature. While freezing damage often occurs about 5-15<br />
microns into the worms, some animals are very well frozen throughout. The frozen samples are held at<br />
low temperature and freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic<br />
resin and cured for thin sectioning. For HPF, we have tried two methods to concentrate live animals into<br />
small metal planchette, either holding the animals within fine strands of dialysis tubing (C. Lavin, pers.<br />
comm.), or mixing them into a slurry of yeast paste to form a space-filling solid support (McDonald, 1999).<br />
Examination of fast-frozen specimens by TEM reveals excellent views of membrane events and<br />
organelles. For instance, we see many omega figures on coelomocytes which are indicative of active<br />
endocytosis, events which are not commonly captured by chemical fixation. Synaptic active zones and<br />
vesicles are well preserved, as are their relationships to microtubules. A network of microtubules can also<br />
been seen extending to the periphery of hypodermis. Basal laminae look strikingly different, much looser<br />
and more mesh-like when compared to chemical fixation. Sample images are shown on our website<br />
[www.aecom.yu.edu/wormem/new.html].<br />
These two preparation methods, HPF and MMF, also hold great promise for high resolution immuno-EM.<br />
By reducing the osmium content and adding a dilute aldheyde fixation to the freeze substitution medium,<br />
we can obtain better resolution than is currently possible by our microwave technique. We have<br />
successfully localized epitopes in thin sections from HPF samples. MMF equipment is available here at<br />
Einstein campus. We are conducting HPF trials with the help of Stan Erlandson and Ya Chen at the<br />
University of Minnesota. As our skills improve, we will be happy to offer such services to the C. <strong>elegans</strong><br />
community.<br />
For further information on HPF, we recommend the following sources:<br />
Colleen Lavin’s website at www.geology.wisc.edu/~uwmr/caoting.html<br />
Martin Muller’s website at www.em.bio.ethz.ch/<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Human Press) 1999.<br />
In the U.S., there are HPF machines open to the outside users in Madison, Berkeley, Minneapolis and<br />
Albany.<br />
153
ROLE OF THE CAENORHABDITIS ELEGANS HOMOLOGS OF<br />
CDK5 AND P35 IN MIGRATION AND AXON OUTGROWTH<br />
Thomas Harbaugh, Gian Garriga<br />
Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720<br />
While cdk5 was originally identified in vertebrates based on its homology to the cell cycle regulated cdc2,<br />
further study has demonstrated a function not in the cell cycle but instead in post-mitotic neurons. Mouse<br />
knockouts of cdk5 and its activator, p35, result in defects in the pattern of neuronal migration in the cortex<br />
(1,2). Studies in cultured rat neurons demonstrated that the cdk5/p35 kinase activity can promote neurite<br />
outgrowth (3).<br />
The C. <strong>elegans</strong> homolog of cdk5 is 74% identical to mouse, and the p35 homolog contains a 159 AA<br />
region that is 54% identical. Translational GFP reporter constructs for cdk5 and p35 are expressed in the<br />
cytoplasm of most neurons beginning around the two-fold stage of embryogenesis and continuing into<br />
adulthood.<br />
Simultaneous overexpression of both cdk5 and p35 produces a strongly uncoordinated phenotype, with<br />
defects in fasciculation and axon pathfinding. Along with the expression pattern, these phenotypes<br />
suggest a role for cdk5 and p35 in neuronal development.<br />
The mig-18(k140) mutation was identified based on defects in distal tip cell migration (4) and may<br />
represent a Ce-cdk5 allele. Extrachromosomal arrays containing the Ce-cdk5 genomic region can<br />
partially rescue the distal tip cell migration defect of mig-18(k140). Furthermore, sequencing of the<br />
Ce-cdk5 genomic region revealed a point mutation predicted to change a conserved histidine into a<br />
tyrosine. To confirm that k140 is a Ce-cdk5 allele, we plan to isolate additional mig-18 mutants and<br />
determine whether they contain Ce-cdk5 molecular lesions. Also, we will screen a deletion library for<br />
alleles of Ce-cdk5 and its activator Ce-p35.<br />
(1) Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K, J Neurosci, 18:6370-7 (1998)<br />
(2) Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai LH, Neuron, 18:29-42 (1997)<br />
(3) Nikolic M, Dudek H, Kwon YT, Ramos YF, Tsai LH, Genes Dev, 10:816-25 (1996)<br />
(4) Nishiwaki K, Genetics, 152:985-97 (1999)<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
154
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
REGULATION OF EGG-LAYING BY SENSORY CUES<br />
Laura Anne Hardaker, William R. Schafer<br />
Dept. of Biology, University of California at San Diego, La Jolla, CA 92093-0349<br />
Egg-laying in C. <strong>elegans</strong> is regulated by sensory cues. For instance, it has been found that food<br />
deprivation increases the length of the inactive phase of egg-laying. However, the exact mechanisms by<br />
which this occurs are not known. We are investigating this phenomenon by focussing on two pathways,<br />
the flp-1 pathway and the type II TGF-beta dauer pathway.<br />
The gene flp-1 encodes a precursor of FMRFamide related neuropeptides, and it was shown by Chris Li<br />
to be expressed in a specific subset of head neurons. She found that flp-1 knockouts affect a variety of<br />
behaviors, including egg-laying. When we tested flp-1 worms, we found that they had a lengthened<br />
inactive phase (similar to the food-deprived worms). In addition, their egg-laying rate did not change<br />
depending on the presence or absence of food. Ablation experiments have shown that the effects of flp-1<br />
encoded peptides on egg-laying are independent of the presence of the HSN. Thus, we believe that the<br />
effects of FLP-1 may be mediated, at least in part, by a hormonal mechanism.<br />
Jim Thomas’ lab showed that the type II TGF-beta daf (dauer formation) mutants also displayed defects in<br />
egg-laying. As the Ruvkun and Riddle labs found that the TGF-beta pathway functions in sensory<br />
neurons, it seems to be a reasonable candidate to be involved in the regulation of egg-laying by sensory<br />
cues. Interestingly, we found that the egg-laying patterns of the Daf-c mutants (daf-1, daf-4, daf-7, and<br />
daf-8) are similar to that of flp-1: all have a lengthened inactive period of egg-laying. In addition, the<br />
egg-laying of daf-4 and daf-8 (but not daf-1 or daf-7) is unable to be modulated by the presence or<br />
absence of food. Furthermore, we found that the Daf-d mutation in daf-3 does not cause a defect in food<br />
modulation, and in fact has the ability to suppress the food insensitivity of daf-4.<br />
These results suggest that there may be two hormonal pathways involved with relaying sensory cues to<br />
the egg-laying circuitry, one involving flp-1 and the other involving the Daf-c genes. We are currently<br />
investigating connections between the flp-1 and daf pathways by constructing double mutants, and the<br />
results will be presented.<br />
155
CHARACTERIZATION OF THE C. ELEGANS<br />
SEROTONIN-SYNTHETIC AROMATIC AMINO ACID<br />
DECARBOXYLASE GENE BAS-1<br />
Emily Hare, Curtis M. Loer<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Biology, Univ. of San Diego, 5998 Alcala Park, San Diego, CA 92110<br />
We are attempting to identify mechanisms by which neurons choose a neurotransmitter during<br />
development. We are currently characterizing genes used by the serotonergic neurons in C. <strong>elegans</strong> to<br />
learn how they are regulated. <strong>Worm</strong>s with mutations in the bas-1 gene (biogenic amine synthesis<br />
abnormal) are serotonin- and dopamine-deficient. Wildtype serotonin can be restored by the application of<br />
exogenous serotonin, but not its immediate precursor 5-HTP; this phenotype is consistent with the loss of<br />
serotonin-synthetic aromatic amino acid decarboxylase (AAADC) activity. We have previously shown that<br />
wildtype serotonin immunofluroescence can be rescued in bas-1 mutants by injection of a 15.1 kb<br />
genomic clone (C05D2XN) containing two adjacent AAADC homologous sequences. The genomic<br />
structure of the region suggests the two AAADC sequences (called C05D2.4 and C05D2.3) will be<br />
transcribed together as an operon (e.g., the two coding regions are less than 400 bp apart). The pair of<br />
sequences must be from an ancient gene duplication since the predicted amino acid sequences are only<br />
66% identical. Interestingly, the second sequence (C05D2.3) is missing a highly conserved segment of<br />
protein, suggesting it may not be able to function as an AAADC. The existence of a partial cDNA for<br />
C05D2.3, however, does demonstrate the gene is expressed. We are currently attempting to determine<br />
which of the two predicted sequences is necessary for serotonin synthesis and how the AAADC gene is<br />
expressed and regulated. Two bas-1 mutant alleles sequenced to date contain point mutations in<br />
C05D2.4 coding sequence resulting in premature stop codons. Furthermore, a construct in which<br />
C05D2.4 is mutated does not rescue bas-1 mutants whereas a construct mutated in C05D2.3 does<br />
rescue bas-1 mutants. Our preliminary results strongly suggest that C05D2.4 constitutes the bas-1 gene;<br />
we are still attempting to determine what role C05D2.3 might play. This work is supported by the Fletcher<br />
Jones Foundation and an NIH (AREA) Grant.<br />
156
XOL-1 FILES<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Christian A. Hassig, Barbara J. Meyer<br />
HHMI and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720<br />
Sexual differentiation in C. <strong>elegans</strong> is mediated by the developmental switch gene xol-1. XOL-1 represses<br />
one or more of the hermaphrodite-specific sdc genes or products, thereby directing the male modes of<br />
sex-determination and dosage compensation. XOL-1 has no known homologs and the primary structure<br />
offers no clues towards understanding its molecular function. Experiments are under way to determine the<br />
precise molecular mechanism(s) of this biological alien.<br />
The primary sex-determination signal (X:A ratio) is transduced by a set of signal elements, including sex-1<br />
and fox-1, that determine the level of XOL-1 protein. This X chromosome counting mechanism results in<br />
10-fold higher XOL-1 levels in XO versus XX embryos. Genetic studies suggest that both sex-1 and fox-1<br />
function as xol-1 repressors even in XO embryos. Consistent with these results, our antibodies detect<br />
XOL-1 in XO embryos carrying reduced copies of sex-1 (and 2 extra copies of xol-1), but not in wildtype<br />
XO embryos. Staining occurs in nearly all nuclei of embryos between the 100-500 cell stage. Similar<br />
experiments are being performed with fox-1 mutants. These results will establish a direct correlation<br />
between X-signal element dose and XOL-1 levels.<br />
Experiments are consistent with a role for XOL-1 as a repressor of sdc-2. Several features distinguish<br />
SDC-2 from other SDC proteins and make it a likely target for XOL-1 regulation: 1) SDC-2 directs the<br />
assembly of the dosage compensation complex on the hermaphrodite X chromosome, 2) SDC-2 is<br />
undetectable in wildtype XO embryos, 3) overexpression of SDC-2 causes male-specific lethality. These<br />
results suggest that XOL-1 could interfere with the expression of sdc-2 and thereby inhibit the formation of<br />
the dosage compensation complex in XO embryos.<br />
Our experiments using transgenic strains further support a role for XOL-1 in repressing sdc-2.<br />
Hermaphrodite lethality caused by overexpression of XOL-1 is partially suppressed by SDC-2 expressed<br />
from a heterologous promoter. This suppression is not seen with overexpression of SDC-3 and suggests<br />
that XOL-1 might regulate sdc-2 at the transcriptional level. Transgenic lines will be used to determine if<br />
XOL-1 associates with the sdc-2 locus in vivo. Results from these experiments will direct future inquiries<br />
into the XOL-1 Files.<br />
157
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Y41G9A.1, THE C. ELEGANS HOMOLOGUE OF TG737, IS<br />
EXPRESSED IN CILIATED NEURONS<br />
Courtney J. Haycraft 1 , Patrick D. Taulman 1 , Stephen M. Krum 1 ,<br />
Bradley K. Yoder 2<br />
1MCLM 639, 1530 3rd Ave. South, Birmingham, AL 35294-0005<br />
2MCLM 656, 1530 3rd Ave. South, Birmingham, AL 35294-0005<br />
The C. <strong>elegans</strong> gene Y41G9a.1 encodes the homologue of the mouse gene Tg737 which was identified<br />
through a BLAST search of the C. <strong>elegans</strong> genome database. Y41G9a.1 (CeTg737) was isolated from a<br />
mixed stage C. <strong>elegans</strong> cDNA library. CeTg737 encodes a predicted 823 amino acid protein with 45%<br />
identity to the mouse protein (Polaris). As determined for Polaris, CeTg737 contains two blocks of<br />
tetratricopeptide motifs (TPR) thought to be involved in protein-protein interactions. Northern blot analysis<br />
reveals a 2.6 kb transcript with the highest level of expression during embryogenesis and early larval<br />
stages. Transcriptional Y41G9a.1::GFP fusions using 400 bp upstream of the predicted ATG show<br />
expression in ciliated neurons including the amphids and phasmids. Analysis of the promoter region<br />
identified a potential X-box sequence approximately 100 bp upstream of the ATG. X-boxes are targets for<br />
the DAF-19 transcription factor and have been identified in the promoters of several genes involved in<br />
sensory cilia formation or maintenance. The role of DAF-19 in regulation of CeTg737 is currently being<br />
explored. Consistent with GFP expression analysis, monoclonal antibodies raised against recombinant<br />
CeTg737 detect a protein in the dendrites and cilia of sensory neurons and the sensory rays of the male<br />
tail. Overall the results in the worm are in agreement with our analysis in the mouse where Polaris<br />
localizes to the base of cilia and its disruption results in a loss of cilia.<br />
158
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
LET-381 IS A FORKHEAD GENE<br />
Marika Hellqvist-Greberg 1 , Ann M Rose 2 , David L Baillie 1<br />
1IMBB, Simon Fraser University, Burnaby, B.C, V5A 1S6, Canada<br />
2Dep.of Medical Genetics, UBC, 6174 University Blvd., Vancouver, B.C V6T 1Z3 Canada<br />
Forkhead genes encode transcription factors characterized by a highly conserved region of 100 amino<br />
acids necessary for DNA binding. Members of the forkhead gene family have been identified in eukaryotic<br />
organisms ranging from yeast to mammals. They have proved to be an important gene family involved in<br />
development and tumorigenesis.<br />
We are studying this gene family in C.<strong>elegans</strong>. We have identified at least 15 members of the forkhead<br />
gene family in the finished sequence of C.<strong>elegans</strong>. 5 of them have known mutants, daf-16, pha-4, pes-1,<br />
unc-130 and lin-31.<br />
RNAi was used for three of the forkhead genes not yet described, T14G12.4, F26B1.7 and K03C7.2.<br />
F26B1.7 had a lethal phenotype that resembled the phenotype of let-381 identified in Ann Roses lab.<br />
Therefore we sequenced one of the alleles of let-381, h107, and found a point mutation in the splice site<br />
of exon 3 in the F26B1.7 gene. We are now analyzing the phenotype and the expression pattern in order<br />
to understand the function and identify target genes for this forkhead protein.<br />
159
GENETIC ANALYSIS OF DYNAMIC SEARCH BEHAVIOR IN C.<br />
ELEGANS<br />
T.T. Hills, F. Adler, A. V. Maricq<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Biology, University of Utah, Salt Lake City, UT 84112<br />
We are interested in identifying genes that control dynamic behavior modification underlying exploratory<br />
search in C. <strong>elegans</strong>. Immediately following removal from food, worms engage in an area-restricted<br />
search (ARS), characterized by an initial high frequency of sharp turns that then decays over time. This<br />
causes the search path to shift over time from local and overlapping to globally directed with infrequent<br />
overlap. ARS is found in a wide variety of animals and is theoretically supported as an efficient search<br />
strategy for finding resources that are distributed in patches. Using motion-sensitive computer analysis,<br />
we observed worms immediately after removal from food and quantified the temporal dynamics of their<br />
search paths including such features as velocity, radial displacement, and turning angle distribution.<br />
These features change continuously with time, but stabilize by approximately 40 minutes. This search<br />
behavior is experience dependent, changing significantly in a predictable way across worms reared in<br />
different resource distributions. <strong>Worm</strong>s grown in liquid also display ARS, suggesting a genetic<br />
predisposition for the behavior.<br />
To identify genes controlling this dynamic behavior modification, we quantified ARS in worms with<br />
mutations in candidate genes. Mutations in the glutamate receptor glr-1 and the amino acid<br />
decarboxylase bas-1 cause significant changes in the dynamic aspects of ARS, suggesting that the<br />
dynamics of ARS are modified by mutations in a single gene. To further identify genes controlling this<br />
behavior, we are developing a genetic screen to isolate mutants that lack the local search following<br />
removal from food. We are isolating worms that disperse to a point significantly farther than wild type<br />
following removal from food. bas-1 mutants, which lack the local search, are being used to calibrate the<br />
distance and time that provides the most efficient separation between wild type and potential mutants. In<br />
conclusion, the area-restricted search phenotype has been quantified in C. <strong>elegans</strong>, shows predictable<br />
interactions with the environment, and appears to be under the control of specific genes which we are<br />
currently in the process of identifying.<br />
160
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MULTIPLE ROLES FOR THE RAS-MAPK SIGNAL<br />
TRANSDUCTION PATHWAY IN CHEMOTAXIS TO<br />
ODORANTS?<br />
Takaaki Hirotsu 1 , Satoshi Saeki 2 , Yuichi Iino 1<br />
1Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo 113-0033, JAPAN<br />
2Present address: Tokyo Research Laboratories, Kyowahakkokogyo Co. Ltd., Machida 194-8533,<br />
JAPAN<br />
We have previously reported that mutants affected in the Ras-MAPK pathway show defects in chemotaxis<br />
to volatile odorants. Experiments in which let-60 ras was expressed using a heat shock promoter and a<br />
cell-specific promoter indicated that the normal activity of LET-60 Ras is required in mature olfactory<br />
neurons.<br />
To determine how the Ras-MAPK pathway is activated in olfactory neurons, we observed accumulation of<br />
activated MAPK by immunofluorescence. The activation of MAPK in the AWC neurons was detected after<br />
10 seconds of application of isoamylalcohol (which is sensed by AWC) at 10 -4 dilution. This activation of<br />
MAPK was dependent on the function of the G protein alpha subunit ODR-3, cyclic-nucleotide gated<br />
channel TAX-2/TAX-4 and the voltage-activated calcium channel subunit UNC-2. These results suggest<br />
that odorant-induced neuronal activity is essential for the activation of the Ras-MAPK pathway.<br />
When animals were exposed to higher concentration (10 -2 dilution) of isoamylalcohol for 10 seconds and<br />
stained for activated MAPK, the AWB neurons, as well as AWC, were stained. Interestingly, the activation<br />
of MAPK in AWC was shut off by longer exposure (for 5min) to 10 -2 isoamylalcohol, while the activation in<br />
AWB was sustained. On the other hands, the application of 10 -4 isoamylalcohol induced activation of<br />
MAPK in AWC, but not in AWB.<br />
AWB neurons are known to mediate avoidance of volatile odorants. Our immunofluorescence analysis<br />
indicated the possibility that high concentration of isoamylalcohol might induce avoidance behavior.<br />
Indeed, worms show avoidance behavior when large amount of undiluted isoamylalcohol is presented.<br />
The let-60(gf), lin-45(lf) and mek-2(0) mutants exhibited defects in this behavior, suggesting that the<br />
pathway is also important for avoidance of odorants.<br />
When wild-type worms are pre-exposed to isoamylalcohol at 10 -4 dilution for 5min, they show avoidance<br />
of isoamylalcohol at the normally attractive concentration. The let-60(lf) mutants showed defects in this<br />
behavior. The Ras-MAPK pathway may also play roles in this behavioral plasticity.<br />
161
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MAB-26 ENCODES THE C. ELEGANS EPHRIN EFN-4<br />
Thomas Holcomb, Sean E. George, Ian Chin-Sang, Mei Ding, Andrew<br />
Chisholm<br />
Department of Biology, University of California, Santa Cruz, California 95064<br />
In C. <strong>elegans</strong>, Eph signaling functions in embryonic neuroblast movements after gastrulation, and in<br />
epidermal enclosure. The C. <strong>elegans</strong> genome encodes one Eph receptor, VAB-1, and four ephrins:<br />
VAB-2/EFN-1, EFN-2, EFN-3, and EFN-4. mab-26 mutants have defects in male tail morphogenesis<br />
(Chow and Emmons 1994). The predicted ephrin gene efn-4 (aka F56A11.3), was located in the same<br />
region as mab-26. A genomic clone containing F56A11.3 rescued Mab-26 phenotypes in transgenic<br />
arrays. These data combined with identification of mutant alleles demonstrate that mab-26 and efn-4 are<br />
the same gene.<br />
The EFN-4 protein encodes a divergent ephrin. EFN-4 overall is most similar to murine Ephrin-B2 (27%<br />
identity and 45% similarity). Like other worm ephrins, EFN-4 is predicted to be GPI-anchored. Unlike all<br />
other ephrins EFN-4 contains a 24 amino acid insert in the putative receptor-binding domain.<br />
The mutation bx80 is a deletion of second exon of efn-4, and thus is likely a null mutation. Three other<br />
alleles from the Cambridge collection cause alterations in efn-4: e36 causes an early nonsense mutation,<br />
and the weak alleles e660 and e1746 (provided by Jonathan Hodgkin) cause missense alterations in the<br />
receptor binding domain.<br />
The phenotypes of efn-4 mutants indicate that like other eph signaling proteins it is required for embryonic<br />
morphogenesis. Genetic interactions with the other ephrins and vab-1 are inconsistent with EFN-4 acting<br />
only in the VAB-1 pathway (see abstract by Moseley and Chisholm). These observations have led us to<br />
examine the the EFN-4/VAB-1 binding interaction using cell culture experiments. Previously the EFN-1<br />
ephrin was shown to bind VAB-1::AP fusion proteins with high affinity in mammalian cell culture assays<br />
(Chin-Sang et al 1999). We are using parallel approaches to test EFN-4/VAB-1 interactions.<br />
An EFN-4::GFP reporter gene is expressed in a subset of neurons. This expression pattern suggests that<br />
EFN-4/VAB-1 signaling in the embryo occurs between neurons and is thus required non-autonomously for<br />
epidermal development. We will test this idea using genetic mosaic analysis and tissue-specific rescue<br />
experiments.<br />
162
SYD-8, A NEW PLAYER IN AXON GUIDANCE.<br />
Xun Huang, Mei Zhen, Yishi Jin<br />
Department of Biology, University of California, Santa Cruz, CA95064<br />
Growth cones sense and respond to various extracellular cues to be guided to their targets. Many such<br />
cues and their receptors have been identified in recent years, including netrins, slits, and ephrins.<br />
However, the signal transduction pathways of these cues are not well understood. We describe here a<br />
gene that may function in the downstream signaling of axon guidance.<br />
syd-8 (ju39) was isolated in a genetic screen for abnormal synapse mutants 1 . syd-8 (ju39) animals have<br />
wild-type mating, moving and egg-laying behaviors. In syd-8 (ju39) animals, the synaptic GFP puncta are<br />
discontinuous in the dorsal cord. Analysis of axonal morphology in syd-8 mutant revealed several axonal<br />
defects in DD and VD neurons: 1) commissures terminate prematurely before reaching the dorsal cord, 2)<br />
commissures branch or wander to the lateral region, 3) axons defasciculate in both dorsal and ventral<br />
cords. Similar defects are also observed in the DA and DB axons, but to lesser extents. We also observed<br />
defects in pathfinding by the HSNs. In wild type animals, HSNs send out processes along the ventral<br />
nerve cord to the nerve ring . Normally, HSNL extends along the left small bundle of ventral cord. HSNR<br />
extends along the large right bundle of ventral cord. In syd-8(ju39) animals, HSNs have two types of<br />
defects: 1) HSN, mostly the HSNL, wander in the lateral region, and 2) HSNs fasciculate on the ventral<br />
cord. In 20% of syd-8 (ju39) animals, the HSNs are in the large right bundle of ventral cord. The touch<br />
neurons appear to be normal as judged by the Pmec-7-GFP marker.<br />
ju39 is a recessive, partial loss-of-function mutation. We have mapped syd-8 on Chromosome V between<br />
lin-25 and him-5. arDf1, itDf1 and yDf8 complement syd-8; yDf12 and nDf42 fail to complement it. Cloning<br />
of syd-8 is underway and we will report further molecular analysis of syd-8 at the meeting.<br />
1. Zhen, M and Jin, Y. Nature 1999<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
163
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ANALYSIS OF GCY-31, A PUTATIVE SOLUBLE GUANYLYL<br />
CYCLASE GENE IN CAENORHABDITIS ELEGANS<br />
Martin L Hudson 1,2 , David S. Karow 1,2 , Michael A. Marletta 1,2 , David<br />
B. Morton 1,2<br />
1 Department of Biological Structure and Function, Oregon Health Sciences University, 611 SW Campus<br />
Drive, Portland, OR. 97201<br />
2 Howard Hughes Medical Institute and Department of Cell & Molecular Biology, University of Michigan,<br />
Ann Arbor, MI 48109<br />
Soluble guanylyl cyclases (sGCs) are involved in many cellular and physiological roles including neuronal<br />
signaling, axon guidance and maintenance of vascular tone. Many of these functions are carried out<br />
through activation of sGCs by the gaseous signaling molecule, nitric oxide (NO). Recently, a non-NO<br />
activated sGC (MsGCb-3) has been isolated from the developing larval nervous system of the tobacco<br />
hawk moth, Manduca sexta. By BLAST analysis, its closest homologue in C. <strong>elegans</strong> is the putative sGC,<br />
gcy-31. Previous work by Yu et al. failed to demonstrate any expression data for this gene using<br />
GFP-reporter constructs. In our hands, gcy-31::GFP constructs show expression in a bilaterally<br />
symmetrical pair of cells in the C. <strong>elegans</strong> head. These have processes that lead anteriorly to the tip of<br />
the pharynx, as well as distinctive circular projections posteriorly into the nerve ring. From this<br />
morphology, we have tentatively identified these as either OLL or IL1 neurons.<br />
cDNAs corresponding to gcy-31 have been isolated and show multiple splice variants. However,<br />
heterologous expression of the cDNAs in COS-7 cells has so far failed to show any cyclase activity. Apart<br />
from MsGCb3, all sGCs previously isolated have been obligate heterodimers. These are composed of an<br />
a and b subunit, hence an absent subunit partner may explain the lack of activity in this assay. To answer<br />
this, we are cloning and mapping the expression patterns of the remaining sGCs in C. <strong>elegans</strong>. Inverted<br />
repeat constructs under the control of heat-shock promoters are also being made to generate in-situ<br />
dsRNA corresponding to gcy-31, allowing us to generate and analyze a null mutation in this gene.<br />
164
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
USING C. ELEGANS TO DETERMINE THE MECHANISM OF<br />
ACTION OF PHARMACEUTICALS AND PESTICIDES<br />
Tak Hung, Ben Burley, Emery Dora, Dan Elkes, Steve Gendreau,<br />
Denise Jacobus, Rachel Kindt, Mark Lackner, Lisa Moore, Scott Ogg,<br />
Dianne Parry, Roxanna Peng, Ellyn Pham, Jenny Kopczynski<br />
Exelixis, Inc., 170 Harbor Way, P.O. Box 511, South San Francisco, CA 94083-0511 (www.exelixis.com)<br />
The primary goal of the mechanism of action program at Exelixis is to identify the cognate targets (binding<br />
partners) of compounds with unknown mechanisms of action. We start the process by identifying<br />
mutations in genes whose activity modulates the response of a model organism -- C. <strong>elegans</strong>, Drosophila,<br />
yeast, algae or plants -- to the drug, insecticide, fungicide or herbicide. We then clone these genes and<br />
their homologs in human or pest species, and use biochemical approaches to distinguish the cognate<br />
targets from the other genes in the response pathways. Once a cognate target has been identified, new<br />
compounds with increased activity, less toxicity and better delivery properties can be developed.<br />
Secondary goals of the program include identifying other genes in the response pathways that may also<br />
be drug or pesticide targets, and identifying genes that mediate undesirable side-effects of drugs.<br />
We have corporate partnerships with Bayer, Pharmacia and Bristol-Myers Squibb to identify the pathways<br />
and cognate targets of pharmaceuticals and agrochemicals. These partnerships typically start with a<br />
feasibility study to establish that a particular model system can be used to determine the mechanism of<br />
action for a particular compound. Roughly half of tested compounds cause genetically tractable<br />
phenotypes in C. <strong>elegans</strong>. Based on the observed phenotypes, mutations and RNAi of candidate genes<br />
in the response pathway are tested for resistance or hypersensitivity to the compound. Also, forward and<br />
reverse genetic screens are carried out to identify target pathways and candidate cognate targets.<br />
In addition to our corporate partnerships, we have an internally funded program to identify the cognate<br />
targets of pharmaceuticals. A significant number of drugs, with annual sales over $100 million, act by<br />
unknown mechanisms. Many more compounds- with unknown mechanisms of action- have demonstrated<br />
promising activities in clinical or pre-clinical studies. The development of second generation drugs with<br />
improved efficacy, fewer side effects, and lower production costs is often hindered by a lack of<br />
understanding of how these compounds act. One advantage of starting with compounds rather than<br />
mutations for target discovery is that we know in advance that at least the cognate targets will be<br />
druggable and therapeutic. We will present our analysis of C. <strong>elegans</strong> phenotypes caused by<br />
oncology-related compounds.<br />
165
IN VIVO CHARACTERIZATION OF THE EFFECTS OF THE<br />
UNC-64(MD130) MUTATION ON ANESTHETIC SENSITIVITY.<br />
Hunt S.J., Mike Crowder<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Anesthesiology, Washington University School of Medicine<br />
We have previously shown that mutations in the neuronal syntaxin gene unc-64 profoundly alter the<br />
volatile anesthetic (VA) sensitivity of C. <strong>elegans</strong>. Two hypomorphic unc-64 alleles confer hypersensitivity<br />
to the VAs isoflurane and halothane but a third unc-64 hypomorph is VA resistant. The difference<br />
between the isoflurane EC 50s (the concentration where the effect is half maximal) of the hypersensitive<br />
and resistant alleles is over 30-fold. The high-level resistance produced by the unc-64(md130) mutation<br />
indicates that VAs act specifically through a single major mechanism to disrupt coordinated locomotion in<br />
C. <strong>elegans</strong>. The md130 mutation is a single base change at the splice-donor site of the sixth intron,<br />
causing some aberrant splicing. By RT-PCR, md130 mutants produce truncated RNAs, in addition to<br />
wild-type unc-64 mRNAs. The predicted truncated forms of syntaxin are believed nonfunctional as they<br />
lack the transmembrane and part of the H3 domains proven vital for syntaxin’s ability to function in<br />
neurotransmitter release. The behavioral phenotype of md130 is recessive and similar to other weak<br />
reduction-of-function unc-64 alleles. However, the VA-resistance phenotype of md130 is semidominant<br />
and behaves as a gain-of-function mutation, consistent with the truncated forms acting dominantly to<br />
block VA action. The current studies attempt to address in vivo the molecular mechanism(s) by which the<br />
md130 mutation confers this resistance. A plasmid was constructed containing the full-length genomic<br />
unc-64(md130) sequence and used to transform wild-type and unc-64(null) animals. These animals are<br />
significantly VA resistant. Next we addressed if a truncated product alone could produce the VA<br />
phenotype. We constructed a plasmid to produce solely truncated and not wild-type unc-64 product by<br />
introducing mutations at both the donor and acceptor splice consensus sequence of the sixth intron. This<br />
plasmid was unable to rescue null syntaxin mutants, consistent with no wild-type product being produced;<br />
however it successfully knocked-in VA resistance in both the wild-type and null/wild-type backgrounds.<br />
Additional plasmids for expressing various syntaxin fragments are currently being constructed to define<br />
the specific region(s) of syntaxin that are regulating VA action.<br />
166
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
REGULATION OF THE C. ELEGANS EPIDERMAL GROWTH<br />
FACTOR HOMOLOG LIN-3<br />
Byung Joon Hwang, Paul W. Sternberg<br />
Division of Biology, California Institute of Technology, Pasadena, California 91125<br />
The C. <strong>elegans</strong> lin-3 gene, a homolog of the epidermal growth factors (EGFs), is required for multiple<br />
aspects of C. <strong>elegans</strong> development. LIN-3 is required for vulval induction, ovulation, and cell fate<br />
specification of the P12 neuroblast of hermaphrodites. It is also required for cell fate specification of B cell<br />
lineage in males, and for viability of males and hermaphrodites.<br />
LIN-3 has an extracellular domain with one EGF motif, a transmembrane domain, and a cytoplasmic<br />
domain that is longer than that of most other growth factors containing an EGF motif. Little is known about<br />
the mechanisms that regulate LIN-3.<br />
We are taking two approaches to understand the mechanisms regulating lin-3 expression. First, deletion<br />
analysis of lin-3 regulatory region has been performed to identify cis-acting elements for its tissue-specific<br />
expression. Second, a cis-acting element of lin-3 which is mutated in e1417 allele of lin-3 has been<br />
identified by sequencing the lin-3 regulatory regions. The wild type sequence of e1417 element inserted<br />
into an enhancer assay vector supports expression of lin-3::gfp in anchor cell, but the e1417 mutation<br />
abolishes enhancer activity in this assay. Currently, we are looking for proteins that bind to this<br />
AC-specific element of lin-3.<br />
167
CHARACTERIZATION OF THE REGULATORY ELEMENTS<br />
REQUIRED FOR NEURON-SPECIFIC EXPRESSION OF<br />
SNAP-25 IN THE NEMATODE<br />
Soon Baek Hwang, Junho Lee<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biology, Yonsei University, Seoul, Korea 120-749<br />
SNAP-25 is a presynaptic protein exclusively expressed in the nervous system of the nematode. We<br />
wanted to characterize the regulatory elements that are required for this tissue-specific expression. We<br />
determined the whole sequence of the C. <strong>elegans</strong> SNAP-25 gene including the 5’ upstream region and<br />
the relatively large first intron. In order to obtain a C. briggsae SNAP-25 homolog, we screened a genomic<br />
library and obtained a fosmid clone named G01P23 that contained the full genomic sequence of the C.<br />
briggsae homolog. We obtained the entire sequence of the fosmid from the genome center. As<br />
transformation of G01P23 into the C. <strong>elegans</strong> SNAP-25 mutants complements the mutant phenotypes,<br />
specific transcription factors of C. <strong>elegans</strong> may be able to bind the cis-acting elements in the C. briggsae<br />
SNAP-25 gene, and the cis-acting elements may be conserved in these two species. By both examining<br />
serially-deleted 5’ upstream regions and the first intron of SNAP-25 and comparing the sequences<br />
conserved both in the C. <strong>elegans</strong> and C. briggsae SNAP-25 genes, we were able to identify the<br />
regulatory elements required for neuron-specific expression of SNAP-25. We defined two sequence<br />
motifs in the promoter region and two cis-acting regulatory motifs in the first intron. The -1096-1058 region<br />
of 5’ upstream was required in motor neurons of the body region and the -207-190 region was required in<br />
amphid and phasmid neurons. A 1.3Kb patch of the first intron may act in pharyngeal neurons and<br />
another 1.6Kb patch may act in mechanosensory neurons. We expect that each motif confers binding site<br />
for transcription activator(s) in different subsets of neuron cells, which may differ in cell lineage and<br />
function.<br />
168
ANALYSIS OF 2&DEG; VULVAL LINEAGE EXECUTION<br />
Takao Inoue, Paul W. Sternberg<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
California Institute of Technology, Division of Biology MC 156-29, Pasadena, CA 91125<br />
During the development of the hermaphrodite vulva, P6.p adopts the 1° cell fate and P5.p and P7.p adopt<br />
the 2° cell fate. Cells that adopt the 2° cell fate divide twice longitudinally, and then in a characteristic<br />
LLTN (P5.p) or NTLL (P7.p) pattern. Five distinct cell types are generated from this lineage; vulA (from<br />
outer L), vulB1 and vulB2 (inner L) vulC (T) and vulD (N). Our goal is to understand the mechanism of 2°<br />
lineage execution; specifically how these cell types are specified through a network of genetic interations.<br />
To facilitate the identification of different vulval cell types, we are assembling a panel of chromosomally<br />
integrated gfp reporter constructs which express in subsets of vulval cells. These markers will be<br />
examined under various conditions (e.g. mutant background and/or laser ablation of key cells) to<br />
determine how they are regulated. Furthermore, to identify genes involved, screens for mutations which<br />
alter the expression pattern will be carried out. Previously, egl-29 mutations were shown to affect the<br />
expression of egl-17::gfp in the 2° lineage (M. Wang and P.W.S). Since this gene may be involved in 2°<br />
lineage patterning, we are cloning it.<br />
169
DEVELOPING A C. BRIGGSAE GENETIC MAP<br />
B. Johnsen 1 , S. Gharib 2 , A. Mah 1 , K. Brown 2 , D. Baillie 1 , P.<br />
Sternberg 2<br />
1Simon Fraser University<br />
2Caltech<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
We are developing a genetic map of <strong>Caenorhabditis</strong> briggsae in order to facilitate comparative studies<br />
with <strong>Caenorhabditis</strong> <strong>elegans</strong>. The two nematode species are morphologically similar but are<br />
approximately 25 - 40 million years apart, which is about one-half the 60 - 90 million year evolutionary<br />
distance between mice and humans. Insights gained by the studies of the two nematode species should<br />
help in the studies of the two mammals. The 100 million base pair C. <strong>elegans</strong> genome is effectively<br />
completely sequenced revealing approximately 19,000 genes. Over 9.4 million base pairs (about 12%) of<br />
the C. briggsae genome has been sequenced and roughly another 4 million bases pair should be<br />
sequenced over the next year (M. Marra pers. comm.). The two nematodes show 30-50% divergence of<br />
unselected nucleotide sequence. Blocks of unaltered sequence are, presumably, under selective<br />
pressure to remain unchanged. The comparisons of the genetic maps of the two species should reveal<br />
how selective pressure constrains evolution of the linkage groups. For example, in C. <strong>elegans</strong>’<br />
chromosomes there are regions of low and high rates of recombination. Highly conserved genes might<br />
more likely be in regions of low recombination whereas rapidly evolving genes more likely reside in<br />
regions of high recombination. If this were true we would expect a similar gene distribution in C. briggsae.<br />
Although the two species are similar there are some differences. We have noted that C. briggsae matures<br />
quicker and lays its first eggs at about age three days, about one-half day sooner than C. <strong>elegans</strong>. C.<br />
briggsae also lives longer then C. <strong>elegans</strong> (averaging 29.7 days versus 18.6 days @ 20° for our lab’s<br />
strains). We have over 100 C. briggsae visible mutations. So far we have identified 15 cby, 7mip, 1 rot<br />
and 1 sml genes (the C. briggsae versions of dpy, unc, rol and sma respectively). Five of the cby genes,<br />
three of the mips and the rot are on the X-chromosome while the other 10 cbys, 4 mips and sml map to<br />
autosomes. We hypothesize that mip-1 is C. briggsae unc-22 and cby-4 is dpy-1. We have also shown<br />
that mip-1, mip-3, mip-6, cby-7 and cby-8 are linked.<br />
170
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
COENZYME Q AND AGING IN THE NEMATODE<br />
CAENORHABDITIS ELEGANS.<br />
Tanya Jonassen 1 , Pamela L. Larsen 2 , Catherine F. Clarke 1<br />
1UCLA Department of Chemistry and Biochemistry, Los Angeles, CA 90095.<br />
2The USC Andrus Gerontology Center, Los Angeles, CA 90089.<br />
The nematode <strong>Caenorhabditis</strong> <strong>elegans</strong> has been used as a model for the genetic studies of aging.<br />
Mutations in the clk-1 gene of C. <strong>elegans</strong> result in slowed development and rhythmic behaviors, and an<br />
extended life span. The C. <strong>elegans</strong> clk-1 gene is a homologue of COQ7, a gene in Saccharomyces<br />
cerevisiae required for the biosynthesis of ubiquinone (coenzyme Q). Coenzyme Q is a redox active lipid<br />
that functions in the electron transport chains of mitochondria and plasma membranes, and plays an<br />
important role as an antioxidant. The nematode, rat and human homologues of clk-1/COQ7 all function to<br />
restore coenzyme Q biosynthesis in the yeast coq7 null mutant. Given the functional conservation of<br />
yeast rat, human and C. <strong>elegans</strong> CLK-1/Coq7 polypeptides, it is crucial to test whether changes in the<br />
level of coenzyme Q may be responsible for the slowed development, behavior and rate of aging in the<br />
nematode model. We have examined whether mutations in the clk-1 gene effect the level of coenzyme Q<br />
in C. <strong>elegans</strong>. We have found conditions where the level of coenzyme Q affects developmental timing,<br />
behavior and lifespan. The studies to be presented show that the clk-1 mutations do impact the level of<br />
coenzyme Q in the nematode system. This system provides a model that is ideal for evaluating the<br />
relationship between coenzyme Q and aging. These studies also indicate that C. <strong>elegans</strong> provides a<br />
metazoan model uniquely suited to address questions regarding Q uptake, metabolism and redistribution.<br />
171
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
OSM-9 SIGNALING: WHO’S INVOLVED?<br />
Amanda H. Kahn, David Tobin, Cornelia I. Bargmann<br />
UCSF, 513 Parnassus Ave, San Francisco, CA 94143-0452<br />
osm-9 encodes a predicted cation channel that is involved in multiple C. <strong>elegans</strong> sensory modalities. Loss<br />
of osm-9 function eliminates chemotaxis to the AWA-sensed odorant diacetyl, compromises adaptation to<br />
a subset of AWC-sensed odorants, and diminishes avoidance of ASH-sensed noxious stimuli. osm-9<br />
mutants also exhibit reduced AWA expression of a Green Fluorescent Protein (GFP) transgene. OSM-9 is<br />
similar to sensory channels in other species, including the Drosophila TRP/TRPL phototransduction<br />
channels and a novel fly homolog. OSM-9 bears closest homology to the mammalian nociceptive<br />
receptors VR1/VRL-1. Although signaling through the TRP/TRPL channels has been well-studied,<br />
relatively little is understood about the mechanisms of osm-9 signaling. Thus, we are using forward and<br />
candidate genetic approaches to elucidate osm-9 signaling pathways.<br />
We employed the osm-9 AWA GFP phenotype in a visual screen for molecules that interact genetically<br />
with osm-9. The screen utilized an allele of osm-9 containing a charge substitution in a conserved residue<br />
of an ankyrin protein-protein interaction motif. In anticipation of identifying gain-of-function mutations, we<br />
screened for dominant suppressors of this allele -- that is, F1 worms with restored expression of the AWA<br />
GFP transgene. At present, we are mapping and characterizing three highly penetrant dominant<br />
suppressors of osm-9. Interestingly, one mutant (ky440) is suppressed for both the AWA GFP and ASH<br />
avoidance phenotypes of osm-9. We will present recent progress in characterizing and mapping these<br />
mutants.<br />
We have also taken a candidate approach to studying osm-9 signaling, using existing C. <strong>elegans</strong><br />
signaling mutants. The osm-9 AWA GFP phenotype can be suppressed by overexpression of the G_<br />
protein odr-3 and by a gain-of-function allele of the calcium-calmodulin dependent kinase unc-43. Our<br />
provisional model is that ODR-3 is an upstream activator of OSM-9, which allows Ca2+ entry to regulate<br />
UNC-43 and gene expression. We are presently characterizing the effects of these suppressors on osm-9<br />
mediated behaviors.<br />
These studies will intertwine novel and well-studied transduction components in sensory signaling<br />
pathways, with the eventual goal of understanding how C. <strong>elegans</strong> employs similar -- or different -elements<br />
in individual sensory neurons.<br />
172
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
LOOKING FOR SYNERGY WITH PHA-4 ON THE MYO-2<br />
PROMOTER<br />
John Kalb 1 , Pete Okkema 2 , Jim McGhee 1<br />
1Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, CANADA<br />
2Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois<br />
PHA-4 is a fork head transcription factor that is essential for pharyngeal development. Forced expression<br />
of PHA-4 outside of the pharynx can activate pharynx-specific genes. For example, it has been previously<br />
shown that PHA-4 activates myo-2, pharyngeal myosin, through the C-subelement in the myo-2 promoter.<br />
Ectopic PHA-4 expression leads to strong ectopic expression of a C-subelement regulated reporter gene.<br />
However, it only weakly activates endogenous myo-2; with ectopic expression of PHA-4 throughout the<br />
embryo, ectopic expression of myo-2 is only detected in body wall muscles, showing factors in addition to<br />
PHA-4 are necessary for myo-2 expression. Thus, we suggest that PHA-4 works with co-factors to<br />
activate gene transcription. One likely candidate to be a co-factor with PHA-4 is PEB-1. PEB-1 is a novel<br />
DNA binding protein expressed in the pharynx and was identified by its ability to bind to the C-subelement<br />
in the myo-2 promoter (Thatcher, Fernandez, Beaster-Jones, Haun and Okkema). The binding sites for<br />
PHA-4 and PEB-1 overlap in the C-subelement. To test if PHA-4 and PEB-1 act synergistically via the<br />
C-subelement to activate gene expression, we have been assembling the transcriptional regulatory<br />
network in yeast. We find that both PHA-4 and PEB-1 are (rather weak) transcriptional activators on their<br />
own. However, when both PHA-4 and PEB-1 are expressed together, we find no synergism, either<br />
positive or negative. To continue our investigation into the biochemical basis of PHA-4 action, we have<br />
produced all three forms of PHA-4 in baculovirus to define preferred binding sites.<br />
173
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
INITIAL CHARACTERIZATION OF SOLUBLE GUANYLATE<br />
CYCLASES IN C. ELEGANS<br />
David Karow 1 , Jennifer Chang 2 , Scott Nicholls 2 , Ronald Ellis 3 ,<br />
Martin Hudson 4 , David Morton 4 , Michael Marletta 5<br />
1Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109<br />
2Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109<br />
3Department of Biology, University of Michigan, Ann Arbor, MI 48109<br />
4Department of Biological Structure and Function, Oregon Health Sciences University, Portland, OR<br />
97201<br />
5Cellular and Molecular Biology, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI<br />
48109<br />
Soluble guanylate cyclases (sGCs) catalyze the conversion of GTP to cGMP. In organisms other than C.<br />
<strong>elegans</strong>, biochemically characterized sGCs are heterodimers composed of a1 and b1 subunits. The b1<br />
subunit is responsible for binding heme, which is ligated to a histidine residue (His 105). Nitric oxide (NO)<br />
binds to the heme and stimulates the enzyme 400-fold, increasing cGMP levels.<br />
In contrast to the previously characterized heterodimers described above, analysis of the completed C.<br />
<strong>elegans</strong> genome revealed seven putative sGC b subunits but no a subunits. Each of the putative b<br />
subunits contains the residues necessary for catalysis, binding GTP and binding heme (His 105).<br />
However, no open reading frame for nitric oxide synthase was found. This finding raises the possibility<br />
that these putative b subunits might function as novel NO-insensitive isoforms.<br />
Our preliminary data are consistent with this hypothesis. We have identified NO-insensitive sGC activity in<br />
C. <strong>elegans</strong> lysates. For further analyses, we have begun by focusing on one subunit -- T04D3.4.<br />
Preliminary characterization of the over-produced putative heme domain from T04D3.4 suggests that it<br />
does bind heme. In addition, promoter::GFP fusion studies suggest that this subunit is expressed in<br />
specific ciliated sensory neurons and possibly some mechanosensory neurons.<br />
These cyclases are conserved in Drosophila. They are also more homologous to the relatively<br />
uncharacterized mammalian b2 subunit than to the b1 subunit. If our preliminary evidence proves correct,<br />
these sGCs would define the first class of heme-binding cyclases that are insensitive to NO.<br />
174
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MULTIPLE REGULATORY ELEMENTS ACTIVATE END-1<br />
EXPRESSION IN THE E LINEAGE<br />
Jodie J. Kasmir, Morris Maduro, Joel H. Rothman<br />
University of California-Santa Barbara, Santa Barbara, CA 93106<br />
end-1 encodes a GATA factor that is sufficient to specify the identity of the sole endoderm precursor, the<br />
E blastomere. By dissecting the end-1 regulatory region, we have identified elements that control the<br />
levels and specificity of end-1 expression.<br />
end-1 is activated by at least one maternal factor, SKN-1. Heat-shock-driven expression of SKN-1 results<br />
in widespread end-1 expression. In addition, end-1 appears to be activated by zygotically expressed<br />
GATA factors: ectopic expression of either END-3, the genetically redundant partner of END-1, or MED-1,<br />
another zygotic GATA factor (see abstract by Maduro and Rothman), also result in widespread end-1<br />
expression. We are testing whether conserved SKN-1 and GATA consensus binding sites are directly<br />
responsive to SKN-1, END-3 and MED-1 action.<br />
While embryos lacking SKN-1 do not express detectable MED-1, end-1 is still expressed, albeit at low<br />
levels, and gut is still made 30% of the time. This observation suggests the existence of yet another<br />
activator of end-1. Previously, we reported that POP-1, a Lef-1 like protein that represses end-1<br />
expression in MS, appears to activate end-1 and gut differentiation in the E lineage in response to the<br />
MAPK/Wnt signal that induces endoderm. However, we have been unable to identify a single region in<br />
the end-1 promoter that is necessary for this POP-1-dependent activation of end-1. Indeed, our genetic<br />
and biochemical data indicate that multiple sites may be necessary for activation of end-1 in the E lineage<br />
by Wnt/MAPK-activated POP-1.<br />
Our further analysis has uncovered several important regulatory elements that are necessary to establish<br />
normal levels of end-1 expression. Some of these elements contain no consensus sites for known<br />
regulators, further indicating the existence of multiple positive inputs.<br />
175
MUTATIONS THAT PERTURB THE EFFECT OF<br />
OCTOPAMINE/SEROTONIN ON PHARYNGEAL ACTIVITY.<br />
John Keane, Leon Avery<br />
University of Texas Southwestern Medical Center, Department of Molecular Biology, Dallas, TX<br />
75390-9148, USA<br />
We have identified some mutants which increase the rate of pharyngeal pumping in C. <strong>elegans</strong>, most<br />
notably during the dauer larval stage. Pharynxes of these mutants (unc-9, unc-7 and goa-1) also respond<br />
abnormally to the application of exogenous octopamine. The neuromodulators serotonin and octopamine<br />
have previously been reported to act antagonistically (1). It is possible that these mutants are either<br />
hypersensitive to serotonin (normally stimulates pumping) or resistant to octopamine (inhibits pumping) or<br />
both. To address this we are examining the effect on pharyngeal activity of mutations which are reported<br />
to reduce octopamine (osm-3, che-3) and serotonin (tph-1) levels.<br />
Efforts to identify where unc-7, unc-9 and goa-1 mediate the observed effect have involved combining<br />
these mutants with eat-2, a mutant that knocks out excitatory synaptic communication between motor<br />
neuron MC and pharyngeal muscle. The double mutants reveal that MC is necessary for the observed<br />
increase in pumping rates. As yet, we have not determined whether this means that the motor neuron is<br />
more active in these mutants or whether the muscle has become more receptive to neuronal stimulation.<br />
As the increase in dauer pumping rate in these mutants is small compared to wild type, it is unlikely that<br />
octopamine has a major role in inhibiting pharyngeal activity during this developmental stage.<br />
(1) Horvitz R. et al. Science 216: 1012-1014 (1982)<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
176
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
PHEROMONE REGULATION OF NEUROENDOCRINE<br />
OUTPUTS IN C. ELEGANS<br />
Scott Kennedy, Gabriel Hayes, Gary Ruvkun<br />
Dept. of Molecular Biology, Massachusetts General Hospital and Dept. of Genetics, Harvard Medical<br />
School, Boston MA 02114<br />
A major regulator of the decision to enter the dauer stage is the environmental concentration of a<br />
constitutively secreted dauer promoting pheromone. One mechanism by which dauer pheromone controls<br />
dauer formation is via the regulation of the expression levels of the TGF-b like ligand DAF-7. To<br />
determine the mechanism(s) by which pheromone regulates the expression of DAF-7 we undertook a<br />
genetic screen to identify genes that when mutated would lead to both a loss of DAF-7 expression and a<br />
Daf-c (dauer constitutive) phenotype. In order to monitor DAF-7 expression we have utilized a daf-7p::gfp<br />
reporter construct, which is expressed predominantly in the ASI amphid neuron. From an initial screen of<br />
20,000 haploid genomes we identified one mutation, mg295. Mapping data indicates that mg295 maps to<br />
LGV, very near the daf-11 locus. In addition, mg295 fails to complement daf-11 (m47). Consequently we<br />
conclude that mg295 is very likely to be a mutation in daf-11. Daf-11 encodes a guanylyl cyclase , an<br />
enzyme that converts GTP to the intracellular second messenger cGMP. This suggests the possibility that<br />
pheromone signaling in C. <strong>elegans</strong> involves the second messenger cGMP, a molecule previously<br />
identified as essential for mammalian photoreceptor signal transduction.<br />
To further our understanding of the mechanism(s) by which pheromone and daf-11 control daf-7<br />
expression we have begun two additional genetic screens. The first screen seeks to identify mutants that<br />
lead to both a loss of daf-7p::GFP expression and a Daf-c phenotype under more stringent environmental<br />
conditions (27É). We anticipate that this screen will identify additional genes involved in the regulation of<br />
dauer formation and daf-7 expression. Progress on this screen will be reported. We have also begun a<br />
genetic screen to identify mutations that both suppress the daf-11 Daf-c phenotype and lead to a<br />
reversion in daf-7p::GFP expression. From an initial screen of 250,000 haploid genomes we have<br />
identified two mutations; tentatively named PollyAnna-1 (mg296), and PollyAnna-2 (mg297), that satisfy<br />
these criteria. Progress of this screen and on the characterization and mapping of mg296 and mg297 will<br />
be reported.<br />
177
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CALCIUM IMAGING IN EXCITABLE CELLS OF C. ELEGANS.<br />
Rex Kerr 1 , Varda Lev-Ram 2 , Roger Y. Tsien 2 , William R. Schafer 1<br />
1Dept. of Biology, UCSD, La Jolla, CA 92093-0349<br />
2Dept. of Pharmacology, UCSD, La Jolla, CA 92093-0617<br />
It is desirable in behavioral studies to record the activity of neurons and muscles, but this has traditionally<br />
been difficult in C. <strong>elegans</strong>. We are using a transfectable calcium indicator, cameleon [Miyawaki et al.,<br />
Nature 388:882], to overcome this barrier.<br />
To develop the technique, we focused on the pharyngeal muscle. The pharynx has been previously<br />
characterized behaviorally and electrically, primarily by the Avery lab, and is large enough to simplify<br />
imaging. In addition, calcium influx in muscles causes contraction, which provides an independent<br />
measure of activity. Using the myo-2 promoter, we expressed cameleon in the pharynx and recorded<br />
distinctive calcium transients coupled to muscle contraction in intact animals. The duration of these<br />
transients varied in mutants of egl-19, a voltage-gated calcium channel, consistent with previous electrical<br />
recordings [Lee et al., EMBO 16:1066]. In mutants of unc-36, a channel-associated a2 subunit, we found<br />
that transients were substantially increased in magnitude, suggesting an inhibitory role for the wild-type<br />
UNC-36 protein. This was surprising as coexpression studies of vertebrate homologues in Xenopus<br />
oocytes had found that the a2 had many relatively subtle effects, including increasing expression levels of<br />
the pore-forming subunit. We are currently extending our study of muscular calcium transients to<br />
investigate the effects of neurotransmitters and mutations on the vulval muscles and their role in<br />
egg-laying behavior.<br />
In addition, we expressed cameleon using the pan-neuronal promoter unc-119. We provided direct<br />
electrical stimulation through an extracellular electrode inserted through the cuticle and positioned near<br />
the nerve ring. Transients were observed coupled to the stimulation, indicating that calcium influx through<br />
voltage-gated calcium channels can be observed in neurons using this method. This raises the possibility<br />
of constructing a functional map of neuronal connectivity by electrically stimulating single neurons and<br />
recording the activity of their synaptic partners using cameleon.<br />
178
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A GENETIC ANALYSIS OF THE EFFECTS OF ETHANOL ON<br />
EGG LAYING<br />
Hongkyun Kim, M. Christina Yu, James Kim, Steven L. McIntire<br />
Gallo Center and Program in Biological Sciences, Department of Neurology, UCSF, CA 94608<br />
Ethanol causes dose-dependent suppression of egg laying in C. <strong>elegans</strong>. To elucidate the neuronal<br />
targets of ethanol, we screened for mutants showing ethanol-resistant or -inducible egg-laying behavior in<br />
a plate assay. F2 progeny of mutagenized animals were exposed to a dose of ethanol that strongly<br />
suppresses egg laying of wild type animals. Eggs were collected and allowed to hatch in the absence of<br />
ethanol. Once the F3 animals had matured to adulthood, the process was repeated for 2 cycles. By using<br />
this enrichment strategy, we isolated four different classes of mutants. Class I mutants are hyperactive<br />
and exhibit hyperforaging behavior and are aldicarb-hypersensitive. At least 4 mutants of class I belong to<br />
the same complementation group and are alleles of slo-1. Class II mutants show high amplitude<br />
sinusoidal tracks and slightly exaggerated head movement in the direction of the forward movement. This<br />
class exhibits variable aldicarb responses and normal foraging behavior. This class includes at least three<br />
complementation groups. The finding of the same behavioral defects in the different mutants of this class<br />
suggests that the same, ethanol-sensitive pathway may be affected by each of the mutations. Class III<br />
mutants have egg-laying defects in the absence of ethanol. Class IV mutants do not have any obvious<br />
phenotype. Egg laying of class I and II mutants is induced at relatively low ethanol concentrations that<br />
moderately suppress egg laying of N2. More eggs are laid in the presence than in the absence of ethanol<br />
in these mutants. Further mapping and characterization of these mutants may lead to the identification of<br />
the molecular targets of ethanol in C. <strong>elegans</strong>.<br />
179
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GENES AFFECTING THE ACTIVITY OF NICOTINIC<br />
RECEPTORS INVOLVED IN EGG-LAYING BEHAVIOR<br />
Jinah Kim 1 , Daniel S. Poole 2 , Laura E. Waggoner 2 , Alexandra<br />
Treschow 2 , William R. Schafer 2<br />
1Group in Neuroscience, University of California at San Diego, La Jolla, CA 92093-0349<br />
2Dept. of Biology, University of California at San Diego, La Jolla, CA 92093-0349<br />
Egg-laying behavior in C. <strong>elegans</strong> is regulated by multiple neurotransmitters, including acetylcholine and<br />
serotonin. Agonists of nicotinic acetylcholine receptors such as nicotine and levamisole stimulate<br />
egg-laying; however, the genetic and molecular basis for cholinergic neurotransmission in the egg-laying<br />
circuitry is not well understood. We have examined the egg-laying phenotypes of eight known levamisole<br />
resistance genes which may mediate or regulate nicotinic receptor activity in the egg-laying<br />
neuromusculature. Five "strong" levamisole resistance genes, including unc-63, unc-74, and the nicotinic<br />
receptor subunit genes unc-29, unc-38, and lev-1, were essential for the stimulation of egg-laying by<br />
levamisole. In the absence of drug these mutants retained the characteristic biphasic pattern of<br />
egg-laying and caused only subtle shifts in the timing of egg-laying behavior. <strong>Worm</strong>s mutant for two or<br />
three of these nicotinic receptor genes also had a generally wild-type pattern of egg-laying. These genes<br />
appear to encode components of a levamisole-sensitive nicotinic receptor that promotes egg-laying but is<br />
not necessary for egg-laying muscle contraction. unc-29 and unc-38 mutants were also hypersensitive to<br />
the stimulation of egg-laying by serotonin, suggesting that nicotinic receptors might negatively regulate<br />
serotonin response pathways in the egg-laying muscles or neurons. Three "weak" levamisole resistance<br />
genes, lev-8, lev-9, and lev-10, had effects on egg-laying that were distinct from those of the levamisole<br />
receptor genes. Phenotypic analysis indicated that lev-8, lev-9, and lev-10 may encode regulatory<br />
molecules that control the functional activity of nicotinic receptors, since their loss of function attenuates<br />
nicotinic receptor function without seeming to eliminate the receptors themselves.<br />
180
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SENSORY AXON GUIDANCE DEFECTS IN C. ELEGANS<br />
Susan Kirch, Gage Crump, Cori Bargmann<br />
HHMI and Department of Anatomy; UCSF; San Francisco, CA 94131<br />
<strong>Worm</strong>s depend on taste, touch and smell to sense and explore their environment. Appropriate responses<br />
to information received through these different modalities depends on precise connectivity between<br />
sensory neurons and downstream effector neurons in the nerve ring. Little is known about how axons<br />
migrate through this nerve ring neuropil during development to find their partners.<br />
How is positional information interpreted by an extending axon so that desired synaptic partners are<br />
found? We focused on the guidance of the ASI chemosensory neurons to their appropriate positions in<br />
the nerve ring. A mutant screen led to the isolation of 16 candidate mutant strains that appeared to have<br />
ASI axon guidance and termination phenotypes. These mutations define at least five new genes which<br />
we have named sax-10-14 (sensory axon guidance). We have isolated five alleles of sax-10. The<br />
canonical allele, ky297, has axon defects in several axons in the nerve ring including the chemosensory<br />
neurons AWB, ASI and ASH and the interneuron PVQ, as well as an altered expression pattern of the<br />
AWC- specific gene str-2. ky297 is normal for the VD, DD and HSN motoneurons, the chemosensory<br />
neuron ADL and the phasmids. sax-12 suffers from defects in ASI and ADL, but is normal for AWC<br />
structure. sax-13 displays abnormal axon structures when we examine ASI, ADL, AWB, ASH and PVQ,<br />
but appears wild-type for AWC. Mutants display either one or a combination of phenotypes (including:<br />
premature termination, branching, thickening, wandering and inappropriate pathfinding) with a penetrance<br />
that ranges from 13% to 100%. Dye filling of sensory neurons with the fluorescent dye DiI, and<br />
examination of neurons with other cell-specific GFP constructs in these mutants reveals that the overall<br />
morphology of the nerve ring is normal in many sax-10, sax-12, and sax-13 animals, and that the<br />
mutations disrupting ASI are probably in genes specifically required by ASI or a subset of neurons.<br />
sax-14, however, has more severe dye filling phenotypes that are being characterized further. To facilitate<br />
genetic analysis and cloning of these new sax genes, we are mapping the genes and currently trying to<br />
rescue the sax-10 phenotype by cosmid injections and germline transformation.<br />
181
ISOLATION OF A THIRD LIN-4 ALLELE FROM A LIN-3A<br />
OVEREXPRESSION LINE<br />
Martha Kirouac, Paul Sternberg<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HHMI and Division of Biology, 156-29, Caltech, Pasadena, CA 91125<br />
In order to investigate the role of LIN-3 in cell fate specification, we conducted a F2 screen using a<br />
reduction of function lin-3(n378) strain which contains an integrated transgene that carries a mutation that<br />
presumably eliminates lin-3B splicing and should, therefore, only produce lin-3A. The line carrying the<br />
integrated transgene is fertile and 100% multivulva. Animals that lack lin-3A are sterile (Jing Liu & PWS,<br />
unpubl.). To potentiate rescuing any lines that are sterile because they lack lin-3A, lfe-1 was incorporated<br />
into the strain background. lfe-1(sy290) is a gain of function allele that partially rescues the sterility<br />
caused by let-23 loss of function animals. The strain is fertile and 100% multivulva. From these screens,<br />
we looked for animals which were non-Muv and isolated two mutations.<br />
One of these mutations still displayed some induction, but displayed neither invagination nor<br />
morphogenesis. Since these animals were 100% vulvaless, we artifically created a hole connecting the<br />
outside to the uterus using a typical injection scope and needle. After obtaining cross progeny and<br />
outcrossing this mutant, it displayed characteristic heterochronic defects; extra molts, late divisions, and<br />
no adult cuticle. Having preliminarily mapped this mutation to linkage group II, we tested the hypothesis<br />
that it was a lin-4 allele by performing single worm PCR on the homozygous animals using primers to<br />
lin-4. Indeed, there was a mutation in the lin-4 gene, C516T. Though there are only two other alleles of<br />
lin-4 ,this is the second time that this mutation has been isolated (the other allele was isolated in Victor<br />
Ambros lab and is known as ma161). Lin-4 is a heterochronic retarded mutant which is critical to the<br />
controlled coordination of cell division in postembryonic divisions. What is most interesting about lin-4 is<br />
that it encodes a 22 nt. RNA form to block translation of other proteins such as lin-14 and lin-28. The fact<br />
that this allele has been isolated twice is indicative of the importance of this site in the function of the lin-4<br />
RNA. It has been hypothesized (Ha, I. and colleagues) that this C forms a bulge which is recognized by<br />
another, as of yet unknown, factor. When this C is replaced with a T, this bulge does not form, and does<br />
not inhibit the translation of lin-14 and lin-28.<br />
182
ELT-5 AND ELT-6 ARE ESSENTIAL FOR DEVELOPMENT OF<br />
SEAM CELLS, THE VULVA, AND THE MALE TAIL.<br />
Kyunghee Koh, Joel H. Rothman<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Neuroscience Research Institute, University of California, Santa Barbara, CA 93106<br />
Patterning of the embryonic epidermis into the major cell types (syncytial, P, and seam cells), and<br />
regulation of their post-embryonic development, involve a complex series of regulatory events. We<br />
previously described the role of elt-5 and -6, adjacent genes encoding GATA transcription factors, in<br />
embryonic seam cell development (WCWM, 1998). We since found that they perform a variety of<br />
essential functions in embryonic and post-embryonic ectodermal development.<br />
elt-5 and -6 show complex and dynamic expression patterns. GFP reporters express 1) in many anterior<br />
and ventral cells during embryogenesis, 2) in seam cells, 3) in a subset of sheath and socket cells, 4) at<br />
low levels in the P cells and higher levels in the descendants of the vulval precursor cells, and 5) in the<br />
developing male tail. Expression patterns of elt-5:gfp and elt-6:gfp overlap, but are not identical. ELT-5<br />
and -6 antibodies corroborate some of these observations.<br />
High levels of elt-5 dsRNA result in penetrant late embryonic or L1 lethality, in which the affected animals<br />
are lumpy, dumpy, uncoordinated, and show fusion of seam cells with epidermal syncytia. Anterior seam<br />
cells fuse more frequently than those in the posterior, suggesting that an A/P patterning system in the<br />
seam or surrounding syncytia regulates their propensity for fusion. Many, but not all seam-specific<br />
markers are eliminated in the affected animals, and elt-3, normally expressed in non-seam epidermis, is<br />
occasionally expressed in unfused seam cells.<br />
At lower doses of dsRNA, surviving vulvaless adults are observed. Examination of the vulval precursor<br />
cells shows that some inappropriately fuse with hyp7. In addition, migration of the gonad is aberrant, and<br />
rays in the male tail are often missing.<br />
Ectopic expression of elt-5 or -6 during mid-embryogenesis results in an excess of seam cells, while<br />
ectopic expression in L1s and L2s often results in adult males with missing rays and hermaphrodites that<br />
herniate through a malformed vulva.<br />
Collectively, these findings suggest that ELT-5 and -6 may collaborate with A/P patterning systems and<br />
inductive processes to regulate development of the embryonic ectoderm and post-embryonic genitalia.<br />
183
A GENETIC SCREEN FOR GENES INVOLVED IN GUT<br />
DEVELOPMENT AND DIFFERENTIATION IN<br />
CAENORHABDITIS ELEGANS<br />
Jay D. Kormish, James D. McGhee<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1<br />
Canada<br />
Our lab is currently interested in studying genes, in particular transcription factors, that direct the<br />
development and differentiation of these cells into a functioning organ. I have developed a genetic screen<br />
to isolate zygotically expressed genes that when mutated result in a gut obstructed "gob" defect. When<br />
mutagenized worms are fed a mixture of bacteria and polystyrene fluorescent microspheres, mutant<br />
worms with an obstructed gut can be identified under the dissecting microscope because of the highly<br />
fluorescent beads that collect in the terminal bulb of the pharynx and in the anterior intestine. The<br />
indication that screening for such a phenotype could isolate genes specifically involved in gut<br />
development came from our observation that mutations in a gut specific GATA transcription factor gene<br />
elt-2 resulted in the gob phenotype. I am currently in the middle of a saturation screen; five thousand<br />
genomes have already been screened and the remaining five thousand are expected to be completed in<br />
the near future. During the characterization of my initial round of candidates, I have found three strains<br />
that appear to give gut specific defects. The strain with the highest degree of penetrance, 26-3(8), has<br />
been chosen for mapping and cloning of the gene. 26-3(8) has been mapped to the X chromosome and is<br />
being tested for complementation with elt-2. Electron microscopy of lumen cross sections and gut<br />
morphology markers are being used to describe the lumen morphology of the mutants.<br />
184
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
AN E1-LIKE ACTIVATING ENZYME IS INVOLVED IN CELL<br />
DIVISION PROCESSES IN THE EARLY C. ELEGANS<br />
EMBRYO.<br />
Thimo K. Kurz, Danielle R. Hamill, Bruce Bowerman<br />
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403<br />
In a screen for temperature-sensitive embryonic lethal mutations, we isolated a mutant called or198ts that<br />
has several defects in early C. <strong>elegans</strong> embryos. In or198ts mutant embryos, the first mitotic spindle often<br />
is delayed in aligning along the anterior-posterior axis, and shortly after the spindle begins to elongate, it<br />
is no longer clearly visible by DIC videomicroscopy. In about half of or198ts mutant embryos, the first<br />
attempt at cytokinesis fails. In embryos in which cytokinesis succeeds, we observe penetrant defects in<br />
nuclear positioning and in mitotic spindle orientation at the two cell stage. In addition, in interphase partial<br />
ectopic cleavage furrows may form; there is extra pinching and blebbing at the cell membranes, and the<br />
cells are misshapen.<br />
or198ts is on LGIII at approximately --0.8 m.u., and corresponds to the Genefinder locus F11H8.1. We<br />
have identified a single base lesion in the mutant that converts a valine to alanine at amino acid position<br />
269. F11H8.1 encodes an E1-like activating enzyme with around 30% amino acid identity to its yeast and<br />
human homologs. E1-like activating enzymes are involved in conjugating ubiquitin-related proteins (e.g.<br />
SUMO-1/Smt3p) to a small subset of other cellular proteins in a manner similar, although not identical to<br />
ubiquitination. In contrast to ubiquitin, which targets proteins for degradation, post-translational<br />
modification by ubiquitin-like proteins may regulate the activity and intracellular localization of the proteins<br />
they modify. In both yeast and mammals, Smt3p/SUMO modifies multiple proteins. Perhaps the<br />
pleiotropic defects observed in or198ts embryos reflect multiple targets of this post-translational<br />
modification. To our knowledge, this is the first example of a ubiquitin-like modification being important for<br />
cell division processes in multicellular eukaryotes.<br />
185
OLFACTORY ADAPTATION<br />
Noelle L’Etoile, Cori Bargmann<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HHMI/Dept. Anatomy, UCSF, San Francisco, CA 94143<br />
To extract information from a complex environment, an organism continuously adjusts its sensitivity to<br />
different stimuli. One strategy for integrating olfactory information over time is to down-regulate odorant<br />
perception at the level of the sensory neuron. Another strategy requires a circuit in which interneurons<br />
provide feedback inhibition. I have been focusing my efforts on examining adaptation to odorants sensed<br />
by the olfactory neuron AWC, a process that may take place within the sensory neuron itself.<br />
The pair of AWC neurons allows the animal to respond to at least five different odorants. The signal<br />
transduction pathway within the AWCs includes putative seven transmembrane G-protein coupled<br />
odorant receptors, at least two heterotrimeric G-proteins, at least two guanylyl cyclases and a<br />
cGMP-gated cation channel that may open in response to cyclase stimulation. Intriguingly, adaptation to<br />
each odorant is independent of the others sensed by this pair. I have identified a mutant, pkg-1, that fails<br />
to adapt to all AWC-sensed odorants tested. I mapped the mutation and was able to obtain rescue of the<br />
phenotype with an extrachromosomal array of a cosmid containing one of the two C. <strong>elegans</strong> genes<br />
encoding cGMP-dependent protein kinases (PKGs) present in the genome.<br />
I am attempting to identify the molecular lesion by sequencing the locus that encodes the PKG in our<br />
existing allele. I am also generating additional alleles using a non-complementation screen.<br />
What is the target of PKG-1 phosphorylation in olfactory adaptation? I am taking a candidate approach to<br />
this question by mutating potential PKG phosphorylated residues within members of the signal<br />
transduction pathway. Animals that express only the mutated form of the protein should be adaptation<br />
defective if that protein’s function is regulated by phosphorylation. I hope to examine the phosphorylation<br />
state of these candidates in extracts made from both N2 and pkg-1 worms. An important conundrum<br />
remains: how does the worm utilize a shared adaptation component to specifically down regulate its<br />
response to one of five odorants sensed by the same pair of neurons? This will be addressed by<br />
identifying PKG-1’s targets, binding partners and examining their localization within the pair of AWC<br />
neurons during adaptation.<br />
186
YOU CAN’T GET THERE FROM HERE: A GENE REQUIRED<br />
FOR PHARYNGEAL EXTENSION.<br />
SK Lange, JR Saam, SE Mango<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Huntsman Cancer Institute Center for Children and Department of Oncological Sciences, University of<br />
Utah, Salt Lake City, UT 84112<br />
During pharyngeal extension, a ball of pharyngeal precursors is converted into a linear tube connected to<br />
the buccal cavity anteriorly and the midgut posteriorly. Time-lapse videomicroscopy has shown that this<br />
process depends on the reorientation of the pharyngeal epithelial cells and their adhesion to neighboring<br />
arcade cells in the buccal cavity (see abstract by Portereiko and Mango). Intriguingly, mutants that disrupt<br />
cell-cell or cell-substratum adhesion do not affect pharyngeal extension.<br />
In a survey of homozygous deficiencies, we discovered one locus, mnDf90, with defective pharyngeal<br />
extension. Nevertheless, homozygous mutant embryos form a pharyngeal primordium, differentiate<br />
normally and produce the correct number of PHA-4+ cells. This phenotype suggests that cell fate<br />
specification is unaffected and that the defect is specific for morphogenesis. We have identified three<br />
EMS-generated alleles that phenocopy the pharyngeal extension phenotype and map near mnDf90. The<br />
map position and phenotype resemble cdl-1 (Cell Death Lethal), discovered by the Yamamoto lab, and so<br />
we have provisionally called our locus cdl-1.<br />
Homozygous cdl-1 embryos arrest at the 3-fold or L1 stage with an unattached pharynx. This phenotype<br />
suggests that the locus is not required for at least two other morphogenetic processes: ventral closure<br />
and embryonic elongation. Surprisingly, two of our alleles also accumulate cell corpses. This phenotype is<br />
likely to reflect a defect in cell corpse engulfment, a process thought to depend on cytoskeletal<br />
reorganization to extend filopodia around the corpse. Since mnDf90 homozygotes do not show the cell<br />
corpse defect, one possibility is that cdl-1 is not required for engulfment of corpses, but that our cdl-1<br />
alleles produce altered proteins that interfere with normal engulfment. We suggest that cdl-1 is involved in<br />
regulation or function of the cytoskeleton.<br />
Our current goal is to determine what aspect of pharyngeal extension is disrupted by cdl-1 and whether<br />
other morphogenetic processes (e.g. migration) are also compromised. In addition, we are using standard<br />
approaches to clone cdl-1 and initiate a molecular analysis of pharyngeal extension.<br />
187
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SIGNALING BY THE VAB-1 EPH RECEPTOR<br />
INTRACELLULAR DOMAIN<br />
Kristoffer Larsen,, Sean George, Andrew Chisholm<br />
Department of Biology, University of California, Santa Cruz, CA 95064<br />
The VAB-1 Eph receptor tyrosine kinase functions in embryonic morphogenesis and axon guidance. The<br />
VAB-1 intracellular domain (ICD), like those of other Eph receptors, includes a conserved juxtamembrane<br />
motif containing phosphotyrosines, a tyrosine kinase domain, and a C-terminal region with weak similarity<br />
to SAM domains. Genetic analysis suggests that VAB-1 has both kinase-dependent and<br />
kinase-independent functions. We are using two-hybrid and genetic screens to identify components of the<br />
VAB-1 kinase-dependent pathway.<br />
The full-length VAB-1 ICD could not be used as bait in two-hybrid screens because it caused reporter<br />
gene activation in the absence of prey. After extensive subcloning we identified fragments of the VAB-1<br />
ICD that did not cause self-activation. One of these contains the complete kinase domain and C-terminal<br />
tail, and has been the basis for a pilot screen from which we identified several positive clones. We will<br />
present our analysis of these potential interactors.<br />
Several proteins have been previously identified as binding to activated Eph receptor ICDs. These include<br />
SH2-domain containing adaptor proteins (Nck, etc), and other proteins whose mode of interaction is less<br />
clear. We have begun testing C. <strong>elegans</strong> homologs to ask if these interactions have been conserved.<br />
Finally, we are using genetics to ask whether the VAB-1 kinase-dependent pathway shares components<br />
with other RTK signaling pathways characterized in C. <strong>elegans</strong>. We have found that activating mutations<br />
in LET-60 Ras do not significantly suppress vab-1 loss-of-function phenotypes, suggesting that VAB-1<br />
does not signal via Ras activation. We will present results of additional double mutant experiments.<br />
188
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MDF-1 SUPPRESSORS THAT MAY PLAY A ROLE IN THE<br />
METAPHASE TO ANAPHASE CHECKPOINT<br />
Elaine Law, Risa Kitagawa, Ann M. Rose<br />
Department of Medical Genetics, University of British Columbia, Vancouver V6T 1Z3<br />
The spindle assembly checkpoint is an evolutionarily conserved mechanism that ensures accurate<br />
chromosome segregation. This mechanism inhibits cell-cycle progression in response to a signal<br />
generated by mitotic spindle damage or by kinetochores that have not attached to microtubules prior to<br />
sister-chromatid separation. Such arrest allows cells to complete essential events for the maintenance of<br />
genetic fidelity. The spindle checkpoint mechanism has been studied in yeast and mammalian tissue<br />
culture systems, but little is known about it in multi-cellular organisms. We have identified mdf-1 and<br />
mdf-2 (mitotic arrest defective) as homologues of yeast spindle checkpoint genes MAD1 and MAD2 in<br />
<strong>Caenorhabditis</strong> <strong>elegans</strong>. Both genes are essential for the long-term survival and fertility of C. <strong>elegans</strong>.<br />
Loss of function of either gene leads to the accumulation of a variety of defects, which ultimately results in<br />
genetic lethality. To better understand how the checkpoint functions in C. <strong>elegans</strong>, and to identify<br />
additional components of the checkpoint, we screened for suppressors capable of rescuing the mdf-1<br />
(gk2) loss of function phenotype. Sixteen suppressors were recovered, which increased the number of<br />
fertile progeny and the survival of the genetic strain. The suppressors are being genetically mapped. One<br />
dominant suppressor was recovered, h1983. We have positioned this mutation and are currently<br />
examining the molecular basis for the suppression. One hypothesis, which we are now testing, is that the<br />
suppressor gene product is a downstream target of MDF-1 and acts compensatory to the spindle<br />
checkpoint defect to reduce chromosome missegregation.<br />
189
CHARACTERIZATION OF A C. ELEGANS DEFECATION<br />
MUTANT<br />
Anne Lehtela, Garry Wong<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A.I. Virtanen Institute, Kuopio University, PL 1627, Kuopio 70211, FINLAND<br />
Defecation in C. <strong>elegans</strong> is a tightly regulated and complex behavior. The defecation behavior is<br />
generated from neuronal signals and is transmitted into movement of multiple body muscles in a highly<br />
coordinated fashion. Defecation occurs in 3 steps: contraction of posterior body muscles (pBoc);<br />
contraction of anterior body muscles (aBoc); expulsion of intestinal contents (exp). The defecation cycle<br />
occurs at regular 50-60 second intervals when wildtype Bristol N2 worms are feeding.<br />
A C. <strong>elegans</strong> defecation mutant was identified in a mutagenesis screen. Six-thousand genomes were<br />
mutagenized with 50 mM ethyl methanesulfonate for 4 h. F2’s were selected for resistance to 0.7 mM<br />
aldicarb, an acetylcholine esterase inhibitor. A single mutant, which appeared to be constipated, was<br />
cloned and then outcrossed to N2.<br />
The defecation mutant, in comparison to N2 per 30 min, had fewer pBOC (15.3 ± 2 vs. 32.8 ± 1), fewer<br />
exp (6.5 ± 1 vs. 31.8 ± 0), and fewer successful complete defecation motor program events (32 ± 9% vs.<br />
97 ± 2%), respectively. When pBOC was followed by an exp event, the latency appeared longer in the<br />
mutant (8-10 sec) compared to N2 (2-3 sec). On the few occasions when the complete defecation motor<br />
program was intact in mutants, the interval was longer (80-90 sec) than N2 (55-60 sec). Genetic analysis<br />
has tentatively identified the location of the mutation on chromosome III.<br />
These results, describing a derangement of the defecation motor program, combined with identification of<br />
the gene mutation responsible for this phenotype, should provide insights into the control of complex<br />
neuronal and neuromuscular controlled behaviors.<br />
190
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ORGANOGENESIS OF THE C. ELEGANS INTESTINE<br />
Benjamin Leung 1 , Greg J. Hermann 2 , James R. Priess 3<br />
1MCB Program, University of Washington and Fred Hutchinson Cancer Research Center, Seattle, WA<br />
98109.<br />
2Fred Hutchinson Cancer Research Center, Seattle, WA 98109<br />
3MCB Program, University of Washington and Fred Hutchinson Cancer Research Center, HHMI, Seattle,<br />
WA 98109.<br />
The C. <strong>elegans</strong> intestine is a bilaterally symmetric tube of 20 polarized epithelial cells derived from a<br />
single early blastomere called E. Cell polarity in the intestine begins with reorganization of the microtubule<br />
cytoskeleton, followed by migration of the intestinal nuclei apically, and other organelles basally. Small<br />
apical membrane separations coalesce into a continuous compartment to form the lumen of the intestine.<br />
The early primordium consists of two tiers of polarized cells; specific cell intercalations rearrange the<br />
primordium into a single elongated tier.<br />
An E blastomere cultured in isolation produces a cyst of polarized cells, but the aggregate lacks the<br />
bilateral symmetry seen in the normal intestine. Thus the ability to polarize appears to be an intrinsic<br />
property of intestinal cells, but bilateral symmetry requires interactions with cells that normally surround<br />
the intestine. Cell killing experiments suggest these interactions must occur at an early stage in intestinal<br />
morphogenesis. We are currently using the technique of RNAi to determine whether genes implicated in<br />
epithelial polarity in other systems are required for proper development of the C. <strong>elegans</strong> intestine.<br />
191
EXPRESSION AND REGULATION OF DAF-16::GFP<br />
CONSTRUCTS<br />
Kui Lin, Cynthia Kenyon<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biochemistry & Biophysics, University of California at San Francisco, San Francisco, CA<br />
94143-0448<br />
Wild-type C.<strong>elegans</strong> ages rapidly, undergoing development, senescence, and death in less than 3 weeks.<br />
In contrast, mutants with reduced activity of daf-2, a homolog of the insulin and insulin-like growth factor<br />
(IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al.,<br />
1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since<br />
severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the<br />
lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the<br />
activity of daf-16, which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997).<br />
We have created GFP-tagged daf-16 constructs and used them to study the expression and regulation of<br />
daf-16. We will report our progress on this study.<br />
192
IDENTIFICATION OF NOVEL UNC-64 (SYNTAXIN) ALLELES<br />
Christine Liu, C. Michael Crowder<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO 63110-1093<br />
unc-64 encodes a homolog of vertebrate syntaxin 1A. Syntaxin is expressed ubiquitously in the nervous<br />
system of the nematode. unc-64 contains a high degree of homology with human and Drosophila<br />
syntaxin, suggesting that this molecule is conserved across species and performs similar functions.<br />
Syntaxin is involved in membrane fusion of synaptic vesicles and controls neurotransmitter release, in<br />
part through its H3 helical domain, which interacts with SNAP-25 and synaptobrevin. Very few viable<br />
syntaxin alleles have been identified in any animal, including C. <strong>elegans</strong>. In C. <strong>elegans</strong>, the null allele,<br />
js115, confers larval lethality: the nematode completes embryogenesis, but dies as L1 larva. Currently,<br />
only four viable syntaxin alleles have been identified in C. <strong>elegans</strong>: e246, md1259, js21, md130. js21 and<br />
e246 contain different C to T missense mutations in exon 7; md130 and md1259 contain G to A splice<br />
site mutations at the splice donors of intron 6 and intron 3, respectively. All these previously identified<br />
mutations affect the H3 domain of syntaxin or cause reduced expression. While all viable syntaxin alleles<br />
appear to reduce neurotransmitter release, large allelic differences are seen in their sensitivities to volatile<br />
general anesthetics: md130 is resistant to volatile anesthetics while js21 and md1259 are hypersensitive.<br />
In addition to the H3 domain, syntaxin also contains N-terminal H ABC domains and a hinge region that are<br />
also thought to regulate membrane fusion events. However, no mutations have been identified in these<br />
regions. To better define these portions of syntaxin that regulate transmitter release and anesthetic<br />
sensitivity, a non-complementation screen using EMS as the mutagen was conducted using the unc-64<br />
(e246) allele. Three potentially new unc-64 alleles have been isolated out of approximately 4800 F1<br />
genomes screened. These animals exhibit phenotypes consistent with syntaxin hypomorphic alleles,<br />
including being uncoordinated and failure on re-testing to complement e246 allele. Other experiments to<br />
characterize these putative new viable syntaxin alleles are currently in progress.<br />
193
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MUTATIONS THAT CAUSE NEURITE SPROUTING OF THE<br />
DVB MOTOR NEURON<br />
Loria, P., Boulin, T., Conte, S., Hobert, O.<br />
Columbia University, College of Physicians & Surgeons, Center for Neurobiology and Behavior, New<br />
York, NY 10032<br />
Neuroanatomical studies in vertebrates long ago revealed that certain forms of insult such as axotomy or<br />
activity blockade result in the outgrowth of additional neurites. These observations suggest that<br />
postmitotic neurons contain an intrinsic capacity to assess their integrity and to modulate their anatomy<br />
accordingly. In C.<strong>elegans</strong>, several genetic lesions have been described that lead to the outgrowth of<br />
additional neurites ("neurite sprouting")(1,2). We have found that a null mutation in the lim-6 LIM<br />
homeobox gene causes neurite sprouting in the DVB motor neuron (3). Using this observation as a<br />
starting point, we have set out to understand the molecular components of neurite sprouting and to further<br />
dissect the role of lim-6 in this process. First, we used a candidate gene approach to test mutations in<br />
proteins involved in different aspects of neuron and muscle function. Second, we performed an unbiased<br />
genetic screen for mutants that phenocopy the lim-6 sprouting defect in DVB.<br />
Since activity blockade at the neuromuscular junction in vertebrates and flies causes neurite sprouting,<br />
we tested whether loss of the neurotransmitter GABA causes sprouting of the GABAergic DVB<br />
motorneuron. We indeed find that mutations in unc-25 cause neurite sprouting of DVB. These defects can<br />
be enhanced by mutations in unc-31, which presumably abolishes peptinergic neurotransmission.<br />
Furthermore, blocking the activity of the enteric muscle targets of DVB with an egl-2(gf) mutation, which<br />
activates a K-channel (4), leads to neurite sprouting of DVB, as does the complete removal of the enteric<br />
muscles in hlh-8/twist mutants. We also find that a gain-of-function mutation in CamKII/unc-43, a gene<br />
whose role in neurite outgrowth has been described extensively in vertebrates, causes neurite sprouting<br />
in DVB.<br />
Using a clonal, fluorescence microscope-based screen of 3300 haploid genomes, we identified nine<br />
mutants, ot1 through ot9, that affect neurite sprouting of DVB. These mutants fall into at least 4<br />
complementation groups and are not allelic to any of the genes described above. The mutants do not<br />
cause sprouting in other defined sensory or interneurons tested. We also find that the as yet uncloned<br />
unc-122 gene causes specific neurite sprouting of DVB. We are in the process of mapping and cloning<br />
these genes.<br />
194
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A B -TUBULIN GENE, TBB-2, FUNCTIONS AS AN ACTIVATOR<br />
OF MEI-1 AND MEI-2 IN FEMALE MEIOTIC SPINDLE<br />
FORMATION IN CAENORHABDITIS ELEGANS.<br />
Chenggang Lu, Martin Srayko, Paul E. Mains<br />
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N<br />
2T9<br />
C. <strong>elegans</strong> female meiosis requires two meiotic-specific genes, mei-1 and mei-2. Loss of mei-1 or mei-2<br />
function blocks female meiotic spindle formation while the subsequent mitotic cleavages are normal. In a<br />
mei-1 gain-of-function mutant, however, the mitotic cleavages are disrupted after normal meiotic divisions.<br />
Wild-type MEI-1 and MEI-2 localize to the meiotic, but not mitotic spindle. 1,2 However, MEI-1(gf) and<br />
MEI-2 ectopically localize to mitotic spindles in mei-1(gf) embryos. mei-1 and mei-2 encode homologs of<br />
p60 and p80 subunits of the sea urchin microtubule severing protein katanin, respectively, and MEI-1 and<br />
MEI-2 together disassemble interphase microtubules in a HeLa cell system. 2 We propose that MEI-1 and<br />
MEI-2 form a complex in meiosis and regulate meiotic spindle formation by katanin-like activities.<br />
In a mei-1(gf) suppressor screen, we recovered three extragenic suppressors. One of them, sb26, was<br />
found to be a missense mutation in a b -tubulin gene, tbb-2. tbb-2(sb26) also enhances a weak mei-2(lf)<br />
allele. Together, these results suggest that tbb-2 is required for the function of mei-1 and mei-2. Antibody<br />
staining shows that TBB-2 is widely expressed during worm development. Neither tbb-2 nor tbb-1<br />
(another b -tubulin highly similar to tbb-2) RNAi has severe effects during early development. However,<br />
tbb-2 and tbb-1 double RNAi results in 100% dead eggs indicating that they act redundantly during<br />
embryogenesis. We are doing experiments to examine the interactions of MEI-1/MEI-2 with tbb-2 and<br />
tbb-1.<br />
1. Clark-Maguire, S. and Mains, P.E. (1994). J. Cell Biol. 126, 199-209.<br />
2. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J., and Mains, P.E. (2000). Genes Dev. 14 (9).<br />
195
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GLOBAL PROFILE OF GENE EXPRESSION DURING AGING<br />
James Lund 1 , Pamela Larsen 2 , Pat Tedesco 3 , Thomas Johnson 3 ,<br />
Stuart Kim 1<br />
1Stanford University<br />
2University of California Los Angeles<br />
3Institute for Behavioral Genetics, University of Colorado, Boulder<br />
Aging is a physiological phenomenon characteristic of metazoan life. As animals age, they suffer a broad<br />
functional decline and an exponentially increasing probability of death. Many aspects of the aging<br />
phenotype are present in animals from C. <strong>elegans</strong> to mammals, including a build-up of lipofuscin<br />
deposits, mitochondrial DNA deletions, and degradation of organ function. The life span of an animal is<br />
thought to be set by the balance between deleterious events and repair mechanisms. A number of C.<br />
<strong>elegans</strong> mutants live significantly longer than wildtype, making C. <strong>elegans</strong> an attractive model for studies<br />
of aging.<br />
DNA microarrays can be used to profile the expression levels of a large number of genes in parallel.<br />
Using our array, which contains a PCR product representing every C. <strong>elegans</strong> gene, we are profiling<br />
expression patterns during the normal aging process. We isolated RNA from synchronized cultures of<br />
sterile animals at various ages from young adult to old age. We hybridized this RNA to DNA microarrays,<br />
and are now analyzing the data to identify groups of genes with expression levels which change as<br />
reproduction ceases and the worms age. These expression profiles with be compared with the partial<br />
profiles of aging mice and humans that have been published. The expression profile of normal aging will<br />
be used as a reference to understand the expression changes underlying the extended life spans in<br />
longevity mutants.<br />
196
CONDITIONAL MUTATIONS AFFECTING MITOTIC SPINDLE<br />
POSITIONING AND POLARITY IN THE C. ELEGANS EMBRYO<br />
Rebecca Lyczak, Bruce Bowerman<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403<br />
Early polarity in the C. <strong>elegans</strong> embryo requires proper regulation of the cytoskeleton.<br />
Cytoskeletal-dependent processes such as pronuclear migration, centrosome rotation, and spindle<br />
positioning must be properly accomplished to ensure an asymmetric first cleavage. To investigate the role<br />
of the cytoskeleton in regulating asymmetric cell division, we have undertaken a screen for temperature<br />
sensitive embryonic lethal mutations affecting spindle positioning in the early embryo. We have identified<br />
mutations in two classes that affect P0 spindle positioning: 1) misorientation of the spindle axis 2)<br />
mispositioning of the spindle along the A-P axis.<br />
We have identified several mutants that show a defect in pronuclear migration and P0 spindle orientation.<br />
These include alleles of mel-26 and zyg-9 as well as at least three novel loci. In or346ts embryos, the<br />
oocyte pronucleus does not always migrate to the posterior of the embryo before initiation of the first<br />
mitotic spindle. The centrosome/nuclear complex remains in the posterior of the embryo and does not<br />
rotate. Thus, the first mitotic spindle sets up transverse to the A-P axis. The misoriented spindle results in<br />
a missegregation of P-granules and daughter cells of aberrant size and developmental potentials.<br />
Terminally differentiated or346ts embryos also show striking patterning defects, often making extra<br />
intestinal cells.<br />
We have also identified at least two mutants in which the P0 spindle is mispositioned along the A-P axis.<br />
In or282ts mutants, both pronuclei migrate toward the center of the embryo where they meet before<br />
setting up a spindle along the A-P axis. This spindle varies in its position along the A-P axis resulting in a<br />
randomization of cell size in the daughters. This mispositioning of the spindle is coupled with<br />
mislocalization of P-granules and a loss of polarity in the daughter cells. Terminally differentiated or282ts<br />
embryos show patterning defects, often making extra pharyngeal cells and lacking intestine. In or358ts<br />
embryos, the P0 spindle is displaced too far posterior resulting in an excessively asymmetric first division.<br />
Analysis of spindle positioning mutants should provide valuable insights into the link between<br />
establishment of polarity and the cytoskeleton.<br />
197
ROLE OF PDZ DOMAIN PROTEINS IN ESTABLISHING GUT<br />
EPITHELIAL POLARITY<br />
Kathleen E. Mach, Stuart K. Kim<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Developmental Biology, Stanford University, Stanford, CA 94305<br />
Epithelial cells are polarized cells that set up and maintain distinct basolateral and apical membrane<br />
domains. The PDZ protein motif, originally recognized in the postsynaptic density protein PSD-95, the<br />
Drosophila discs-large (Dlg), and the tight junction protein ZO-1, mediates protein-protein interactions and<br />
is found in many proteins having a central role in localizing proteins to the basolateral membrane domain<br />
of epithelial cells or to neuronal synapses. For example, our work has shown that three proteins with PDZ<br />
domains (LIN-2, LIN-7 and LIN-10) are involved in basolateral localization of the LET-23 EGF receptor in<br />
the vulval precursor cells. We are currently testing the hypothesis that other PDZ proteins may also be<br />
involved in epithelial cell polarity. Analysis of the C. <strong>elegans</strong> genome revealed 58 open reading frames<br />
predicted to encode PDZ domains, 51 of which represent genes that have not yet been characterized. To<br />
examine the role of each of the PDZ proteins, the loss of function phenotype each gene is being<br />
determined by RNAi. To date, the RNAi phenotype of over half the PDZ genes has been determined and<br />
several of these genes have a defects in the polarity of the gut epithelia. These phenotypes fall into 2<br />
distinct classes: 1. those where epithelial cell junctions form but cell organization is disrupted and 2. those<br />
where cell junctions fail to form or are disrupted. Two genes in the latter group are the C. <strong>elegans</strong><br />
homologues of Drosophila Dlg (dlg-1) and scribble (scb-1). Early embryonic cell divisions are normal in<br />
dlg-1(RNAi) and scb-1(RNAi) but embryos arrest at the two fold stage. MH27 antibody staining indicated<br />
that the epithelial cell junctions begin to form in these animals but are not completed leading to<br />
disconnected segments of junctional material.<br />
198
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GENETIC ANALYSIS OF NMDA RECEPTOR EXPRESSION IN<br />
C. ELEGANS<br />
David M. Madsen, Chingju Lin, Penelope J. Brockie, Andres V. Maricq<br />
Dept. of Biology, University of Utah, Salt Lake City, UT 84112<br />
NMDA-type glutamate receptors are of great interest because of their roles in synaptic plasticity and<br />
neuronal excitotoxicity. We have identified two genes, nmr-1 and nmr-2, that encode NMDA-type subunits<br />
(see abstract by J. Mellem). Using the reporter molecule GFP, we have shown that nmr-1 and nmr-2 are<br />
co-expressed in a small subset of interneurons that contribute to the locomotory control circuit (AVA,<br />
AVD, AVE and PVC). Analysis of nmr-1 deletion mutants reveals defects in the timing of locomotion.<br />
Despite their important roles in most nervous systems, we do not understand which genes are required<br />
for the expression of NMDA receptors.<br />
We performed a visual-based genetic screen for mutations that affect the development and differentiation<br />
of NMDA expressing interneurons. We mutagenized a transgenic strain that expresses GFP under the<br />
control of the nmr-1 promoter and screened ~20,000 haploid genomes for aberrant GFP expression. One<br />
class of mutants showed a marked decrease of GFP expression in the PVC neuron. This neuron is still<br />
present, suggesting that the mutation affects steps late in differentiation.<br />
We identified this mutation as a nonsense mutation in ceh-14, a LIM-homeobox gene. Based on GFP<br />
reporter constructs, ceh-14 expression is observed in several neurons, including PVC. In the ceh-14<br />
mutant, expression of other glutamate receptor subunits (glr-1, glr-2, and nmr-2) is also decreased or<br />
absent in PVC. We are currently testing for cell autonomous rescue using PVC specific promoters to drive<br />
expression of ceh-14. Besides the defects in glutamate receptor expression, ceh-14 mutants exhibit a<br />
mechanosensory defect. We hypothesize that this is due to diminished or loss of PVC function in the<br />
locomotory control circuit. In addition, ceh-14 animals also exhibit a thermal avoidance defect (Tav) and a<br />
phasmid dye-filling defect. Introduction of a wild-type copy of ceh-14 into transgenic worms rescued the<br />
mutant phenotypes.<br />
199
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
LARGE SCALE REVERSE GENETIC APPROACH USING RNAI<br />
Sarah Mahoney, Alex Phan, Mark Maxwell, Candace Swimmer,<br />
Jonathan Heller, Brett Milash, Kate McKusick, Monique Nicoll<br />
Exelixis, Inc. 170 Harbor Way, S. San Francisco, CA 94083-0511<br />
One of our primary missions at Exelixis is the discovery of new drug targets for human pharmaceutical or<br />
agrochemical research through the use of model organism genetics. To rapidly survey for genes that<br />
function in disease-related pathways, we have undertaken several reverse genetic strategies, including<br />
development of a genome wide RNAi library. First we data mine, which involves compiling predicted<br />
cDNAs from GeneFinder in a database and sorting and categorizing them by predicted protein motifs.<br />
Next, we employ an automated primer picking program created at Exelixis to generate primer pairs<br />
against portions of the predicted coding sequence. PCR is then preformed on cDNA and the products are<br />
cloned into a modified version of pCCM113 (kindly provided by C. Mello). Individual clones are<br />
sequenced in an automated fashion and clones are validated by automatic blast via an inhouse database.<br />
This has allowed us to correlate GeneFinder predictions with expression data. In our initial examination of<br />
more than 1000 PCR reactions using cDNA as the template, we have observed that approximately 85 %<br />
of the predicted genes are expressed.<br />
In parallel to automating the cloning process, we are assessing the effectiveness of dsRNA delivery<br />
methods. We initiated these experiments with a set of 30 genes and have compared injection, soaking,<br />
and feeding. In addition, we have tested several parameters, including concentration dependence,<br />
pooling effectiveness, and tissue specificity of RNAi. Our results indicate that RNAi is concentration<br />
dependent, titratable, and that pooling of up to three dsRNAs gives us phenotypic results that are<br />
comparable to RNAi of each gene individually. Initial RNAi soaking results from more than 100 predicted<br />
genes suggests that a quarter of predicted genes have highly penetrant RNAi phenotypes in wild type<br />
worms by dissection scope analysis.<br />
200
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
SEQUENCE CONFIRMATION OF 182 SNPS BETWEEN C.<br />
ELEGANS N2 AND CB4856 STRAINS AND PLANS FOR<br />
GENERATION OF 1000 NEW SNPS.<br />
Penny Mapa, Kathryn Swan, Mike Ellis<br />
Exelixis Inc., 170 Harbor Way, South San Francisco, CA 94080<br />
Exelixis Inc. is a leader in model systems genetics and comparative genomics. As part of our C. <strong>elegans</strong><br />
mapping and cloning effort we have sequence confirmed 182 snps, evenly spaced throughout the<br />
genome, between the N2 and CB4856 (Hawaiian) strains. These snps are a subset of the 1416 potential<br />
snps initially identified by the Genome Sequencing center at the Washington University School of<br />
Medicine (http://genome.wustl.edu/gsc/CEpolymorph/snp_chrom.shtml). We have successfully used<br />
these genome wide, confirmed snps to map mutants. Additionally, to aid in fine scale mapping, we have<br />
begun efforts aimed at identifying another 1000 new genome wide snps between these same strains. As<br />
a service to the worm community we plan to release this information to the public.<br />
201
BUILDING A DICTIONARY FOR C. ELEGANS PROMOTER<br />
SEQUENCES<br />
Steven McCarroll, Hao Li, Cori Bargmann<br />
U.C. San Francisco<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
We would like to understand how regulatory sequences encode the expression patterns of genes. The<br />
availability of complete genomic sequence allows us to try to identify candidate transcriptional control<br />
sequences based on statistical criteria. We have used a dictionary algorithm (Bussemaker, Li, and Siggia,<br />
manuscript in preparation) to partition C. <strong>elegans</strong> promoter sequences into "words" -- discrete sequences<br />
that are distributed statistically as if they represent a coherent functional unit. We have built a dictionary<br />
for the upstream sequences of 850 C. <strong>elegans</strong> G-protein-coupled-receptor genes. When this dictionary is<br />
used to partition these promoter sequences according to maximum-likelihood criteria, 2.1% of the<br />
sequence is covered by words of length 8 or greater. Many of these words appear interesting according to<br />
multiple criteria: (i) non-Poisson distribution across genes, suggesting a tendency to appear in clusters;<br />
(ii) non-random distribution across positions, suggesting a preference for particular locations relative to<br />
the transcriptional start site, and (iii) appearance in genes that have similar expression patterns. We are<br />
using this dictionary in a number of ways:<br />
(i) We have correlated the expression patterns of several dozen chemoreceptor genes to the distributions<br />
of words across these genes, generating testable hypotheses about sequences that may confer<br />
cell-specific gene regulation;<br />
(ii) We are correlating gene-expression microarray data with the distribution of words across genes, to<br />
identify words that correspond to genes whose expression is modulated in particular experiments;<br />
(iii) We are testing candidate control sequences (words) in promoter-gfp fusion experiments, in which a<br />
particular word is deleted from a promoter (to test the necessity of this sequence for wild-type gene<br />
regulation) or added to a promoter (to test its sufficiency for conferring gene regulation).<br />
202
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HIGH PRESSURE FREEZING METHODS FOR C. ELEGANS<br />
EMBRYO ULTRASTRUCTURE AND EM IMMUNOLABELING<br />
Kent L. McDonald 1 , Thomas Mueller-Reichert 2 , Akiko Tagawa 3 , Chad<br />
A. Rappleye 3 , Raffi Aroian 3<br />
1Electron Microscope Lab, UC Berkeley<br />
2Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany<br />
3Dept. of Biology, UC San Diego<br />
Electron microscopic analysis of C. <strong>elegans</strong> has a long and distinguished history, from the early studies<br />
reconstructing the nervous system to recent work by David Hall and his colleagues. However, attempts at<br />
more routine EM studies of C. <strong>elegans</strong> have been hampered by the impermeability of the cuticle of the<br />
worm and of the eggshell of embryos. These structures act as diffusion barriers, preventing the rapid<br />
exchange of fixatives and other solutions that are used in typical EM processing protocols.<br />
Our EM facility specializes in an alternative to conventional EM, high pressure freezing (HPF). In this<br />
technique, 50 or more nematodes at a time are frozen under 2100 bars pressure in 10-20 milliseconds.<br />
This method is useful for excellent preservation of ultrastructure to depths of 200 microns or more,<br />
compared to 10 microns or so for most other freezing methods. Furthermore, this preservation is<br />
independent of the impermeability of the eggshell or cuticle. HPF is particularly exciting for studying early<br />
embryogenesis since the embryos inside hermaphrodites are well preserved. Thus, within each gravid<br />
adult are a row of early embryos of various stages making it much easier than before to fix, find, orient,<br />
and work with embryos. We are currently using this technique for studying early embryogenesis. Recent<br />
work has suggested a new class of embryonic mutants important for early polarity, the polarity osmotic<br />
mutants (see abstracts by Tagawa et al and by Rappleye et al). Our models predict that these genes are<br />
involved in membrane trafficking. We are in the process of testing this at the ultrastructural level by<br />
looking at the general morphology of the mutants and by studying the immunolocalization of one of the<br />
proteins. We will present information of our technique of preservation, fixation, and embedding and some<br />
preliminary results in the early embryo.<br />
203
MOLECULAR IDENTIFICATION OF TRANSCRIPTIONAL<br />
TARGETS OF THE DAF-16 WINGED HELIX TRANSCRIPTION<br />
FACTOR<br />
Joshua J. McElwee 1 , James H. Thomas 1,2<br />
1Program in Molecular and Cellular Biology<br />
2Department of Genetics, University of Washington<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dauer arrest and longevity in C. <strong>elegans</strong> are controlled by an insulin-like signaling pathway transduced by<br />
the winged helix transcription factor DAF-16. Mutations in several genes within this pathway (daf-2, age-1,<br />
and pdk-1) result in constitutive dauer formation (Daf-c) and increased lifespan (Age) phenotypes.<br />
Invariably, both the Daf-c and Age phenotypes of these mutants are suppressed by loss of function<br />
mutations in daf-16. This suggests DAF-16 acts as the principal transcriptional output controlling both<br />
diapause and lifespan governed by this pathway. We are attempting to identify both direct and indirect<br />
transcriptional targets of DAF-16 utilizing several complementary molecular approaches. First, we are<br />
performing in vitro binding site selection to isolate the DAF-16 DNA consensus sequence. Utilizing this<br />
sequence, we will identify putative DAF-16 targets by genomic analysis and a candidate gene approach.<br />
Second, we are performing direct selection for genomic sequences that are bound by this transcription<br />
factor. Third, we are examining DAF-16 transcriptional outputs in vivo. Using cDNA arrays, we will assess<br />
global RNA expression in various Daf mutants and double mutants. Presumably, these experiments will<br />
allow us to begin to identify genes downstream of daf-16 involved in both dauer formation and regulating<br />
lifespan.<br />
204
FUNCTIONAL CONSERVATION OF C. ELEGANS UNC-30 AND<br />
MOUSE PITX2 IN GABAERGIC NEURON SPECIFICATION<br />
Jason McEwen, Yishi Jin<br />
Department of Biology, University of California, Santa Cruz, CA. 95064<br />
The C. <strong>elegans</strong> GABAergic nervous system contains 26 neurons of 5 types. The homeodomain protein<br />
UNC-30 is required for the specification and function of the type-D motor neurons. In unc-30 mutants the<br />
D neurons fail to express GABA, have axon guidance errors, form improper synapses and shrink on<br />
themselves while trying to move backwards. It has previously been shown that UNC-30 directly activates<br />
the expression of unc-25, the glutamic acid decarboxylase enzyme (GAD) and unc-47, the GABA<br />
vesicular transporter, in these neurons (1).<br />
The UNC-30 homeodomain is over 80% identical to those of the newly identified vertebrate Pitx family<br />
and dPtx in Drosophila (2). Pitx proteins are expressed in many tissues and have been implicated in a<br />
variety of developmental functions (2). Pitx2 is also widely expressed in the developing and adult central<br />
nervous system, but its function there is not known. UNC-30 and Pitx2 can activate mammalian GAD67<br />
expression in cultured neurons (Condie B, Pers. Com.), suggesting that Pitx2 and UNC-30 may have an<br />
evolutionarily conserved role in GABAergic neuron specification.<br />
We have shown that mouse Pitx2 can functionally replace UNC-30 in unc-30 mutant worms. Site-directed<br />
mutagenesis experiments on the UNC-30 binding sites in the unc-25 promoter and the Pitx2<br />
homeodomain support that Pitx2 mediated unc-25 activation depends on a homeodomain/UNC-30<br />
binding site interaction. Like UNC-30, ectopic expression of Pitx2 under the control of heat shock<br />
promoters activates unc-25 expression in other tissues (3). Our data supports that UNC-30 and Pitx2 are<br />
likely functional homologues.<br />
UNC-30 also regulates the expression of genes required for axon guidance and synapse formation but<br />
these target genes have yet to be found. To address this we have generated modified UNC-30 proteins<br />
using the activation domain of VP16 and the repressor domain of Engrailed. The modified UNC-30<br />
proteins have shown corresponding changes in transgenic studies using the Punc-25::GFP reporter.<br />
These constructs will be used to sensitize future screens for UNC-30 target genes.<br />
References:<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1.<br />
2. Eastman et al. (1999) J. Neuroscience 19(15), 6225-6234<br />
3. Gage et al.(1999) Mamm. Gen. 10, 197-200<br />
4. Jin et al. (1994) Nature Vol 372, 780-783<br />
205
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GENES INVOLVED IN NICOTINIC NEUROTRANSMISSION IN<br />
THE PHARYNX<br />
Jim McKay 1,2 , David Raizen 3,4 , Leon Avery 1,5<br />
1Department of Molecular Biology. University of Texas Southwestern Medical Center. Dallas, Texas<br />
75390-6148<br />
2jim@eatworms.swmed.edu<br />
3Department of Neurology. University of Pennsylvania School of Medicine. Philadelphia, Pennsylvania<br />
19104<br />
4raizen@mail.med.upenn.edu<br />
5leon@eatworms.swmed.edu<br />
MC is the main excitatory motorneuron of the pharynx and is required for the large increase in pumping<br />
rate in response to food. We identified eat-2 and eat-18 in genetic screens for worms incapable of rapid<br />
pharyngeal pumping. Both eat-2 and eat-18 lack MC neurotransmission but have no other obvious<br />
defects. eat-2 encodes a non-alpha nicotinic receptor subunit. It is expressed in pharyngeal muscle and is<br />
localized to a region where we think the MC synapse is. We conclude that MC is cholinergic and acts<br />
directly on pharyngeal muscle to stimulate pumping. We observed allele specific genetic interaction<br />
between eat-2 and eat-18 indicating that the gene products physically interact. In an eat-18 mutant<br />
background, EAT-2 is expressed and correctly localized but the channel is not functional. We are<br />
attempting to clone eat-18 by transformation rescue. We have narrowed down to a 3 kb piece of genomic<br />
DNA that can rescue eat-18 mutants and we are sequencing this region from two eat-18 mutant alleles to<br />
help identify the coding region.<br />
206
GENETIC ANALYSIS OF THE FUNCTIONS OF A<br />
GSK-3&SZLIG; HOMOLOG CALLED SGG-1 AND A<br />
&SZLIG;-TRCP/SLIMB HOMOLOG DURING C. ELEGANS<br />
EMBRYOGENESIS<br />
Marc Meneghini 1 , Greg Ellis 1 , Ann Schlesinger 2 , Bruce Bowerman 1<br />
1Institute of Molecular Biology, University of Oregon, Eugene OR, 97403<br />
2Whitehead Institute, Cambridge MA<br />
During C. <strong>elegans</strong> embryogenesis, a four cell stage blastomere called P2 uses Wnt signaling to induce<br />
anterior-posterior polarity in its sister blastomere EMS. In embryos defective for the function of Wnt<br />
pathway components the posterior EMS daughter E, which normally produces endoderm, develops like<br />
its anterior sister MS. We have previously reported the characterization of a C. <strong>elegans</strong> GSK-3ß homolog<br />
called sgg-11, which shares this phenotype suggesting that sgg-1 acts positively with the Wnt pathway to<br />
regulate a-p polarity in EMS. This was somewhat surprising because in other systems, GSK-3ß functions<br />
to repress Wnt pathway outputs and is negatively regulated by Wnt signaling to relieve this repression.<br />
We also reported that sgg-1 mutant embryos produce an ectopic E-like cell that is derived from C, a<br />
daughter of P2. Further analysis has shown that the source of this ectopic endoderm is Cp, the posterior<br />
daughter of C. Double mutant analysis shows that Wnt pathway components also are required for the<br />
production of Cp-derived endoderm in sgg-1 embryos. This result suggests that Wnt signaling functions to<br />
make Cp different from Ca and that sgg-1 is required in Cp to prevent it from adopting an E-like fate.<br />
Furthermore, sgg-1 synergizes with the Wnt pathway in EMS indicating that sgg-1 and the Wnt pathway<br />
have some non-overlapping functions. Rather than functioning with the Wnt pathway during polarity<br />
induction, perhaps sgg-1 functions at the level of blastomere identity to make Cp and E, or perhaps EMS<br />
and C, different from each other. Like the Wnt pathway, sgg-1 is required for the orientation of the mitotic<br />
spindle in EMS1 suggesting an overlap with the Wnt pathway during induced polarity. We are also<br />
studying the function of a ß-TRCP/slimb related gene which we call slm-1. ß-TRCP/slimb related proteins<br />
are known to function with GSK-3ß to target proteins for proteolytic degradation. Interestingly, slm-1<br />
embryos have many phenotypic similarities with sgg-1 embryos. We are performing more phenotypic<br />
analyses to better understand the functions of sgg-1 and slm-1 during C. <strong>elegans</strong> embryogenesis.<br />
1 Schlesinger et al., Genes & Dev. (1999)<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
207
THE EFFECT OF NONIMMOBILIZERS ON C. ELEGANS<br />
Laura B. Metz 1 , Mike Crowder 1,2<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1Department of Anesthesiology Washington University, St. Louis, MO<br />
2Department of Molecular Biology/Pharmacology, Washington University, St. Louis, MO<br />
The mechanism of action of volatile anesthetics (VAs) is still uncertain. The Meyer - Overton hypothesis<br />
tries to correlate anesthetic potency directly to lipid solubility. Yet this theory does not explain why some<br />
lipophilic compounds in many homologous series of anesthetics are devoid of any anesthetic potency. F6<br />
and F8, so called nonimmobilizers, are two such compounds that should have anesthetic action but don’t.<br />
Here, we test whether C. <strong>elegans</strong> is also unaffected by nonimmobilizers and whether mutants<br />
hypersensitive to VAs are sensitized to nonimmobilizers. Behavioral assays including locomotion and<br />
mating assays were all performed with concentrations of F6 and F8 at 3-4 times the EC 50 (concentration<br />
when effect should be half-maximal) predicted by the Meyer-Overton hypothesis. The nonimmobilizers did<br />
not significantly effect N2 in any behavioral assay, even after 24-hr exposure. Strains extremely<br />
hypersensitive to bona fide VAs were tested with F6 and F8. At 4 times the EC 50, F8 had a slight effect<br />
on ric-4(js20) while F6 showed none. Both F6 and F8 affected unc-64(js21) at those same high<br />
concentrations. These data suggest that reduced neurotransmission somehow sensitize animals to these<br />
drugs. To test whether F6 and F8 alter cholinergic neurotransmission as has been shown for true VAs, we<br />
measured the effect of F6 and F8 on aldicarb sensitivity (which correlates with the level of cholinergic<br />
neurotransmission). Neither F6 nor F8 had an effect on aldicarb sensitivity, and neither antagonized<br />
VA-induced aldicarb resistance. Thus, as in vertebrate models, nonimmobilizers do not affect the<br />
behavior of the wildtype C. <strong>elegans</strong>; however, the drugs do have some effect when synaptic transmitter<br />
release is reduced.<br />
208
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ISOLATION AND CHARACTERIZATION OF MUTATIONS<br />
THAT ENHANCE LET-23(SA62GF) DURING VULVAL<br />
DEVELOPMENT<br />
Nadeem Moghal, Paul W. Sternberg<br />
Dept. of Biology, California Institute of Technology. Pasadena, CA 91125<br />
In C. <strong>elegans</strong> hermaphrodites, adoption of vulval fates by P5.p-P7.p is dependent on activation of the<br />
LIN-3/LET-23 signaling pathway. This pathway is structurally and functionally related to EGF receptor/Ras<br />
signaling pathways described in other systems. Although the major positive effectors of this pathway<br />
including EGF(lin-3), the EGFR(let-23), GRB2(sem-5), SOS(let-341), Raf(lin-45), MEK(mek-2), and<br />
MAPK(sur-1/mpk-1) have been identified, little is known regarding modulation or negative regulation of<br />
this pathway in C. <strong>elegans</strong> or in any system.<br />
To identify modulators and negative regulators of this pathway, a genetic screen was undertaken using<br />
an allele of let-23, sa62, that encodes a ligand-independent activated receptor. In wildtype<br />
hermaphrodites or animals harboring one copy of sa62, three Pn.p cells adopt vulval fates. However, in<br />
the presence of two copies of sa62, on average, four cells acquire vulval fates. sa62 heterozygous<br />
animals were mutagenized and screened for mutants with enhanced vulval induction. To date, at least<br />
seven different loci have been isolated, and six have been assigned linkage to specific chromosomes.<br />
The best characterized mutation, sy598, also enhances the activity of a reduction in function allele of<br />
let-23, sy1, but has no effect on its own or in sensitized backgrounds harboring loss of function mutations<br />
in the negative regulators sli-1 or gap-1, or a gain of function mutation in let-60. Laser microsurgery<br />
experiments indicate sy598 does not regulate the production of LIN-3(EGF) in the somatic gonad. These<br />
data suggest that sy598 may control LET-23 levels in the Pn.p cells. sy598 has been mapped to linkage<br />
group IV, and its identity is being sought.<br />
209
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE TRAMPOLINE ASSAY: A NEW METHOD FOR<br />
MEASURING THE STEP RESPONSE OF THE CHEMOTAXIS<br />
MECHANISM IN C. ELEGANS.<br />
Moravec, M.L, Cervantes, J., Lockery, S.R.<br />
Institute of Neurosci., Univ of Oregon, Eugene, OR 97403<br />
Chemotaxis in C. <strong>elegans</strong> involves a series of abrupt turns (pirouettes) triggered by movement down a<br />
gradient of chemical attractant (Pierce-Shimomura, J.T., et al.,J. Neurosci. 19:9557-9569, 1999). Analysis<br />
of the time series of concentration change experienced by a chemotaxing worm, together with its<br />
pirouette record, suggests a three-stage model in which instantaneous attractant concentration is<br />
differentiated, smoothed by low-pass filter, and thresholded by a sigmoidal function relating filter output to<br />
pirouette probability. This model predicts that a sudden decrease in attractant concentration will produce<br />
a sudden increase in pirouette probability. Moreover, the increase in probability should decay<br />
approximately exponentially with a time constant that reflects the worm’s memory for concentration<br />
changes in the recent past. To test these predictions, we have devised an apparatus that allows us to<br />
stimulate an unteathered worm with a nearly instantaneous (step-wise) change in the concentration of<br />
soluable attractants such as NaCl. The apparatus consists of a thin (10 mm) agarose film suspended over<br />
a buffer-filled chamber, resembling trampoline placed over a swimming pool. The underside of the<br />
agarose film contacts the surface of the buffer solution, while the top side of the film contacts the air. The<br />
worm is placed on the top of the film and allowed to adapt to a buffer containing a high concentration of<br />
attractant for 5 min. The chamber is then drained and quickly refilled with buffer containing a low<br />
concentration of attractant. Preliminary results indicate that a step-wise decrease in attractant<br />
concentration causes an immediate increase in pirouette probability, as predicted by the model. We plan<br />
to use the step response to investigate the time course of the worm’s memory for concentration changes,<br />
and how this memory is affected by mutations and neuronal ablations. Supported by NIMH MH51383,<br />
and NSF IBN9458102,<br />
210
IDENTIFICATION OF GENES REGULATING BODY LENGTH IN<br />
THE DBL-1 PATHWAY BY DIFFERENTIAL HYBRIDIZATION<br />
OF ARRAYED CDNAS<br />
Kiyokazu Morita 1 , Makoto Mochii 1 , Yukiko Sugihara 1 , Satoru<br />
Yoshida 1 , Yo Suzuki 2 , William B. Wood 2 , Yuji Kohara 3 , Naoto Ueno 1<br />
1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology,<br />
Okazaki 444-8585, JAPAN<br />
2 Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, USA<br />
3 National Institute of Genetics, Mishima 411-8540, JAPAN<br />
dbl-1 has been shown to regulate C. <strong>elegans</strong> body length and male tail ray patterning 1, 2). To identify<br />
downstream target genes of DBL-1 signaling, we screened arrayed cDNAs using differential hybridization<br />
(HDF) analysis. C. <strong>elegans</strong> cDNAs representing 7,584 independent genes were arrayed on a nylon<br />
membrane at a high density and hybridized with 33P-labeled DNA probes synthesized from mRNAs,<br />
which were isolated from L3-stage worms. We compared signals from dbl-1(-) worms with those from N2<br />
worms3), dbl-1(++) worms that carry multiple copies of a dbl-1 genomic fragment, or dbl-1(-); HSP::dbl-1<br />
worms that express DBL-1 protein upon heat shock. Many genes positively or negatively regulated by the<br />
dbl-1 signal were identified. dsRNAi experiments were performed with all the clones identified. Several<br />
clones caused Sma or Lon phenotypes by dsRNAi. These results demonstrate that analysis with arrayed<br />
cDNA combined with dsRNAi gene inactivation is a powerful approach to identify genes that are directly<br />
or indirectly regulated by an extracellular signal.<br />
1)K. Morita et al., (1999) Development 126, 1337-1347<br />
2)Y. Suzuki et al., (1999) Development 126, 241-250<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
3)M. Mochii et al., (1999) Proc. Natl. Acad. Sci. USA 96, 15020-15025<br />
211
MUTATIONS IN THE EPHRIN MAB-26/EFN-4 CAUSE<br />
DEFECTS IN CLOSURE OF THE GASTRULATION CLEFT AND<br />
IN EPIDERMAL ENCLOSURE<br />
Sarah L. Moseley, Andrew Chisholm<br />
Department of Biology, UC Santa Cruz, CA 95064<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Signaling involving the Eph receptor tyrosine kinase VAB-1 and its ephrin ligand VAB-2/EFN-1 is required<br />
for normal epidermal and neuronal morphogenesis. Our lab has shown that mab-26, identified by virtue of<br />
its role in male tail morphogenesis, corresponds to another C. <strong>elegans</strong> ephrin ligand, efn-4 (see abstract<br />
by Holcomb et al.).<br />
mab-26/efn-4 null mutants display incompletely penetrant defects in epidermal morphogenesis.<br />
Approximately 10% arrest during embryogenesis, and about 20% arrest as larvae. Surviving mab-26<br />
adults do not display the head morphology defects (’Notched head’) characteristic of vab-1 or vab-2<br />
mutants, but are instead typically defective in morphogenesis of the posterior body and tail epidermis.<br />
Using 4-D microscopy we have found that mab-26 embryos, like vab-1 and vab-2 mutants, are defective<br />
in the closure of the ventral gastrulation cleft by neuroblast movements, and in later enclosure of the<br />
embryo by epidermal cells. Interestingly, almost all mab-26 embryos show delays in gastrulation cleft<br />
closure, although in only some cases does this result in later morphogenetic defects. Thus, the defects of<br />
mab-26 mutants are different from (although partly overlapping with) those of vab-1 and vab-2 mutants.<br />
mab-26 mutations also display unexpected synthetic-lethal interactions with all vab-1 and vab-2 mutations<br />
tested. Double mutants show completely penetrant morphogenetic defects, in striking contrast to the<br />
incompletely penetrant vab-1 null phenotype. We are currently analyzing the phenotypes of these double<br />
mutants.<br />
The differences in mutant phenotypes and the synthetic lethal interactions are inconsistent with EFN-4<br />
acting only as a ligand for VAB-1. If EFN-4 is not signaling through the VAB-1 receptor, how is it<br />
functioning? To identify other genes required for mab-26 function we are screening for new mutations that<br />
are synthetic-lethal with a weak vab-1 allele. Results of a pilot screen will be presented.<br />
212
CELLULAR AND DEVELOPMENTAL EVENTS REQUIRED TO<br />
GENERATE FUNCTIONAL MUSCLE IN C. ELEGANS.<br />
K. Norman, S. Cordes, G. Mullen, P. Rahmani, T. Rogalski, D.<br />
Moerman<br />
Dept of Zoology, UBC<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
In C. <strong>elegans</strong> a physical linkage between the myofilament lattice of the body wall muscle and the cuticle is<br />
required to allow myofilament contraction to result in the movement of the animal. This complex linkage<br />
system is composed of integrin containing adhesion-complexes within the body wall muscle cells that<br />
anchor the myofibrils to the muscle cell membrane, a specialized basement membrane underlying the<br />
muscle quadrant, and hemidesmosomal-like structures present in the epidermis. The epidermis has<br />
specialized faces, one to anchor the epidermis to the basement membrane and the other to secrete and<br />
anchor the cuticle. The nematode provides a very elegant system to study the interaction between the<br />
body wall muscle and the epidermis during development. Through genetic and molecular approaches we<br />
have identified (1) several components involved in assembly, localization, and maintenance of adhesion<br />
complexes within muscle (the unc-52, unc-97, unc-112, dim-1 and spc-1 gene products), (2) components<br />
that are involved in structuring the underlying basement membranes (the let-268 gene product), and (3)<br />
others that function in regulating epidermal attachment structures (the unc-23 gene product). The role of<br />
these molecules in muscle and epidermal development during C. <strong>elegans</strong> morphogenesis will be<br />
described. For more information on unc-23 and unc-97 see posters by P. Rahmani and S. Cordes,<br />
respectively.<br />
213
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
IS THE DAG KINASE DGK-1 AN EFFECTOR OF GO ALPHA<br />
(GOA-1)?<br />
Stephen Nurrish, Michael Dybbs, Joshua Kaplan<br />
Molecular and Cell Biology, University of California, Berkeley, CA 94720<br />
We (and others) have previously described two competing G-protein pathways acting in motor neurons to<br />
either facilitate or inhibit synaptic transmission at neuromuscular junctions (NMJs) [1-4]. Facilitation of<br />
release occurs via a pathway composed of a Gq alpha (EGL-30), a phospholipase C beta (EGL-8), and<br />
the diacylglycerol (DAG) binding protein UNC-13 [2,4]. The Gq alpha pathway can be activated by<br />
muscarinic agonists leading to the formation of the membrane bound second messenger DAG by EGL-8<br />
and the subsequent recruitment of UNC-13 to sites of Acetylcholine release [2]. UNC-13 is essential for<br />
neurotransmitter release and it’s enrichment at release sites correlates with an increase in<br />
neurotransmitter release. Addition of serotonin agonists inhibits acetylcholine release at NMJs. Inhibition<br />
of synaptic transmission by serotonin requires Go alpha (GOA-1) and a diacylglycerol kinase (DGK-1)<br />
which phosphorylates DAG to Phosphatididic Acid which is unable to bind UNC-13. Serotonin antagonists<br />
and goa-1 mutations result in the recruitment of UNC-13 to NMJs suggesting that the Gq and Go<br />
pathways converge to regulate UNC-13 localization, most likely by regulating levels of DAG [1,2]. As yet<br />
the Go signaling pathway is not well defined. Go may reduce DAG levels by activating DGK-1, by<br />
negatively regulating the Gq signaling pathway, or by an as yet undefined pathway. We are currently<br />
testing the first of these models. Co-expression of DGK-1 and activated Go alpha in human tissue culture<br />
cells (HEK cells) has no effect on the kinase activity of DGK-1. This suggests that there is no direct<br />
interaction between Go alpha and DGK-1. However, it is possible that Go alpha indirectly regulates<br />
DGK-1 via intermediates that are not conserved in HEK cells. Therefore we have generated a rescuing<br />
MYC tagged GFP::DGK-1 construct. The myc-DGK-1 protein can be immunoprecipitated from adult<br />
animals and possesses DAG kinase activity. We are currently testing whether the DGK-1 DAG kinase<br />
activity changes in response to levels of Go alpha signaling. We are also testing whether Go regulates the<br />
subcellular localization of DGK-1.<br />
1.<br />
2. Nurrish et al.,(1999) Neuron:24 p231-242<br />
3. Lackner et al.,(1999) Neuron:24 p335-346<br />
4. Hajdu-Cronin et al.,(1999) Genes Dev 13: 1780-93<br />
5. Miller et al.,(1999) Neuron 24: 323-33<br />
214
TRANSFORMING NEMATODES INTO INSECTS:<br />
UNDERSTANDING BT-RESISTANCE<br />
Johanna O’Dell, Raffi Aroian<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
University of California, San Diego, La Jolla, CA 92093<br />
The utilization of transgenic crops expressing Bacillus thuringiensis (Bt) toxins is becoming a prevalent<br />
alternative to chemical pesticides. Bt toxins have enormous potential as they are not harmful to<br />
vertebrates, demonstrate a high degree of species-specific toxicity, and are a more environmentally<br />
friendly pest control agent. Currently genetically engineered corn and cotton are being extensively used in<br />
agriculture and express highly similar toxins. It is predicted that resistance to these toxins will develop<br />
within the next 10 years.<br />
We are interested in identifying the cellular machinery underlying Bt-toxicity and resistance and are using<br />
the nematode <strong>Caenorhabditis</strong> <strong>elegans</strong> as a model system. The physiological mode of Bt action has been<br />
elucidated from insect studies. Following ingestion by a susceptible animal, Bt toxin is solubilized and<br />
activated by proteolytic processing. Active toxin interacts with receptors in the gut and becomes inserted<br />
into the apical membrane creating a channel, ultimately leading to death. Despite our knowledge of this<br />
process, we know very little about the molecular components required for toxin action.<br />
Our laboratory has shown that C. <strong>elegans</strong> is susceptible to some Bt toxins and has successfully isolated<br />
mutants resistant to one Bt toxin (see abstract by Marroquin et al). One of these mutants, called bre-2 for<br />
Bacillus toxin resistant mutant, maps to the right arm of chromosome III. Three-factor mapping<br />
experiments position bre-2 very close to dpy-18. We are performing transgenic rescue experiments and<br />
additional mapping by polymorphisms to ascertain the molecular identity of this gene.<br />
To determine whether our studies are directly applicable to understanding pest resistance to transgenic<br />
crops we are attempting to make a C. <strong>elegans</strong> strain susceptible to Cry1Ac, the major Bt toxin used in<br />
transgenic cotton. As toxin specificity is thought to result from binding to species-specific receptors, our<br />
strategy involves the creation of transgenic worms that express the known Cry1Ac receptor,<br />
aminopeptidase N (APN). If APN-expressing worms prove to be sensitive to Cry1Ac toxin, we will<br />
determine whether the bre genes are required for a common pathway of Bt toxicity or for a Cry5B-specific<br />
pathway.<br />
215
THE CYTOSKELETAL PROTEIN ZK370.3 MAY CONTRIBUTE<br />
TO OOCYTE DEVELOPMENT AND FERTILIZATION<br />
Alex Parker, Ann M. Rose<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Medical Genetics, University of British Columbia, 6174 University Blvd., Vancouver, B.C.,<br />
V6T 1Z3, Canada<br />
The cytoskeleton is a dynamic structure that facilitates many events within a cell. We are using the model<br />
organism <strong>Caenorhabditis</strong> <strong>elegans</strong> to study a cytoskeletal gene in the context of animal development. The<br />
C. <strong>elegans</strong> gene product, zk370.3, is conserved through evolution, from yeast to humans. Work in yeast<br />
has determined a cellular role for this gene in the maintenance of the actin cytoskeleton, as well as in<br />
endocytosis. However, very little is known about the role of this gene in the development of higher<br />
organisms. We have used RNA interference to investigate the loss of function phenotype for this gene<br />
product (protocol after Tavernarakis et al. 2000). Treated animals lay significantly more unfertilized<br />
oocytes than controls. This experiment suggests that silencing of the endogenous zk370.3 gene product<br />
results in developmental arrest prior to oocyte fertilization. We are working to determine where the defect<br />
resides. Using a portion of the cDNA fused to GFP under control of the zk370.3 promoter we observed<br />
expression in the proximal gonad, spermatheca and pharynx. Expression in the spermatheca is observed<br />
when gametogenesis is occurring, at late larval stages and early adulthood. Pharyngeal expression can<br />
first be observed in early larval stages, and is maintained through adulthood. We have raised a polyclonal<br />
antibody to zk370.3 for use in immunofluorescence microscopy experiments. The localization of zk370.3<br />
protein detected by the antibody is in agreement with our expression pattern analysis.<br />
216
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
OXIDANT STRESS RESPONSES IN C. ELEGANS<br />
Farhang Payvar, Andrew DeMatteo, Tom Hazinski<br />
Department of Pediatrics, Vanderbilt University Medical School, Nashville, TN, 37232<br />
We are intrigued by the genetic events triggered with hyperoxia and how they might differ from oxidative<br />
stress per se. Hyperoxia can influence gene expression by transcriptional (JCI 96:2083-2089, 1995)<br />
and/or post-transcriptional mechanisms leading directly or indirectly to oxidant stress (OS). In C. <strong>elegans</strong>,<br />
insulin-like signaling pathway regulates life span and sensitivity to OS. For example, animals with<br />
mutations in DAF-2 (insulin-like receptor) or AGE-1 (PI3K) are relatively resistant to OS and have<br />
extended life span; mutations in DAF-16 (fork head transcription factor) suppress the phenotypes of daf-2<br />
and age-1 mutants, suggesting that DAF-16 is target of negative regulation by upstream AGE-1 and<br />
DAF-2.<br />
Two forms of OS-induction not previously explored in C. <strong>elegans</strong> were examined. The wild type (N2) &<br />
mutant (daf-2, age-1, daf-16) animals were exposed to 3.5 days of sodium nitroprusside (NP), an OS<br />
inducer, in normoxic (21 % oxygen) or hyperoxic (95% oxygen) environments. Survival rates were<br />
measured, and the LD50 for NP treatment with and without hyperoxia was calculated.<br />
Our results indicate that survival in wild-type animals is reduced by NP but not by hyperoxia. However,<br />
hyperoxia increased the sensitivity to NP by ~ 3-fold. This increase in NP sensitivity suggests that the<br />
genetic events mediating the effects of hyperoxia are, at least in part, distinct from those for NP in C.<br />
<strong>elegans</strong>, and that there is cross-talk between hyperoxia and NP signaling pathways. Moreover, response<br />
to NP, alone or in combination with hyperoxia is altered in several mutant C. <strong>elegans</strong> strains. Consistent<br />
with the conventional DAF-16 pathway model, age-1 mutants are relatively NP-resistant, suggesting that<br />
AGE-1 is involved in NP signaling. Unexpectedly, age-1 mutants are only ~ 17 % reduced in their<br />
sensitivity to hyperoxia (+NP), pointing to alternate players/pathways for transmission of hyperoxic signal.<br />
Surprisingly, daf-2 mutants are hypersensitive to NP & have nearly lost their NP + hyperoxia-response<br />
phenotype. These findings are consistent with the notion that NP and hyperoxia signals are relayed via<br />
DAF-2 to downstream molecules that are distinct from AGE-1.<br />
217
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
PHARYNGEAL PUMPING DEFECTS IN UNC-103 MUTANTS<br />
Christina I. Petersen 1 , David J. Reiner 2 , Elizabeth M. Newton. 3 ,<br />
James H. Thomas 3 , Jeffrey R. Balser 4<br />
1 Dept. of Anesthesiology, Room 560B MRB II, Vanderbilt University, Nashville, TN 37232-6600<br />
2 Howard Hughes Medical Institute and Dept. of Molecular And Cell Biology, 401 Barker Hall #3204,<br />
University of California, Berkeley, CA 94720-3204<br />
3 Dept. of Genetics, Box 357360, University of Washington, Seattle, WA 98195<br />
4 Dept. of Anesthesiology, Room 560 MRB I, Vanderbilt University, Nashville, TN 37232-6600<br />
Human ether-a-go-go (HERG) potassium channels play a critical role in cardiac repolarization and have<br />
been genetically linked to an inherited arrhythmia, the long QT syndrome. unc-103 encodes the worm<br />
ortholog of HERG with 70 % amino acid identity in the conserved transmembrane and pore regions.<br />
unc-103 gain-of-function (gf) mutants display a variety of phenotypes reflecting reduced muscle<br />
excitation. These phenotypes include defective egg-laying, paralysis and defects in defecation. Because<br />
the worm pharynx displays many similarities to the mammalian heart, we examined unc-103 gf mutants<br />
for defects in pharyngeal pumping. We find that these mutants display lengthy pauses in pharyngeal<br />
pumping (as long as 7 seconds), reminiscent of cardiac bradydysrhythmias. Notably, in wildtype worms,<br />
HERG-specific blockers such as dofetilide and d-sotalol slow the rate of pharyngeal pumping, but do not<br />
induce pauses. Promoter-GFP fusion constructs reveal that unc-103 is expressed in I1, I2 and NSM<br />
neurons, which innervate the pharynx. The molecular nature of the unc-103 gf mutant is an A to T change<br />
at position 334 in the S6 domain, a region important for potassium channel gating. To understand the<br />
diverse effects of the unc-103 gf mutation and HERG-specific blockers, we are undertaking an<br />
electrophysiological characterization of the wild type and UNC-103 gf mutant using a mammalian<br />
heterologous expression system. These studies may clarify whether complex ion channel gating effects<br />
underlie differences between the unc-103 gf phenotype and the effects of HERG blockers in the pharynx.<br />
Furthermore, comparing the electrophysiological properties of wt UNC-103 with HERG in our expression<br />
system will also allow us to address structure/function differences using chimeric constructs of worm and<br />
human channels.<br />
218
A REQUIREMENT FOR C. ELEGANS RHO-BINDING KINASE<br />
IN EARLY CLEAVAGE<br />
Alisa J. Piekny, Paul E. Mains<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Genes and Development Research Group, Department of Biochemistry and Molecular Biology, University<br />
of Calgary, Calgary, AB. CANADA T2N 4N1<br />
Our lab is investigating genes that regulate the actin-mediated cell shape changes that drive C.<strong>elegans</strong><br />
embryonic elongation. let-502 encodes a Rho-dependent kinase and strong antimorphic mutations display<br />
early larval arrest due to failed elongation 1 . We recently isolated hypomorphic homozygous viable let-502<br />
mutations. Some of these alleles sufficiently decrease maternal expression to reveal a new, early<br />
embryonic phenotype. The embryos either do not complete any cell cleavages or form cleavage furrows<br />
that regress and result in multinucleated embryos. Some of these embryos also show defects in<br />
asymmetry, some appear larger than wild-type embryos and this may indicate defects in oocyte<br />
formation.<br />
Consistent with a role in cleavage furrow formation, LET-502 localizes to the furrow during early cell<br />
divisions. mel-11 encodes a myosin phosphatase and suppresses let-502 elongation defects 1,2 . A<br />
hypomorphic mel-11 mutant also suppresses the cleavage defects caused by let-502 mutants suggesting<br />
that mel-11 also may play a role in cytokinesis. However, these mel-11 mutant embryos do not show<br />
cleavage defects on their own.<br />
mlc-4 encodes a nonmuscle regulatory light chain and is a downstream target for both let-502 and mel-11<br />
in embryonic elongation. mlc-4 previously has been shown to play a role in early cleavage 3 . Together,<br />
these results suggest that the pathway regulating embryonic elongation may be similar to the pathway<br />
regulating early cleavage. In higher eukaryotes nonmuscle myosin, Rho, RhoGEF and the Rho-binding<br />
kinase effector, Citron-kinase, have been shown to regulate cytokinesis and cleavage furrow formation.<br />
Since the predicted C. <strong>elegans</strong> homologue for Citron has no associated kinase domain, in C. <strong>elegans</strong><br />
LET-502 could be the kinase required for early cleavage. let-502’s role in cytokinesis is currently being<br />
investigated using various molecular and genetic tools.<br />
1. Wissmann, A., J. Ingles, J.D. McGhee and P.E. Mains, 1997. Genes Dev. 11:409-422.<br />
2. Wissmann, A., J. Ingles and P.E. Mains, 1999. Develop. Biol. 209:111-127.<br />
3. Shelton, C. A., J.C. Carter, G.C. Ellis and B. Bowerman, 1999. J. Cell. Biol. 146: 439-451.<br />
219
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
FUNCTION OF THE RECEPTOR TYROSINE KINASE<br />
CAM-1/KIN-8 IN COORDINATED MOVEMENT<br />
S. Poulson, D. Madsen, A.V. Maricq<br />
Department of Biology, University of Utah, Salt Lake City UT 84112<br />
Receptor Tyrosine Kinases (RTKs) are important for many types of cell-to-cell signaling. Many<br />
developmental processes, including the development of synapses, are mediated by signal transduction<br />
through RTKs. Recently, a Ror-like RTK, KIN-8, was identified in C. <strong>elegans</strong>. Forrester and Garriga<br />
isolated a kin-8 (renamed cam-1) mutation and described the role of CAM-1 in normal migration of the<br />
CAN neurons. Koga and Ohshima later addressed the daf-c phenotype of cam-1 mutants. Independently,<br />
we had studied the expression pattern of kin-8/cam-1, found that it was expressed in many neurons as<br />
well as muscles and muscle arms, and generated a deletion mutation (ak37) in the gene. The mutant<br />
worms were severely uncoordinated, defective in mechanosensation, and showed a kinked head and<br />
withered tail. We are interested in why these worms are so defective in locomotion.<br />
Mechanosensory defects combined with locomotory defects suggested kin-8/cam-1 may be required for<br />
the function of interneurons that subserve locomotion. Using nmr-1::GFP to visualize the command<br />
interneurons (AVA, AVB, AVD, AVE, and PVC), we found that 85% of kin-8/cam-1(ak37) mutants exhibit<br />
aberrant axon outgrowth in these interneurons. We are currently investigating whether kin-8/cam-1 is<br />
required in the commnand interneurons for axon outgrowth. Uncoordination could also result from defects<br />
in synaptic or muscle function. Using electrophysiological recording techniques, we plan to record<br />
ligand-gated currents from identified neurons and muscles.<br />
220
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE AUTOSOMAL SEX SIGNAL IN C. ELEGANS?<br />
Jennifer R. Powell, Barbara J. Meyer<br />
HHMI and Department of Molecular and Cell Biology, University of California at Berkeley 94720.<br />
In C. <strong>elegans</strong>, sex is determined by the ratio of X chromosomes to sets of autosomes. The low X:A ratio<br />
in XO diploid worms permits high expression of the developmental switch gene xol-1, and hence male<br />
development ensues. Conversely, a high X:A ratio in XX animals results in low xol-1 activity and<br />
hermaphrodite development. The X portion of the sex signal is comprised of several X-signal elements<br />
(XSE) that repress the activity of xol-1 in a dose-dependent manner. The nature of the autosomal<br />
component of the sex signal is unknown, but presumably increases the activity of xol-1. It is possible that<br />
discrete autosomal signal element genes (ASE) exist that are analogous to, but act in opposition to, the<br />
XSEs. To test this hypothesis, we are performing a genetic screen to isolate loss-of-function mutations in<br />
ASEs, if they exist.<br />
This screen is based on the fact that the sex signal is cumulative; therefore, we expect loss-of-function<br />
mutations in ASEs to rescue the XX-specific lethality caused by removing XSEs. Specifically, XX worms<br />
homozygous for mutations in the two XSEs fox-1, an RNA-binding protein, and sex-1, a nuclear hormone<br />
receptor, are completely inviable. We are screening for suppressor mutations that rescue this lethality;<br />
these are candidate ASE mutations. The fox-1 sex-1 starting strain can be maintained as viable<br />
hermaphrodites if these worms also overexpress sdc-2(+), a downstream gene in the pathway, from an<br />
extrachromosomal array. Mutagenized worms that contain a suppressor mutation will no longer depend<br />
on the array for viability, so we screen through F1, F2, and F3 generations to look for live<br />
non-array-bearing hermaphrodites that potentially contain dominant, recessive, or maternal effect<br />
suppressor mutations.<br />
In initial rounds of mutagenesis, we have screened approximately 2000 haploid genomes and isolated 10<br />
suppressor mutations. They include autosomal and X-linked mutations, and show a range of suppression<br />
phenotypes. There are several classes of suppressors that could be recovered from this screen, including<br />
ase (lf), xol-1 (lf), sdc-2 (gf), xse (gf), and fox-1 or sex-1 revertants. We are currently characterizing our<br />
existing mutants and continuing to screen for additional suppressors.<br />
221
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
GOT THE BLUES? TRY ANOTHER GENETIC SCREEN!<br />
Chad Rappleye 1 , Rebecca Lyczak 2 , Bruce Bowerman 2 , Raffi Aroian 1<br />
1University of California, San Diego, La Jolla, CA 92093<br />
2University of Oregon, Eugene, OR 97403<br />
To better understand the process by which cell polarity is established, we are analyzing genes required<br />
for the characteristic asymmetries of the early C. <strong>elegans</strong> embryo. Recently, we showed that the pod-1<br />
locus represents a new class of polarity genes as mutation of pod loci results in osmosensitive embryos in<br />
addition to loss of polarity (’pod’=polarity and osmotic defective). The identification of pod-2 by our lab<br />
(see abstract by A. Tagawa) further underscores the importance of the pod class of mutants. The<br />
osmosensitivity of pod mutants appears to result from permeability of the egg shell surrounding the<br />
embryo. Understanding the connection between polarity establishment and egg shell formation should<br />
begin to reveal the molecular mechanisms underlying cellular asymmetry.<br />
To further identify the set of components responsible for this aspect of polarity establishment, we have<br />
initiated a study of mutants that have osmosensitive phenotypes. Among previously identified osmotically<br />
sensitive mutants, emb-8 and emb-30 mutants also exhibit the ’Pod’ phenotype. In these, as well as a<br />
pod-1 null mutant, the polarity defect is not completely penetrant suggesting that multiple, parallel<br />
pathways might contribute to overall cell polarity. However, analysis of double mutants between pod-1<br />
and emb-8 or emb-30 indicate that these three function in a common genetic pathway.<br />
Through two different genetic screens, one of which directly identifies osmosensitive embryos, we have<br />
isolated a number of other mutations that represent new loci of the pod class. This collection includes<br />
genes which when mutated cause near 100% penetrant loss of polarity. We are currently pursuing the<br />
molecular identities of these loci which will enable us to refine our models for how cellular asymmetry is<br />
controlled.<br />
222
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
IDENTIFICATION OF COMPONENTS OF THE MEIOTIC<br />
MACHINERY IN C. ELEGANS<br />
Kirthi Reddy, Monica Colaiacovo, Gillian Stanfield, Anne Villeneuve<br />
Dept. of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305<br />
Meiosis is the specialized form of cell division that diploid cells undergo to produce gametes with a<br />
haploid chromosome number. During prophase of meiosis I, homologous chromosomes locate each other<br />
(pair), associate tightly along their lengths (synapse), and undergo recombination. Failure to complete any<br />
of these processes can result in improper chromosome segregation. We are interested in the molecular<br />
mechanisms that are involved in chromosome pairing, synapsis and crossover formation. We have<br />
initiated a functional genomics strategy to identify components of the C. <strong>elegans</strong> meiotic machinery (see<br />
abstract by Colaiacovo et al). Microarray analysis has identified a set of genes that are upregulated in the<br />
germline and are not differentially expressed in male and female germ cells (V. Reinke, S. Kim and<br />
collaborators). We are screening a subset of these genes to identify those for which RNAi elicits defects<br />
in meiosis.<br />
We initially focused on genes encoding proteins with predicted coiled-coil domains, since several proteins<br />
in this class have been implicated in chromosome structure and behavior. We have identified two genes,<br />
F26D2.2 and F39H2.4, that appear to be involved in homologous chromosome synapsis. Both have RNAi<br />
phenotypes characteristic of animals with severe chromosome segregation defects -- affected<br />
hermaphrodites produce a high percentage of dead embryos, and among the few survivors, there is a<br />
high incidence of males. Further, affected animals show achiasmate chromosomes late in meiotic<br />
prophase, likely reflecting a failure to form crossovers. Earlier in prophase, when chromosomes should be<br />
aligned and synapsed, affected animals exhibit extensive asynapsis. The chromatin morphology seen in<br />
the affected animals is characteristic of sys-1 and sys-2 mutants, which are unable to stabilize<br />
associations between homologs (see abstract by MacQueen and Villeneuve). Indeed, the sys-1(me17)<br />
allele has a stop mutation in the F26D2.2 gene. No previously identified synapsis-defective mutants map<br />
near F39H2.4, which we have designated sys-3.<br />
Our RNAi screen for genes required for meiosis is ongoing. We will report on additional genes that we<br />
identify on the basis of meiotic defects elicited by RNAi.<br />
223
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
NOVEL AND ATYPICAL RECEPTOR TYROSINE KINASES IN<br />
MORPHOGENESIS.<br />
David J. Reiner, Lewis Leng, Barbara J. Meyer<br />
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,<br />
Berkeley, CA 94720.<br />
Most receptor tyrosine kinases (RTKs) fall into one of a number well-characterized growth factor receptor<br />
families. However, in C. <strong>elegans</strong> we have used the genome sequence to identify four apparently novel<br />
receptors and two atypical receptors with mammalian homologs. Over-expression of five of these six<br />
genes results in variable, lumpy lethality, while RNAi of four of these genes results in visible phenotypes.<br />
The RNAi causes mainly morphogenetic phenotypes. For example, RNAi for one of these genes causes<br />
partially penetrant lethality due to extreme hypodermal deformities, and some escaping animals have<br />
pronounced axon guidance and fasciculation defects. The atypical receptors are apparently nematode<br />
homologs of the mammalian Discoidin Domain Receptors (DDRs), RTKs that function as collagen<br />
receptors. The in vivo function of DDRs is unknown, and we hope to address this issue in C. <strong>elegans</strong>.<br />
Candidate mutations have been identified for only one of the six receptors. T14E8.1 rescues the weakly<br />
penetrant tail phenotypes of mab-19, but we were unable to find lesions corresponding to the two mab-19<br />
alleles. A heat-shock promoter driven RNAi foldback construct of T14E8.1 causes moderate, incompletely<br />
penetrant Mab phenotypes comparable to mab-19, plus a novel truncation of certain tail sensory rays. To<br />
identify deletion mutations in these genes, we are currently constructing a large deletion library to be<br />
screened with PCR. Future directions depend on the outcome of these screens.<br />
224
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
DIFFERENTIAL EFFECTS OF HEAT SHOCK AND COLD<br />
SHOCK FOLLOWING MASSED AND DISTRIBUTED<br />
LONG-TERM HABITUATION TRAINING IN C. ELEGANS<br />
Jacqueline Rose 1 , Kenneth Eng 2 , Catharine Rankin 1<br />
1Department of Psychology, University of British Columbia, Vancouver B.C.<br />
2Neuroscience Graduate Program, University of British Columbia, Vancouver B.C.<br />
A new en masse long-term habituation (LTH) training procedure was used to examine the effects of heat<br />
and cold shock on LTH of the tap withdrawal response. In order to induce LTH a distributed training<br />
protocol was used where groups of worms received four blocks of 20 taps separated by one-hour rest<br />
periods and testing each worm individually 24 hours later. LTH following massed training was also<br />
examined by administering one block of 120 taps to worm groups. <strong>Worm</strong>s that received massed training<br />
did not show LTH when tested 24 hours later. Temperature shock was administered by submerging<br />
sealed worm-containing petri plates into either a 32° C (heat shock) or a 0° C bath for the first 40<br />
minutes following training blocks: worms were kept at room temperature for the last 20 minutes. <strong>Worm</strong>s in<br />
the control condition underwent similar submersion during rest periods at 21° C (room temperature). It<br />
was found that when heat shock is administered between distributed training blocks an attenuation of<br />
LTH results. Predictably, heat shock after massed training did not produce LTH. Interestingly, when<br />
worms receive distributed training blocks with cold shock between blocks, LTH of the tap withdrawal<br />
response is at least retained and may be enhanced. Similar to before, LTH did not result from massed<br />
training followed by cold shock. Twelve-hour retention has also been examined with distributed training<br />
and similar findings resulted where LTH was induced after room-temperature submersion and cold shock<br />
but not heat shock. Currently, studies are investigating if massed training results in LTH if measured 12<br />
hours rather than 24 hours later. As well, the effects of heat shock and cold shock on 12-hour retention<br />
following massed training are being examined. The results that have been reported thus far indicate that<br />
differential mechanisms may underlie both LTH resulting from massed versus distributed training and the<br />
effects of heat shock versus cold shock.<br />
Research funded by the Natural Sciences and Engineering Research Council of Canada<br />
225
A NEW EN MASSE TRAINING PROCEDURE TO STUDY<br />
LONG-TERM HABITUATION IN C. ELEGANS<br />
Jacqueline Rose, Catharine Rankin<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Psychology, University of British Columbia, Vancouver B.C.<br />
Past research examining long-term habituation (LTH) of the tap withdrawal response in C. <strong>elegans</strong><br />
required training individual worms with three blocks of 20 tap-trains separated by one hour rest periods<br />
with test tap-trains delivered 24 hours later. This single worm method was time and labor intensive. In an<br />
attempt to increase throughput, a between-groups design was implemented. Groups of worms were<br />
trained at a 60 s interstimulus interval by receiving either 4 blocks of 20 taps with one-hour rest periods<br />
(distributed) or 1 block of 80 taps (massed). Testing was conducted 24 hours after training with single<br />
worms individually being given one block of 10 taps. Trained groups were compared to a control group<br />
that received a single tap on training day and the same individual testing on day two. The results showed<br />
that worms that received distributed training show significantly smaller responses compared to single-tap<br />
controls. <strong>Worm</strong>s that underwent massed training showed no difference in response magnitude compared<br />
to the control group. Since LTH was only found following distributed training a follow-up experiment was<br />
conducted where worms were given either one, two, three or four training blocks separated by one hour<br />
rest periods and were tested individually 24 hours later. It was found that worms that received 4 training<br />
blocks showed the greatest LTH while worms that received a single training block showed no LTH<br />
demonstrating that an effect of accumulation of learning on LTH. Together these results effectively<br />
replicate what has previously been found with the single worm training method showing this new protocol<br />
can now be used to more effectively assess LTH in a variety of situations.<br />
Research funded by the Natural Sciences and Engineering Research Council of Canada.<br />
226
GLOBAL PATTERNS OF EXPRESSION PATTERNS IN<br />
MUSCLE USING MRNA-TAGGING<br />
Peter J. Roy, Stuart Kim<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Developmental Biology, Stanford University, 279 Campus Drive, Stanford, CA 94305<br />
Gene networks that control development must endow each unique cell with distinguishing properties.<br />
Microarray technology along with the completed C. <strong>elegans</strong> genome now makes it possible to know these<br />
gene networks in a comprehensive manner. Our microarrays contain PCR fragments representing nearly<br />
every predicted C. <strong>elegans</strong> gene. By hybridizing labeled cDNA probes from different samples to the<br />
microarray, one can simultaneously determine the expression differences between those two samples for<br />
every gene. A limitation of current microarray technology is that RNA is isolated from whole worms, in<br />
turn, making tissue specific expression patterns difficult to detect. We have developed a strategy to<br />
circumvent this problem of identifying the mRNA profiles in specific tissues or cells by using an approach<br />
we call "mRNA-tagging".<br />
To isolate cell-specific mRNA, we are using previously characterized promoters to drive the expression of<br />
epitope-tagged protein that binds mRNA in a non-biased fashion in a defined set of cells. By<br />
immunoprecipitating the tagged mRNA-binding protein from whole animal lysates, cell-specific mRNA is<br />
co-immunoprecipitated. The abundance of each mRNA species isolated from these distinct cells is then<br />
assayed using microarrays.<br />
To identify genes expressed in muscle, we are using the well-characterized promoter myo-3 (Okkema et<br />
al., Genetics 135, 385-404) to drive the expression of the epitope-tagged mRNA-binding protein in body<br />
wall muscle. To date, we have successfully and reproducibly co-immunoprecipitated muscle-specific<br />
RNA, as assayed through both northern and microarray analysis. Control germ line and neuronal RNA<br />
are not enriched. We are currently optimizing our mRNA-tagging protocol to identify gene expressed in<br />
muscle, and are also extending our studies to identify neuronal and hypodermal genes.<br />
227
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
COOPERATION BETWEEN UNC-26/SYNAPTOJANIN AND<br />
THE DYNAMIN-RELATED PROTEIN DRP-1 DURING<br />
MITOCHONDRIAL DIVISION<br />
Dan Rube 1 , Todd Harris 2 , Erik Jorgensen 2 , Alexander van der Bliek 1<br />
1Department of Biological Chemistry, UCLA School of Medicine, P.O. Box 951737, Los Angeles, CA<br />
90095-1737<br />
2Department of Biology, University of Utah, 257 South 1400 East Salt Lake City, UT 84112-0840<br />
Mitochondria often exist as a dynamic tubular network. Individual mitochondria frequently divide or fuse<br />
with neighboring mitochondria in response to a variety of metabolic and cell division cues. Our laboratory<br />
is interested in the mechanism of mitochondrial division and how this mechanism is related to<br />
endocytosis. A first indication that these processes may be related came from our studies of<br />
dynamin-related protein (DRP-1) in the nematode C. <strong>elegans</strong>. We discovered that DRP-1 is important for<br />
the final stage of division of the mitochondrial outer membrane. This was surprising, because the protein<br />
sequence is very similar to that of dynamin, which we know mediates an early stage of clathrin mediated<br />
endocytosis. We hypothesize that division of the mitochondrial outer membrane is evolutionarily and<br />
mechanistically related to endocytic vesicle formation. The very first endosymbiontic bacterium would<br />
have retained parts of the endocytic machinery to divide the incipient mitochondrial membranes.<br />
To confirm this hypothesis, we are looking for additional proteins that act both in endocytosis and in<br />
division of the mitochondrial outer membrane. We are focusing on two well-known players in endocytic<br />
vesicle formation, synaptojanin and endophilin, which might also be candidates for mitochondrial division.<br />
We tested the effect of the synaptojanin mutant unc-26(e205) on mitochondrial morphology using a GFP<br />
with a mitochondrial leader sequence under control of the myo-3 promoter. The mitochondria in C.<br />
<strong>elegans</strong> body wall muscles appear as a few large clumps rather than as numerous regularly shaped<br />
wild-type mitochondrial bodies. These clumps may arise from impaired mitochondrial division.<br />
Furthermore, this phenotype appears to be partially rescued by overexpression of DRP-1. Our data thus<br />
indicates that in C. <strong>elegans</strong>, synaptojanin itself plays a role in maintaining proper mitochondrial<br />
morphology. Currently, we are seeking to determine whether the morphology defect of unc-26(e205)<br />
mitochondria arises from impaired mitochondrial division or disruption of another mitochondrial process.<br />
228
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CALCIUM DYNAMICS OF FERTILIZATION IN C. ELEGANS<br />
Aravinthan D.T. Samuel 1 , Venkatesh N. Murthy 1 , Michael O.<br />
Hengartner 2<br />
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138.<br />
2Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 11724.<br />
In all animals, fertilization generates a pattern of intracellular calcium dynamics within the oocyte that<br />
constitutes an essential trigger for normal development. The spatiotemporal properties of the calcium<br />
dynamics differ among animals, e.g. cnidarians, nemerteans, fish and frogs have single calcium<br />
transients whereas annelids, ascidians, and mammals have multiple calcium oscillations. However, in all<br />
animals, fertilization-induced calcium dynamics are mediated by release of internal calcium stores by<br />
inositol 1,4,5-triphosphate (IP 3). Although little is known about the signaling pathway intervening<br />
fertilization and the production of IP 3, features of the pathway are likely to be widely shared among<br />
species [1]. Of the animals typically used to study fertilization-induced calcium dynamics, none is as<br />
accessible to genetics and molecular biology as the model organism <strong>Caenorhabditis</strong> <strong>elegans</strong>. Motivated<br />
by the experimental possibilities inherent in using such a well-established model organism to study<br />
fertilization-induced calcium dynamics, we have characterized these dynamics in C. <strong>elegans</strong>. Owing to<br />
the transparency of the nematode, we have been able to study the calcium signal in C. <strong>elegans</strong><br />
fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the<br />
cytosol of oocytes. In C. <strong>elegans</strong>, fertilization induces a single calcium transient that originates at the point<br />
of sperm entry. This calcium elevation immediately spreads throughout the oocyte with an amplitude ~250<br />
nM. The duration of this solitary calcium transient is ~6 min, after which the cytosolic calcium<br />
concentration returns to that preceding fertilization. Among other effects, this calcium signal may trigger<br />
the completion of meiosis and the formation of eggshell.<br />
229
MUTANTS IN THERMOSENSORY NEURON SPECIFICATION<br />
AND FUNCTION<br />
John S. Satterlee, Piali Sengupta<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biology, Brandeis University, 415 South St. Waltham, MA 02454<br />
The AFD neurons are required for thermosensation in C. <strong>elegans</strong>. This pair of ciliated sensory neurons<br />
has a unique morphology and presumably expresses a specific suite of genes which confer<br />
thermosensory function. For example, the promoters of both a nuclear hormone receptor homolog<br />
(nhr-38) and a receptor guanylyl cyclase homolog (gcy-8) drive GFP expression specifically in this neural<br />
pair. We are examining the mechanism by which the fate and functions of the AFD thermosensory<br />
neurons are specified, by identifying genes that work in a cascade to direct expression of these<br />
AFD-specific markers.<br />
Two mutants with reduced expression of nhr-38::GFP in the AFD neurons were found to be allelic to tax-2<br />
and tax-4. The tax-2/tax-4 genes encode subunits of a cGMP-gated channel which function in some<br />
sensory neurons, including AFD. It is possible that the TAX-2/TAX-4 channel is required for<br />
activity-dependent expression of some AFD specific genes. We have made promoter::GFP fusions to<br />
other tax-4-like channels encoded by the worm genome and found that two of these genes are also<br />
expressed in AFD, as well as a number of other sensory neurons.<br />
We have identified mutations in two genes (2 alleles each) with reduced expression of gcy-8::GFP in the<br />
AFD neurons. One of these (sns-6) has been mapped to a small region of LG IV. The other (sns-7) is<br />
allelic to the previously identified thermotaxis mutant ttx-1. Single nucleotide polymorphisms have been<br />
used to map ttx-1/sns-7 to a 130 kb region of LG V which encodes 9 predicted protein products.<br />
Both sns-6 and ttx-1/sns-7 are thermotaxis defective, but are not defective in behaviors mediated by the<br />
AWA, AWC, ASE, and ASH neurons. Mutations in both sns-6 and ttx-1/sns-7 suppress the<br />
dauer-constitutive phenotype of daf-7 mutants at 25ûC.<br />
We have isolated a mutant with a deletion of the gcy-8 guanylyl cyclase domain. This mutation has no<br />
effect on gcy-8::GFP or nhr-38::GFP expression in AFD. Further characterization of this mutant is in<br />
progress.<br />
The molecular characterization of thermotaxis mutants should reveal how AFD function is specified and<br />
may also shed light on how thermosensory signals are transduced.<br />
230
VESICULAR GABA TRANSPORT IN C. ELEGANS REQUIRES<br />
TWO PROTEINS UNC-47 AND UNC-46<br />
Kim Schuske, Erik M. Jorgensen<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biology, University of Utah, Salt Lake City, UT 84112<br />
For neurotransmitter to be released from a neuron, it must be transported into synaptic vesicles from the<br />
cytoplasm where it is produced. Transport proteins are located in the vesicle membrane that bind and<br />
load a specific neurotransmitter into the synaptic vesicle. Previously we cloned the gene unc-47, which is<br />
defective in all GABA neurotransmission, and found that it encodes a transport protein that loads GABA<br />
into synaptic vesicles. We now report that there is a second gene that is phenotypically defective in all<br />
GABA neurotransmission, unc-46, which also appears to be required for loading of GABA into synaptic<br />
vesicles. Overexpression of UNC-47 is capable of rescuing the unc-46 mutant phenotype. This suggests<br />
that an increased level of vesicular GABA transporter (UNC-47) in the neuron can compensate for the<br />
lack of UNC-46 protein. Therefore we believe that UNC-46 may be a modulator of vesicular GABA<br />
transport.<br />
To test this, we have cloned the unc-46 gene. The UNC-46 protein is novel and contains a single putative<br />
transmembrane domain. unc-46 is expressed in GABA neurons and the protein is localized to synaptic<br />
vesicles. UNC-47 protein is also localized to synaptic vesicles even in the absence of unc-46. However,<br />
UNC-46 protein is mislocalized in the absence of unc-47. This suggests that UNC-47 and UNC-46<br />
interact in the synaptic vesicle. The simplest model suggests that UNC-46 may be required for UNC-47<br />
transport activity. Current experiments are aimed at determining whether UNC-46 and UNC-47 physically<br />
interact, testing if UNC-46 can affect transport activity of GABA in cell culture, and identifiying a vertebrate<br />
homolog of UNC-46.<br />
231
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UTILIZING TWO APPROACHES, GENETIC AND GENOMIC,<br />
TO IDENTIFY THE VESICULAR GLUTAMATE TRANSPORTER<br />
Kim Schuske, Dan Williams, Erik M. Jorgensen<br />
Department of Biology, University of Utah, Salt Lake City, UT 84112<br />
Neurotransmitter is loaded into the synaptic vesicles by transport proteins present in the synaptic vesicle<br />
membrane. Transport activity has been characterized for four distinct neurotransmitters in purified<br />
synaptic vesicles from rat brain: GABA, glutamate, acetylcholine, and catecholamines. Of these, only the<br />
vesicular glutamate transporter remains to be identified at the molecular level. We previously showed that<br />
the unc-47 gene encodes the vesicular GABA transporter. Because GABA and glutamate are structurally<br />
similar and their bioenergetics for transport into the vesicle are also similar, we proposed that the<br />
vesicular glutamate transporter is related by sequence to UNC-47. There are fourteen proteins in the C.<br />
<strong>elegans</strong> genome that have sequence similarity to UNC-47. We made GFP reporter constructs for twelve<br />
of these genes, and identified two candidates that are expressed in glutamatergic neurons. We are<br />
currently studying a gene knockout, made by the C. <strong>elegans</strong>gene knockout consortium, and GFP-tagged<br />
protein localization for one candidate cevt6. However, preliminary data suggests that cevt6 is not the<br />
vesicular glutamate transporter.<br />
It is possible that the vesicular glutamate transporter does not resemble the vesicular GABA transporter<br />
by sequence. For this reason we have undertaken a genetic screen for mutants that are likely to be<br />
defective for glutamate neurotransmission. We are therefore looking for mutants resembling eat-4 since<br />
they are defective for all known glutamate neurotransmission. These animals have an Osm phenotype.<br />
We have screened for Osm mutants using a Mos1 transposon (from Drosophila mauritiana) with the goal<br />
of identifying a mutant with a Mos1 insert in a transporter-like protein. We carried out a pilot screen and<br />
identified three Osm mutants. One of these mutants contains a single transposon inserted into exon ten<br />
of the eat-4 gene. The two other mutants are dye filling defective and are therefore not likely to encode<br />
the vesicular glutamate transporter. An increase in the efficiency of Mos1 transposition should allow us to<br />
do a saturation Osm screen in the future.<br />
232
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ACTIN-DEPENDENT PROCESSES IN THE EARLY C.<br />
ELEGANS EMBRYO REQUIRE THE PROFILIN GENE PFN-1,<br />
THE FH GENE CYK-1, AND BEL-1<br />
Aaron F. Severson 1 , Rebecca Lyczak 1 , David L. Baillie 2 , Bruce<br />
Bowerman 1<br />
1University of Oregon, Eugene, OR 97403 USA<br />
2Simon Fraser University, Burnaby, B.C. CANADA V5A 1S6<br />
The actin cytoskeleton is essential for many processes in the early C. <strong>elegans</strong> embryo, including ruffling<br />
of the anterior cortex and pseudocleavage following fertilization, the asymmetric migration of pronuclei<br />
and position of the first mitotic spindle, and cytokinesis. A worm profilin gene, called pfn-1, is required for<br />
all of these processes. In pfn-1(RNAi) embryos, cortical ruffling and pseudocleavage are absent, the<br />
pronuclei meet in the center of the embryo, the first mitotic spindle is centrally positioned, and a cleavage<br />
furrow fails to form during cytokinesis. Profilin regulates the actin cytoskeleton by sequestering actin<br />
monomers and preventing their polymerization into microfilaments. In addition, profilin can stimulate actin<br />
assembly by converting actin into an assembly-competent GTP-bound state. In other systems, profilin can<br />
bind to FH proteins, which may recruit profilin/actin complexes to sites of actin assembly. The C. <strong>elegans</strong><br />
FH protein CYK-1 is required for embryonic cytokinesis (Swan et al, 1998), and embryos produced by<br />
worms mutant for a strong combination of cyk-1 alleles fail early in furrow ingression. We are currently<br />
characterizing anti-PFN-1 antisera to examine PFN-1 localization in wild-type and CYK-1 mutant<br />
embryos. In addition, we are examining actin, myosin, and CYK-1 localization in pfn-1(RNAi) embryos to<br />
determine the effects of profilin inactivation on the actin cytoskeleton.<br />
While PFN-1 may be required for organized contraction of the actin cytoskeleton, a second gene may<br />
function to temporally and spatially restrict contractility in the embryo. In embryos mutant for a<br />
temperature-sensitive, embryonic lethal allele of this gene, waves of contraction sweep across the early<br />
embryo, resembling mobile cleavage furrows. We have named this gene bellydancer, or bel-1. To<br />
determine if the mechanism that underlies these waves is similar to the contractile ring, we are staining<br />
bel-1 mutant embryos with antibodies to actin, myosin, and CYK-1. In addition, we are mapping bel-1,<br />
which we hope to clone by a combination of RNAi phenocopy and transgenic rescue.<br />
233
LIN-12 POST-TRANSCRIPTIONAL DOWNREGULATION<br />
DURING VPC SPECIFICATION<br />
DD Shaye 1 , I Greenwald 2<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
1Dept. of Genetics and Development, Columbia University, New York, NY 10032<br />
2HHMI and Dept. of Biochemistry, Columbia University, New York, NY 10032<br />
In wild-type hermaphrodites, each vulval precursor cell (VPC) adopts one of three distinct fates, 1°, 2° or<br />
3°, in a precise and invariant spatial pattern (reviewed in 1). Analysis of mutants defective in vulval<br />
development have suggested that at least three different signaling events are involved in the correct<br />
specification of VPC fates. An inhibitory signal originates in the hypodermal syncytium. An inductive signal<br />
originates in the gonadal anchor cell and allows VPCs to overcome the inhibitory signal from the<br />
hypodermis. Finally, a lateral signal originates in the presumptive 1° cell and is received by the<br />
presumptive 2° cells through LIN-12.<br />
Studies using a lin-12::gfp fusion have shown that one output of the inductive signal is to influence LIN-12<br />
protein accumulation (2). In wild-type hermaphrodites, all six VPCs initially express LIN-12::GFP.<br />
However, LIN-12::GFP accumulation is reduced specifically in P6.p at the time of vulval induction,<br />
although expression of a lin-12::lacZ reporter remains constant (3). The inductive signal seems to be<br />
necessary and sufficient for this downregulation to occur. These results have led to the proposal that the<br />
downregulation of LIN-12 in the presumptive 1° cell is at least one component of the mechanism by<br />
which the inductive signal influences lateral signaling.<br />
We are interested in elucidating the mechanism by which the inductive signal controls the downregulation<br />
of LIN-12 in P6.p and whether this downregulation plays a role in correctly generating a lateral signal. In<br />
order to answer these questions we are taking two approaches. First, we are examining the effects of<br />
mutations in the inductive pathway and in other genes on LIN-12::GFP protein accumulation. Second, we<br />
are determining the region of LIN-12 that targets LIN-12::GFP for downregulation in P6.p.<br />
1) I. Greenwald, in C. <strong>elegans</strong> II. CSH Press, Cold Spring Harbor, NY, (1997).<br />
2) D. Levitan and I. Greenwald, Development 125, 3101 (1998).<br />
3) H. Wilkinson and I. Greenwald, Genetics 141, 513 (1995).<br />
234
DISTINT AND REDUNDANT FUNCTIONS OF MU1 MEDIUM<br />
CHAINS OF AP-1 CLATHRIN-ASSOCIATED PROTEIN<br />
COMPLEX IN THE NEMATODE CAENORHABDITIS ELEGANS<br />
Jaegal Shim, Junho Lee<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Department of Biology, Yonsei University, Seoul, Korea<br />
The clathrin-associated protein complex I(AP-1 complex) in C. <strong>elegans</strong> is composed of four adaptor<br />
proteins. Of the medium chains in the AP-1 complex of C. <strong>elegans</strong>, the first cloned is unc-101 and the<br />
second chain cloned is apm-1. Also, according to a search of the C. <strong>elegans</strong> genome, unlike the medium<br />
chains, only one each of the beta, gamma and sigma chains of the AP-1 complex exists in C. <strong>elegans</strong>.<br />
These facts support the possibility that the two medium chains in C. <strong>elegans</strong> compose distinct AP-1<br />
complex while sharing the other chains, similar to mammalian mu1a and mu1b. From the RNAi and<br />
expression studies of AP-1 complex in C. <strong>elegans</strong>, we concluded that this hypothesis was correct. Since<br />
the expression of unc-101 and apm-1 is ubiquitous throughout development, and the functions of unc-101<br />
and apm-1 are redundant in embryogenesis and vulval development while becoming distinct during larval<br />
development, we are interested in the distinct and similar functions of these chains as adaptor proteins.<br />
Mediums of the AP complexes are generally known to play a part in cargo selection of clathrin-coated<br />
vesicles, thus we have started experiments to elucidate their respective cargoes using the yeast<br />
two-hybrid system. We screened about 400,000 colonies with full-length apm-1 cDNA as a bait, and<br />
found several positive clones. One of them, F29G6.3A, was the same clone that Marc Vidal group had<br />
found with unc-101 as a bait(Albertha J.M Walhout et al., Science ?????). We have also found several<br />
novel proteins, and will present analyses on the candidate genes<br />
235
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MOLECULAR MECHANISMS OF DAF-12 ACTION:<br />
IDENTIFICATIONOF RESPONSE ELEMENTS AND<br />
FUNCTIONAL ANALYSIS OF THE PROTEIN<br />
Yuriy Shostak 1 , Adam Antebi 2 , Marc R. van Gilst 3 , Kieth R.<br />
Yamamoto 1<br />
1 Program in Biochemistry and Molecular Biology and Department of Cellular and Molecular<br />
Pharmacology, UCSF, San Francisco, CA 94143-0450<br />
2 Max-Planck-Institut fuer molekulare Genetik,D-14195 Berlin, Germany<br />
3 Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94143-0450<br />
Dauer larva formation in <strong>Caenorhabditis</strong> <strong>elegans</strong> is regulated by the intracellular receptor daf-12, which<br />
functions at the convergence of TGF-b, cGMP, and insulin-like signaling pathways. daf-12 has also been<br />
implicated in the regulation of C. <strong>elegans</strong> lifespan and developmental age. Alleles of daf-12 with distinct<br />
protein sequence alteration spartially uncouple its phenotypic effects.<br />
As a step toward understanding the molecular biology of Daf-12 function, we identified specific DNA sites<br />
in the C. <strong>elegans</strong> genome bound by the protein in vitro. We developed an in vitro selection and<br />
amplification method that, using immobilized recombinant daf-12 DNA binding domain, yielded a series of<br />
specific C. <strong>elegans</strong> genomic DNA fragments. We inserted some of these fragments into yeast reporter<br />
plasmids and showed that Daf-12 selectively activated transcription of the reporter genes in<br />
Saccharomyces cerevisiae. Hence, C. <strong>elegans</strong> DNA fragments bound specifically by Daf-12 in vitro<br />
display Daf-12 response element activity in vivo.<br />
Intracellular receptors are multidomain proteins with characteristic DNA binding, putative ligand binding,<br />
and potentially ligand-controlled transcriptional regulatory domains. We used the heterologous yeast<br />
expression system to investigate contributions of different Daf-12 regions to transcriptional regulation. We<br />
found thatDaf-12 derivatives with truncated ligand binding domains are potent transcriptional activators,<br />
and mapped an activation domain to the "hinge region" just downstream of the DNA binding domain.<br />
In future work, we shall determine whether the identified response elements are linked to Daf-12regulated<br />
target genes in C. <strong>elegans</strong>. Similarly, we shall examine the roles of the activation domain in Daf-12<br />
biology and molecular function in the animal.<br />
236
SEROTONIN-RESISTANT EGG-LAYING MUTANTS AND A<br />
RECEPTOR KNOCKOUT IN PROGRESS<br />
Stanley Shyn, William Schafer<br />
UCSD, La Jolla, CA 92093<br />
Serotonin (5HT) stimulates egg-laying in the nematode 1 , and we hypothesize that this occurs by<br />
modulation of vulval muscle excitability from a quiescent mode to an active egg-laying state 2 . The<br />
effectors used to accomplish this are largely unknown, so we are mapping and characterizing catalogued<br />
as well as more recently isolated mutants (5 lines from our own lab) which are 5HT-resistant for<br />
egg-laying. One such mutant is egl-24 (n572) 3 . Further characterization has confirmed its profound<br />
5HT-resistance, but perhaps more intriguingly that, in the absence of any exogenous 5HT, its temporal<br />
pattern of egg-laying is not grossly different from that of N2s. Preliminary data suggests its map interval<br />
may be further narrowed to between -0.54 and -0.79 on LGIII, an interval spanned by 26 cosmids and a<br />
portions of 2 YACs.<br />
From a reverse-genetics approach, we are also preparing to knockout 5HT-Ce, the only known vulval<br />
5HT receptor 4 . Among vertebrate classes, 5HT-Ce shows the most homology to the 5HT 1 subtype,<br />
which typically inhibits adenylate cyclase 5 . This prompted us to look at worms mutant in acy-1, an<br />
adenylate cyclase expressed in virtually all neurons and muscles, including the vulva 6 . nu329 and<br />
pk450::Tc1, both only partial loss-of-function, demonstrated 5HT-resistance despite a baseline with<br />
wildtype parameters. Whether cAMP stimulates or inhibits egg-laying is still unclear, but pharmacological<br />
manipulations are planned to clarify the issue.<br />
Finally, assuming 5HT-Ce inhibits adenylate cyclase, downstream G-protein candidates might include<br />
goa-1 or gpa-7. Yet, functional goa-1 appears to play an inhibitory role in egg-laying 7,8 while testing in our<br />
lab of gpa-7 worms did not uncover significant 5HT-resistance. Recently, the Plasterk lab surveyed the<br />
complete family of genes encoding worm G-proteins 9 . One of these genes, gpa-14, is expressed in the<br />
vulva and appears to be a novel G-protein whose downstream effectors are unknown. gpa-14 (pk347)<br />
mutants were subsequently tested and found to be 5HT-resistant, suggesting that gpa-14 might be part of<br />
a 5HT signalling pathway.<br />
1 Science 216:1012<br />
2 Neuron 21:203<br />
3 Genetics 104:619<br />
4 Tim Niacaris (Avery lab), pers comm<br />
5 J Mol Neurosci 8:53<br />
6 J Neurosci 18:2871<br />
7 Science 267:1652<br />
8 Science 267:1648<br />
9 Nat Genetics 21:414<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
237
A NOVEL GENETIC SCREEN FOR SYNAPTIC<br />
TRANSMISSION GENES ACTING IN THE DIACYGLYCEROL<br />
PATHWAY<br />
Derek S. Sieburth, Wendy Cham, Josh M. Kaplan<br />
UC Berkeley, MCB Dept , 361 LSA, Berkeley CA 94720<br />
Acetylcholine release at the neuromuscular junction is modulated by diacylglycerol (DAG) levels which<br />
are regulated by the opposing effects of two G protein pathways 1-4 . The Gqa pathway produces DAG<br />
through the activation of the egl-8 phospholipase c (PLC) 1,4 , and the Gao pathway reduces DAG levels<br />
possibly through diacylglycerol kinase, encoded by dgk-1 2 . DAG appears to modulate synaptic<br />
acetylcholine release by recruiting UNC-13 to the synapse where it facilitates vesicle release 1,2 .<br />
Mutations in genes acting in these two pathways have opposite effects on acetylcholine release as<br />
assayed by the response to the paralytic effects of the acetylcholine esterase inhibitor, aldicarb 1,4 .<br />
Mutations in the Gqa/PLC pathway result in decreased levels of DAG and resistance to Aldicarb (Ric),<br />
while mutations in Goa/DGK-1 result in increased levels of DAG and hypersensitivity to aldicarb (Hic).<br />
To identify additional components specifically acting the DAG pathway, we have screened for<br />
suppressors of the Hic phenotype of dgk-1 null mutants. Suppressor mutations are likely to define<br />
synaptic transmission genes involved in either DAG production or, like UNC-13 in the response to DAG,<br />
or alternatively in genes acting in a parallel pathway that also modulates acetylcholine release. We have<br />
identified 26 suppressors which each suppress the Hic phenotype of dgk-1 mutants to wild type or to Ric.<br />
22 of these have wild type response to the acetylcholine receptor agonist, levamisole, indicating that they<br />
define genes that are likely acting in the motor neurons. These 22 suppressors define at least eight<br />
genes, including unc-13, an expected target of this screen. Seven suppressors are resistant or partially<br />
resistant to the effects of an exogenously added DAG analogue, PMA, suggesting that they define DAG<br />
targets. Several suppressors have weak or no obvious locomotion or Ric phenotypes in a dgk-1(+)<br />
background, suggesting that they may have regulatory roles in vesicle release. At least two of the<br />
suppressors appear to define new genes not previously implicated in acetylcholine release. Progress on<br />
suppressor characterization will be reported.<br />
1 Lackner et al., Neuron 24: 335-346 1999<br />
2 Nurrish et al., Neuron 24: 231-242 1999<br />
3 Hajdu-Cronin et al., Genes Dev 13: 1780-93 1999.<br />
4 Miller et al., Neuron 24: 323-33 1999.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
238
EVIDENCE OF A MATE-FINDING CUE IN THE FREE-LIVING<br />
SOIL NEMATODE C. ELEGANS<br />
Jasper M. Simon, Paul W. Sternberg<br />
HHMI and Caltech, Division of Biology<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Use of mate-finding cues is well documented throughout the nematode literature, but no assays for C.<br />
<strong>elegans</strong> have come into common practice. Here we report two assays that suggest young adult males<br />
can detect young adult hermaphrodites through some unidentified cue. Accumulation of 14 males (per<br />
trial) to agar conditioned with 5 hermaphrodites (the muscle mutant unc-52 was used to construct a point<br />
source), which were sequentially removed, suggests a cue given off by hermaphrodites and detected by<br />
males [mean number of males observed in 1-cm diameter scoring circles shifted from 3.2+2.2 in control<br />
trials (n=29) to 6.5+2.5 in conditioned-agar trials (n=28); p
UNDERSTANDING C27H5.1: FROM SEQUENCE TO SENSE<br />
Jessica Smith, David Pilgrim<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CW 405 Biological Sciences Bldg., University of Alberta, Edmonton, AB, T6G 2E9<br />
One of the goals of our lab is to determine the function of UNC-119, a protein involved in axon guidance<br />
and outgrowth. In the course of this study we have chosen to look at C27H5.1, a weak homologue of<br />
UNC-119 predicted by the C. <strong>elegans</strong> Genome Sequencing Project. Like UNC-119, C27H5.1 appears to<br />
be expressed early in the developing embryo and pan-neuronally in all subsequent stages of<br />
development, suggesting that it too plays a role in neural development. Unlike UNC-119, C27H5.1 shows<br />
additional expression in pharyngeal and vulval muscles suggesting other roles for this protein.<br />
Unfortunately there are no known mutations in C27H5.1. We are attempting to determine the phenotype<br />
of a C27H5.1 knockout through both deletion screening and in vivo RNA interference.<br />
While C27H5.1 is weakly similar to UNC-119, it is highly conserved (70% identity) with the delta subunit of<br />
mammalian rod phosphodiesterase (PDE6d). This protein was first identified in the retina as a subunit of<br />
rod phosphodiesterase (PDE). However, it has since been shown to be ubiquitously expressed and to<br />
interact with a number of other proteins including Rab13, RPGR, and Arl2. These interactions have not<br />
been fully characterized, but two general observations can be made. PDE6d solubilizes some proteins<br />
and affects the cGMP or GTP binding properties of others. These data suggest that PDE6d plays a<br />
regulatory role through interactions with other proteins. Interestingly, C27H5.1 interacts with and<br />
solubilizes PDE in the same manner as PDE6d suggesting functional conservation between these two<br />
proteins.<br />
To determine the role of the C27H5.1 protein in C. <strong>elegans</strong>, we are in the process of performing a yeast<br />
2-hybrid screen of a C. <strong>elegans</strong> cDNA library using C27H5.1 as bait. We are especially curious to see if<br />
C27H5.1 interacts with proteins similar to those which interact with PDEd in mammals. As well, we are<br />
producing antibodies against C27H5.1 which will allow us to further characterize the function and<br />
expression of this protein.<br />
240
GENETIC SCREENS FOR NOVEL COMPONENTS INVOLVED<br />
IN BLASTOMERE ASYMMETRY IN THE EARLY C. ELEGANS<br />
EMBRYO<br />
Martha Soto, Craig C. Mello<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
University of Massachusetts Medical Center, Worcester, MA 01605, USA<br />
In the early C. <strong>elegans</strong> embryo asymmetric cell divisions result from the partitioning of factors and from<br />
signaling between cells. At the four cell stage the EMS blastomere divides asymmetrically, leading to two<br />
daughters, E and MS, with distinct cell fates. The E blastomere is the only source of gut in the embryo,<br />
while MS makes pharynx and other tissues. This polarization requires a signal from the P2 blastomere to<br />
EMS. In addition, rotation of the spindle of EMS results in only the E daughter contacting P2. Mutations<br />
that disrupt this signal include homologs of WNT signaling factors. We have conducted forward genetic<br />
screens to identify new factors that control the asymmetric divisions of EMS. To date, our screens have<br />
identified many alleles of known WNT components, as well as a few novel genes.<br />
We will discuss a novel mutation involved in regulating cleavage orientation. Several components<br />
involved in spindle orientation have been identified by forward genetics in C. <strong>elegans</strong>, including the Par<br />
genes and let-99, and by reverse genetics, including the small GTPase CDC42 as well as G proteins. In<br />
our mutant, ne236, EMS divides left/right rather than anterior/posterior resulting in embryos in which both<br />
E and MS are in contact with P2. This phenotype is more severe than the slight disruptions in spindle<br />
rotation seen in other WNT mutations. ne236 embryos also have cell fate transformations, including<br />
frequent ectopic gut and pharynx. Interestingly, ne236 interacts with components of WNT signaling,<br />
enhancing partially penetrant gutless mutations. We are attempting to clone ne236, and testing models to<br />
understand how cleavage orientation is regulated in asymmetric cell divisions.<br />
241
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
PAX BE WITH YOU - PATTERNING THE PHARYNX<br />
Jeff Stevenson 1 , Andrew Chisholm 2 , Susan E. Mango 1<br />
1Dept. of Oncological Sciences, Huntsman Cancer Institute Center for Children, University of Utah, Salt<br />
Lake City, UT, 84112<br />
2Dept. of Biology, UC Santa Cruz, Santa Cruz, CA, 95064<br />
Formation of the pharynx consists of four phases: i) specification of pharyngeal precursors during early<br />
embryogenesis, ii) assembly of these precursors into a pharynx primordium, iii) terminal differentiation of<br />
the five pharyngeal cell types, and iv) morphogenesis of the pharynx into its adult form. Establishment of<br />
the pharyngeal precursors requires the pha-4 gene, which encodes a transcription factor expressed in all<br />
cells of the pharynx. In a pha-4 mutant background, no or few pharyngeal cells are generated.<br />
How are five different pharyngeal cell types produced from an apparently homogeneous population of<br />
precursor cells? And what is the role of pha-4 in this process, given its pan-pharyngeal expression<br />
pattern? To address these questions we are studying how one pharyngeal cell type, the marginal cell, is<br />
established during development. We have identified an early marker of marginal cells, PAX-9, a homolog<br />
of the paired transcription factor. C. <strong>elegans</strong> pax-9::GFP is expressed in all nine marginal cells, as well as<br />
three other pharyngeal cells. The involvement of transcription factors in most cell fate decisions suggests<br />
that control of marginal cell specification lies at the transcriptional level. Hence, an understanding of pax-9<br />
promoter elements may reveal the pathways that govern specification of distinct cell types within the<br />
pharynx.<br />
Deletion analysis of the pax-9 promoter shows that proper spatial and temporal expression of a<br />
pax-9::GFP reporter requires only 125 bp of upstream sequence. Within this region, we have identified a<br />
consensus PHA-4 binding site that alone is able to direct expression of a GFP reporter in nearly all cells<br />
of the developing pharynx. Significantly, evidence suggests that PHA-4 binds the pax-9 promoter in vivo,<br />
and that binding is not limited to marginal cells.<br />
Our working model postulates that the binding of PHA-4 to this promoter endows organ (pharyngeal)<br />
identity to pax-9 expression, while the binding of a second, as yet unidentified factor expressed only in<br />
marginal cells, imparts cell type identity. We are presently scanning this region for cis-elements required<br />
for the marginal cell specific expression of our reporter construct.<br />
242
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE EVOLUTION AND EXPRESSION OF FEM-2<br />
Paul Stothard 1 , Dave Hansen 2 , Tamara Checkland 1 , Dave Pilgrim 1<br />
1Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9<br />
2Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110<br />
FEM-2 is a protein phosphatase type 2C (PP2C) that regulates sex determination. We have recently<br />
focused on three aspects of FEM-2 biology. First, why do sex determining proteins in general have an<br />
accelerated rate of evolution? Second, how do mutations in other sex determining proteins affect the<br />
expression pattern of FEM-2? Third, what do the mutant alleles tell us about the function of the protein?<br />
To assess whether different selective pressures act on sex determining proteins we characterized the<br />
orthologs of C. <strong>elegans</strong> PP2C genes from the related nematode C. remanei. Comparison of the C.<br />
remanei sequences with their C. <strong>elegans</strong> orthologs indicates that FEM-2’s PP2C domain is much more<br />
divergent than the same domain in the other proteins. PP2C sequences with no known sex determining<br />
role were also isolated from the zebrafish, Danio rerio, and compared with their previously identified<br />
mouse orthologs. The zebrafish/mouse PP2C domains are more conserved than FEM-2’s PP2C domain,<br />
but less conserved than the other C. <strong>elegans</strong>/C. remanei PP2Cs. Experiments using transgenes suggest<br />
that C. remanei and C. briggsae fem-2 are able to promote male somatic development but not sperm<br />
production in C. <strong>elegans</strong>. To better understand how fem-2 regulates sexual development we have raised<br />
antisera against the FEM-2 protein. The antisera can specifically detect FEM-2 on <strong>West</strong>ern blots, and it<br />
stains sexually dimorphic structures when used on whole animals. We are currently looking for<br />
differences in staining between wild-type animals and animals that carry mutations in other sex<br />
determining genes. Finally, studies of two temperature sensitive missense alleles (b245 and q117) are<br />
underway. The b245 mutation is located in the PP2C domain while the q117 mutation is in the amino<br />
terminus. These two alleles are being characterized in vivo as well as through in vitro protein studies.<br />
243
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
FORMING A GUT: THE VIEW FROM AN ELT AND TWO ODDS<br />
Keith Strohmaier 1 , Morris Maduro 2 , Joel Rothman 2<br />
1Biochemistry and Molecular Biology Program, University of California, Santa Barbara, CA<br />
2MCDB Department, University of California, Santa Barbara, CA<br />
In an effort to understand the genetic events that lead to the formation of the intestine, we are<br />
investigating the network of regulatory factors expressed during gut development. Here, we describe our<br />
studies of three such factors: the GATA-type transcription factor, ELT-7, and two ODD-SKIPPED-like<br />
factors, ODD-1 (formerly EEL-1) and ODD-2.<br />
elt-7::gfp is expressed continuously in the E lineage beginning at the 2E cell stage. This expression<br />
pattern is identical to that of ELT-2, suggesting that these GATA factors may perform overlapping roles in<br />
gut development. Thus, ELT-2 and -7 might act redundantly and immediately downstream of the END-1<br />
and END-3 GATA factors, which redundantly specify E cell identity. However, elt-7(RNAi) embryos show<br />
no discernible phenotype, and the degraded gut phenotype of the elt-2(0) mutation does not appear to be<br />
enhanced by RNAi of elt-7. This contrasts with end-1/end-3 double RNAi, which results in a penetrant<br />
loss of gut not seen with each RNAi alone. Ectopic elt-7 expressed from a heat-shock promoter results in<br />
gut differentiation throughout the embryo, showing that ELT-7 is able to activate gut development.<br />
Ian Hope’s expression screen identified odd-1 as a gut-specific gene. odd-2 was discovered through its<br />
similarity to odd-1. Each encodes a 3 zinc finger protein similar to Drosophila ODD-SKIPPED family<br />
members. odd-1::gfp is expressed in two phases. The first phase corresponds to E lineage expression<br />
from the 4E cell stage through mid-elongation (about 1.5-fold). In the second phase, expression is<br />
continuous throughout development after elongation in intestinal-rectal valve cells and in a decreasing<br />
posterior to anterior gradient in the gut, with increased expression again in the four anterior (int1) cells.<br />
Expression of odd-2::lacZ::gfp is similar to the second phase of odd-1::gfp expression, albeit at lower<br />
levels and with somewhat altered kinetics. Both odd-1(0) and odd-2(RNAi) animals die as L1 larva, with<br />
no obvious effect on gut development.<br />
We are further characterizing the function of these genes to understand their action and regulatory<br />
interrelationships during development of the intestine.<br />
244
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
C. ELEGANS HOMOLOGUE OF PROTEIN PHOSPHATASE 4<br />
IS REQUIRED IN SPERMATOGENESIS<br />
Eisuke Sumiyoshi, Asako Sugimoto, Masayuki Yamamoto<br />
Dept. Biophys. Biochem., Grad. Schl. Sci., Univ. Tokyo, Japan<br />
Protein phosphatase 4 (PP4) is a ser/thr protein phosphatase, which is known to localize to centrosomes.<br />
Analysis using a fly mutant implicated that PP4 is required for the nucleation of microtubules during<br />
mitosis. However, its function in other organisms is not yet understood. In C.<strong>elegans</strong>, two possible<br />
homologues of PP4, namely Y75B8A.30 and Y49E10.3, have been identified by the genome project.<br />
Here we report characterization of Y75B8A.30.<br />
To understand the function of Y75B8A.30, we performed RNAi for it. Although the penetrance varied<br />
among injections, RNAi for Y75B8A.30 at the highest efficiency caused 25% embryonic lethality at F1 and<br />
nearly 100% embryonic lethality at F2.<br />
Y75B8A.30 (RNAi) F2 embryos showed a multi polar spindle with two male pronuclei at 1-cell stage. We<br />
suspected that an extra male pronucleus might result from a defect in spermatogenesis. To confirm this,<br />
we examined the spermatogenesis of the F1 progeny. Observation with Nomarski optics and with DAPI<br />
staining revealed that F1 spermatids often contained multiple nuclei. We also found that the nuclei of<br />
secondary spermatocytes of F1 worms were not properly separated, indicating that Y75B8A.30 is<br />
required for proper chromosome segregation in sperm meiosis. To see whether the multi polar spindle is<br />
a result of the defect in sperm, F1 males were crossed to rde-1 hermaphrodites, which is resistant to<br />
RNAi. 5/16 of cross progeny showed multi polar spindles at 1-cell stage, indicating that multi polar<br />
spindles are indeed caused by the defect of sperm at least in part. Thus, we conclude that PP4 is<br />
required for the segregation of chromosomes and centrosomes during spermatogenesis.<br />
To see whether Y75B8A.30 function is required in embryogenesis, we crossed Y75B8A.30 (RNAi) F1<br />
hermaphrodites with wild type male. Although wild type sperm were supplied in this cross, many cross<br />
progenies died during embryogenesis, showing that Y75B8A.30 is required for embryogenesis. To know<br />
the possible role of Y75B8A.30 in mitosis, we stained F2 embryos with anti-tubulin antibody and DAPI.<br />
Some fraction of 1-cell embryos had condensed chromosomes at the metaphase plate, with microtubules<br />
mislocalized around the nucleus, suggesting that Y75B8A.30 may be involved in the organization of<br />
microtubules during mitosis.<br />
245
TRANSCRIPTIONAL REGULATION OF THE TRYPTOPHAN<br />
HYDROXYLASE GENE TPH-1<br />
Ji Ying Sze<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Anatomy and Neurobiology, Univ. of California, Irvine CA 92697<br />
tph-1 encodes a tryptophan hydroxylase, the key enzyme for serotonin biosynthesis. When I was in Gary<br />
Ruvkun’s lab, I collaborated with Martin Victor in Yang Shi’s lab and isolated a tph-1 deletion mutation,<br />
tph-1(mg280). tph-1(mg280) shows no detectable serotonin based on anti-serotonin antibody staining<br />
and exhibits several behavioral and metabolic defects (Sze et al., 2000). It has been proposed that in<br />
mammals changes in the level of TPH mRNA correlate with long-term changes in the cellular serotonin<br />
level and that the regulation of TPH mRNA tends to be "inducer"- and region-specific (Semple-Rowland et<br />
al., 1996; Clark and Russo, 1997; Siuciak et al., 1998. Bethea, et al., 2000). Thus, the regulation of TPH<br />
expression may be a key step by which the nervous system adjusts its long-term synaptic serotonin<br />
levels.<br />
To identify genes regulating tph-1, I have started a genetic screen for mutations that alter tph-1::gfp<br />
expression in specific neurons. Thus far, mutations identified can be classified into three classes. (1) I<br />
have previously reported that mutations in the POU-homeodomain transcription factor UNC-86 abolish<br />
tph-1::gfp expression in NSM and HSN, but the expression in ADF is not affected. UNC-86 is expressed<br />
in HSN and NSM, but not in ADF. (2) I have identified 18 mutations where tph-1::gfp expression in ADF is<br />
reduced or eliminated. Laser ablation of ADF and ASI causes animals to form dauers (Bargmann and<br />
Horvitz, 1993; Schackwitz et al., 1996) and the tph-1(mg280) mutation downregulates daf-7::gfp<br />
expression and enhances daf-7(e1372) dauer formation at 15 o C. To test the role of ADF-produced<br />
serotonin is important for a non-dauer growth, I am testing if these mutations enhance daf-7(e1372) dauer<br />
formation at 15 o C. (3) I have identified one mutation that has tph-1::gfp expressed in an extra neuron.<br />
These mutants provide me as useful means to characterize the role of serotonin produced in specific<br />
neurons.<br />
246
SEARCHING FOR NEW GENES INVOLVED IN DOSAGE<br />
COMPENSATION<br />
Chun Tsai, Barbara J. Meyer<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,<br />
Berkeley, CA 94720<br />
In C. <strong>elegans</strong> dosage compensation equalizes expression of X-linked genes between males (XO) and<br />
hermaphrodites (XX). This chromosome-wide regulatory process is essential for hermaphrodite<br />
development. A biochemically defined protein complex containing DPY-26, DPY-27, DPY-28 and MIX-1<br />
has been shown to associate with X chromosomes in a sex-specific fashion and is required for global<br />
repression of hermaphrodite X-linked genes. MIX-1 is also required for proper segregation of mitotic<br />
chromosomes. Mutations in mix-1 cause lethality in both sexes and therefore were missed from our<br />
screens for sex-specific lethals. To identify other dosage compensation components that might have been<br />
missed, we used the following observation: mutations in all known dosage compensation genes suppress<br />
the sexual transformation caused by certain sex determination mutations in a dominant fashion, allowing<br />
recessive lethal mutations to be identified by their dominant suppression effect. For example,<br />
heterozygous mix-1 mutations can suppress the masculinization of XX animals caused by homozygous<br />
sdc-3(Tra) mutations. The suppression screen is shown below.<br />
sdc-3(Tra)/+ sdc-3(Tra) 99% Tra, 1% hermaphrodites<br />
m/+; sdc-3(Tra)/+ m/+; sdc-3(Tra) 30-99% hermaphrodites isolate mutants<br />
So far we have screened 12,000 haploid genomes and identified 9 mutations that cause moderate to<br />
strong suppression: two are lethal mutations, two are maternal-effect lethal, one is a mix-1 allele, one is<br />
an sdc-3 null allele, and three show no obvious phenotypes. The two lethal and one maternal-effect lethal<br />
mutations have been mapped extensively and their map positions indicate that they are mutations in<br />
three different genes that have not previously been implicated in dosage compensation.<br />
Immunofluorescence studies showed that the two recessive lethal mutations disrupt the association of<br />
DPY-26 and DPY-27 with the X chromosome. DAPI staining of dead embryos revealed abnormal DNA<br />
morphology and multinucleated cells, suggesting an essential role in chromosome metabolism. These<br />
observations suggest that the mutations isolated in the screen identify new genes that play a role in<br />
dosage compensation as well as chromosome structure. Attempts are ongoing to clone the genes.<br />
247
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CHARACTERIZING THE ROLE OF LET-99 IN SPINDLE<br />
ORIENTATION<br />
Meng-Fu Tsou, Adam Hayashi, Lesilee S. Rose<br />
Section of Molecular and Cellular Biology, University of California, Davis, CA 95616 USA<br />
Cell division plane, which is determined by the orientation of the mitotic spindle, is important for normal<br />
development. The dynein-dynactin complex has been shown to be part of the mechanical force for<br />
spindle orientation in C. <strong>elegans</strong> embryos. Cytoplasmic dynein and dynactin are also essential in a variety<br />
of cell division processes in different organisms, including nuclear positioning and anaphase B spindle<br />
pole separation, partially due to their role in connecting the astral microtubules and cell cortex.<br />
The let-99 gene plays an important role in spindle orientation. We have cloned the let-99 gene and found<br />
it encodes a novel protein that is found in the cytoplasm and at regions of cell-cell contacts in early<br />
embryos. let-99 mutants show three major defects in cell division: first, abnormal spindle positioning in<br />
both the AB and the P cell lineage, second, instability in position of the nuclear-centrosome complex at<br />
prophase (nuclear rocking), and third, poor separation of the spindle poles at anaphase B. Double mutant<br />
analysis indicates that the nuclear rocking activity seen in let-99 embryos requires the function of the<br />
dynein-dynactin complex , suggesting that this behavior may be due to abnormal interaction between<br />
astral microtubules and the cell cortex. However the nuclear rocking phenotype is not due to the<br />
mislocalization of dynein-dynactin complex, because both the localization of DHC-1 (dynein heavy<br />
chain-1) and DNC-1 (p150glued) appear to be normal in let-99 embryos. G protein beta subunit (GPB-1)<br />
was also shown to be required in spindle orientation. Embryos in which the GPB-1 is maternally depleted<br />
show similar phenotypes to that of let-99, suggesting that gpb-1 and let-99 may function in the same<br />
biochemical pathway. Our preliminary result from let99 gpb-1(RNAi) double mutant analysis supports this<br />
idea. The localization of LET-99 in gpb-1(RNAi) embryos appears to be normal, indicating that the<br />
localization of LET-99 does not require the gpb-1 signaling pathway. We are currently exploring the<br />
relationship among LET-99, dynein-dynactin complex and GPB-1 by a variety of techniques, in addition to<br />
searching for LET-99 interacting proteins using the two-hybrid approach.<br />
248
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CHARACTERIZATION AND CLONING OF THE MUSCLE<br />
ACTIVATION GENE UNC-58<br />
Monika Tzoneva, James H. Thomas<br />
Dept. of Genetics, University of Washington, Seattle WA 98195<br />
unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying<br />
muscle in C. <strong>elegans</strong>. unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. Putative<br />
loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These<br />
alleles have no obvious phenotype on their own, suggesting that unc-58(dm) mutants result in an<br />
inappropriately activated gene product. unc-58(dm) animals also frequently flip around their longitudinal<br />
axis. Both the flipping and the hypercontarction phenotypes are partially rescued by the drug endosulfan,<br />
best known as an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal<br />
communication; our unpublished data).<br />
I have cloned unc-58 and found that it encodes a potassium channel of the TWIK family. TWIKs are<br />
distinct from other potassium channels because they have four transmembrane domains (M1 to M4) and<br />
two pore domains. Potassium-selective currents have been recorded from TWIKs in both mammals and<br />
worms.<br />
The three unc-58 gain-of-function mutations cluster in the C-terminal (cytoplasmic) part of the M4<br />
transmembrane domain, which is equivalent to the S6 domain of the voltage-gated potassium channel<br />
family. S6 is thought to form a channel gate that opens in response to voltage changes. The location of<br />
the unc-58(dm) mutations suggests that its M4 helix may also form an activation gate, though the stimulus<br />
that opens it is unknown.<br />
The molecular identity of unc-58 raises several questions. First, how does an activated mutation in a<br />
putative potassium channel lead to muscle hypercontraction? It is possible that the hypercontraction is<br />
due to UNC-58 function in inhibitory motorneurons, while its slow movement may be due in part to its<br />
function in other tissues. We will test this by determining the unc-58 site(s) of action. Second, how is the<br />
unc-58(dm) phenotype rescued by the drug endosulfan? We will attempt to determine whether this rescue<br />
is by direct channel block. Third, how does the UNC-58 channel contribute to overall membrane<br />
excitability? It is not known whether or how UNC-58 activity is regulated and whether it acts alone or in<br />
complex with other subunits. We have isolated extragenic suppressors of unc-58(dm), which may offer<br />
insight into this question.<br />
249
THE STRUCTURE/FUNCTION RELATIONSHIP OF CLK-1 IN<br />
THE NEMATODE CAENORHABDITIS ELEGANS<br />
Antonio Ubach, Siegfried Hekimi<br />
Department of Biology, McGill University, Canada<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The lifespan of the nematode <strong>Caenorhabditis</strong> <strong>elegans</strong> is genetically determined by several classes of<br />
genes. Our laboratory is particularly interested in the clk class of genes, which is composed of clk-1, clk-2,<br />
clk-3, and gro-1. Mutations in these genes have been shown to extend lifespan and to deregulate several<br />
developmental and behavioral processes. For example, clk-1 mutants exhibit an average slowing down of<br />
cell division, pharyngeal pumping, and defecation rates relative to wild-type animals. These phenotypes<br />
are maternally rescued, that is homozygous mutant animals coming from a heterozygous mother are<br />
phenotypically wild type. clk-1 encodes a 187 amino acid mitochondrial protein that is composed of two<br />
homologous TRC domains (TRC for Tandemly Repeated in CLK-1). Interestingly, the yeast homologue of<br />
clk-1, COQ7, has been implicated in ubiquinone biosynthesis. Moreover, coq7 mutants are respiration<br />
defective, yet the respiratory competence can be restored by the addition of exogenous ubiquinone. In<br />
contrast, cellular respiration is only slightly affected in clk-1 mutant worms. However, C. <strong>elegans</strong> clk-1 as<br />
well as the rat and the human homologues are capable of functionally complementing the Dcoq7 mutant.<br />
This observation suggests that the biochemical function of clk-1 have been conserved throughout<br />
evolution.<br />
We have shown previously that a recombinant CLK-1::GFP fusion protein is capable of rescuing the clk-1<br />
mutant phenotype when expressed from transgenic arrays. In the current study, we are taking advantage<br />
of this observation to try to understand the structural requirements for CLK-1 function. In order to address<br />
this, we are using a site-directed mutagenesis approach to generate several new CLK-1 mutant proteins.<br />
We are analysing the effect these CLK-1 mutant proteins have on the general clk-1 phenotype. We are<br />
also examining the subcellular distribution of the mutant fusion proteins in different genetic backgrounds,<br />
such as the wild type, the clk-1 partial loss of function e2519, and the two clk-1 putative nulls qm30 and<br />
qm51.<br />
250
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
CHARACTERIZATION OF TRANSCRIPTIONAL REGULATION<br />
BY A CLASS OF MONOMERIC NUCLEAR RECEPTORS<br />
FOUND IN C. ELEGANS<br />
Marc R. Van Gilst, Keith R. Yamamoto<br />
Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA<br />
94143-0450<br />
Nuclear hormone receptors (NHRs) comprise a large family of metazoan transcription factors that<br />
participate in numerous developmental and metabolic processes. NHRs commonly target their<br />
transcriptional regulation to particular genes through interaction with DNA sequences called hormone<br />
response elements (HREs) and the nature of the transcriptional regulation carried out by a NHR at these<br />
genes is often determined by interaction with a small hydrophobic ligand.<br />
C. <strong>elegans</strong> provides unique advantages over other animal models for studying the physiological<br />
significance of several aspects of NHR function (i). Targeting of NHRs to promoters by HREs (ii)<br />
Characterization of important regulatory domains of NHRs (iii) Interaction of NHRs with protein cofactors<br />
and (iv) Interaction of NHRs with potential ligands.<br />
We have focused our studies on the C. <strong>elegans</strong> homologs of a class of NHRs that can activate<br />
transcription from AGGTCA DNA half-sites, a common binding motif for a mammalian sub-family of<br />
monomeric nuclear receptors that includes ROR, TLX, ERR and SF-1. To this end, we have started our<br />
investigations by utilizing the C. <strong>elegans</strong> receptor CHR3 (nhr-23). CHR3 is closely related to the<br />
mammalian receptor ROR, with nearly 100% conservation in the DNA binding domain, and would be<br />
expected to fall into the class of monomeric NHRs that we wish to study.<br />
Using yeast and worm reporter assays, we have characterized the transcriptional regulatory properties of<br />
CHR3. We found that CHR3 can activate transcription from a single AGGTCA half-site and activates<br />
synergistically from multimerized half-sites. Transcriptional activation occurs in yeast in the absence of<br />
any exogenous ligand. Similar to mammalian receptors, both the N and C terminal domains of CHR3<br />
contain activation functions. Furthermore, CHR3 requires ATP-dependent chromatin remodeling<br />
complexes for activation and can interact with a mammalian coactivator GRIP through a conserved<br />
structural mechanism. We are currently expanding these systems to find and study other C. <strong>elegans</strong><br />
NHRs and to set up screens for the identification of potential protein cofactors and small-molecule<br />
ligands.<br />
251
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UNC-4 TARGETS ACR-5 AND DEL-1: ARE THEY<br />
DETERMINANTS OF SYNAPTIC CHOICE?<br />
Stephen E. Von Stetina, David M. Miller, III<br />
Dept. of Cell Biology, Vanderbilt University , Nashville, TN 37232-2175<br />
Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. EM<br />
reconstruction of an unc-4 mutant revealed that the VA motor neurons fail to receive synapses from their<br />
usual interneuron partners and instead accept inputs normally reserved for their sisters, the VB motor<br />
neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function<br />
together in the VA motor neurons to repress VB-specific genes. Two genes, del-1 (DEG/ENaC sodium<br />
channel subunit) and acr-5 (alpha-like nictonic acetylcholine receptor subunit), are ectopically expressed<br />
in the VA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins and ion channel<br />
components, ACR-5 and DEL-1 are attractive candidates for mediators of synaptic specificity. We have<br />
now performed genetic experiments, however, that rule out a necessary role for either ACR-5 or DEL-1<br />
in the specification of VB-type inputs. Deletion mutants of del-1 and acr-5, as well as the double mutant<br />
(acr-5;del-1), do not perturb forward locomotion as would be expected for a mutation which disrupts<br />
normal inputs to the VBs. Furthermore, assays designed to quantitate locomotion have detected no<br />
differences between wild type and these mutants. Placement of acr-5 and del-1 mutants in an unc-4<br />
background does not suppress the Unc-4 phenotype, suggesting that ACR-5 and DEL-1 are also not<br />
required to promote VB-type inputs in VA motor neurons. A future goal is to ectopically express ACR-5<br />
and DEL-1 in VA motor neurons to determine if these proteins are sufficient to cause miswiring. Also, we<br />
will define the UNC-4 mediated repression domains in the acr-5 and del-1 promoters in an effort to<br />
identify additional UNC-4 targets.<br />
252
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
NICOTINE ADAPTATION: A PROCESS INVOLVING<br />
PKC-DEPENDANT REGULATION OF NACHR PROTEIN<br />
LEVELS.<br />
Laura Waggoner, Kari Dickonson, Daniel Poole, Bill Schafer<br />
Department of Biology, UCSD, 9500 Gilman Dr., La Jolla, 92093<br />
C. <strong>elegans</strong> is capable of undergoing adaptation to prolonged exposure to nicotine, through attenuation of<br />
the activity of nicotinic acetylcholine receptors (nAChRs). We have discovered that in egg-laying behavior,<br />
acute nicotine treatment results in stimulation of egg-laying, but long-term exposure inhibits further<br />
stimulation by cholinergic agonists. Furthermore, we found that this adaptation process involves<br />
UNC-29-containing nAChRs functioning in the vulval muscles. We were interested in determining whether<br />
adaptation was due to a loss or attenuation of theseUNC-29-containing nAChRs; to address this, we<br />
analyzed fluorescence levels of an UNC-29::GFP chimera in the vulval muscles before and after nicotine<br />
treatment. Intriguingly, we found that nicotine treatment in fact decreased receptor protein levels in the<br />
vulval muscles. In addition, we found that this regulation was independent of the unc-29 promoter and 3’<br />
UTR. Lastly, we were interested in identifying other genes involved in this process, and we found that the<br />
protein kinase C homolog encoded by tpa-1 was necessary for the observed decrease in nAChR protein<br />
levels. Thus, it appears that nicotine treatment results in a PKC- dependant down-regulation of<br />
UNC-29-containing nAChRs in the vulval muscles. Further screens are being performed to identify other<br />
genes involved in this process.<br />
253
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ANALYSIS OF GLR-7, GLR-5, IONOTROPIC GLUTAMATE<br />
RECEPTOR SUBUNITS<br />
Craig S. Walker, David M. Madsen, Penelope J. Brockie, Andres V.<br />
Maricq<br />
Department of Biology, University of Utah, Salt Lake City, UT 84112<br />
Of the ten putative ionotropic glutamate receptor subunits identified in the worm, two non-NMDA subunits,<br />
glr-5 and glr-7, show a very restricted expression pattern. When GLR-5 and GLR-7 are fused to GFP they<br />
show expression in only a single cell, the interneuron RIA. RIA receives synaptic input from the neurons<br />
AIY and AIZ. Previous studies have defined this group of cells to form part of the circuit that controls<br />
thermotaxis.<br />
To determine the possible roles of glr-5 and glr-7 in thermotaxis, we have generated deletion mutations in<br />
both genes. These deletions remove a portion of the extracellular domain and the first three<br />
transmembrane domains. In each case the mutations result in a functional null. Analysis of glr-5(ak57)<br />
deletion mutants using thermotaxis assays is underway. glr-5(ak57) worms also appear to have a<br />
diminished brood size, which is more severe at elevated temperatures.<br />
In order to examine the role of RIA in processing thermal information we have generated transgenic<br />
strains which express an activated form of the GLR-1 glutamate receptor subunit, GLR-1 (A/T), under<br />
control of the glr-5 promoter. GLR-1 (A/T) causes constant depolarization when expressed in other cells<br />
(see abstract by Zheng et al.). We are currently characterizing the thermotaxis behavior of these strains.<br />
Any thermotaxic phenotype observed in glr-5(ak57) may be a result of the inability of RIA to receive<br />
signals from the presynaptic cell AIY. Hobert et al. have demonstrated that the gene ttx-3 is expressed<br />
only in AIY. Using ttx-3::VAMP::CFP and glr-5::GLR-5::YFP constructs we are looking at the presynaptic<br />
and post-synaptic components of this synapse to determine if glr-5 could be mediating the signal from<br />
AIY.<br />
254
MICROARRAY ANALYSIS OF GENE EXPRESSION<br />
PATTERNS IN DAUER LARVAE<br />
John Wang, Stuart K. Kim<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept of Developmental Biology, Stanford University, Stanford CA<br />
DNA microarrays containing about 95% of the genome can now be used to analyze gene expression<br />
differences from nearly the entire genome. We are using the full-genome microarrays to profile gene<br />
expression differences associated with the dauer larvae.<br />
Under conditions of starvation or extreme crowding, C. <strong>elegans</strong> enter an alternative arrested stage, the<br />
dauer larva. These larvae exhibit a unique morphology, an extended life span and resistance to<br />
environmental stresses. The decision to form dauers is controlled by TGF-beta and insulin signaling<br />
pathways.<br />
The dauer larvae is an ideal system for microarray analysis. The dramatic morphological and<br />
physiological changes that occur upon entrance into and exit from the dauer larvae suggest global<br />
transcriptional regulation. In principle, by examining the gene expression differences associated with the<br />
dauer larvae on the whole-genome level, we will be able to illuminate the complete set of genes that are<br />
implicated for the dauer-specific attributes. In particular, these candidate genes would provide a<br />
framework for understanding dauer-specific characteristics, such as altered energy metabolism, certain<br />
aspects of aging and longevity, stress resistance, and the coordinated regulation and execution of events<br />
necessary for a complex morphological change.<br />
To identify the genes involved with dauer exit, we are performing DNA microarray experiments with RNA<br />
isolated at different time points after addition of food to a pure dauer population. Each RNA sample is<br />
compared to a common reference RNA, and multiple RNA samples are prepared for each time point. We<br />
will identify those genes that are reproducibly altered during dauer exit and examine their kinetic profiles.<br />
This analysis should reveal the temporal sequence of events that occur during dauer recovery at the<br />
transcriptional level.<br />
255
CHARACTERIZATION OF CAN CELL AND EXCRETORY<br />
CANAL DEFECTS IN MIG-10(CT41) MUTANTS<br />
Nicole Washington, Jim Manser<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Biology, Harvey Mudd College, Claremont CA 91711<br />
We have continued our characterization of mig-10 mutant defects using a ceh-23-gfp reporter strain<br />
(kyIs5 IV, kindly provided by Jennifer Zallen) which allows scoring of CAN cell bodies and axons (see<br />
Manser et al. WCWM 1998 abstracts, p.167). In the current studies, we have focused on the amber<br />
(putative null) mig-10(ct41) allele.<br />
Nearly 90% of CAN cell bodies (61/68) are significantly displaced anteriorward in mig-10(ct41);kyIs5<br />
animals, compared to less than 8% (5/64) for the kyIs5 strain. Manser and Wood (1990) previously<br />
reported a penetrance of 60% for CAN cell body misplacement in mig-10(ct41). The higher penetrance<br />
observed using the gfp reporter probably reflects a synergy between mig-10 and ceh-23-gfp similar to that<br />
reported by Forrester et al. (1997) for other CAN migration mutants.<br />
A significant percentage of posteriorly directed CAN axons in mig-10(ct41);kyIs5 animals (23/67)<br />
terminate in positions significantly anterior to their normal target region near the anus. In contrast, all<br />
anteriorly directed CAN axons appear to extend to their normal target region in the head. This apparent<br />
directional bias is somewhat surprising given that mig-10(ct41) disrupts both anteriorward and<br />
posteriorward cell body migrations (Manser and Wood, 1990). One possibility is that the CAN axon<br />
defects are an indirect result of the shortening of the posterior excretory canals observed in mig-10(ct41)<br />
(Manser and Wood, 1990). Specifically, because CAN axons are closely associated anatomically with the<br />
excretory canals (CAN=canal associated neuron), perhaps the canals serve as guidance cues for axon<br />
outgrowth.<br />
To investigate this possibility, we have scored both CAN axon and posterior excretory canal defects in the<br />
same individuals. In more than 90% of mig-10(ct41);kyIs5 animals scored (28/31), the posteriorly directed<br />
CAN axon extended significantly beyond the point of termination of the posterior excretory canal. This set<br />
includes animals in which the posterior CAN axon extends to the anal region while the posterior excretory<br />
canal terminates significantly anterior to the vulval region. Due to the generally severe truncation of the<br />
posterior canals caused by mig-10(ct41), we have also observed animals in which the CAN cell body is<br />
positioned posterior to the canal terminus. In such animals, anteriorly directed CAN axons were observed<br />
to extend over regions of the anterior-posterior axis from which canals were missing. These observations<br />
do not support the view that the excretory canals serve as guidance cues for CAN axon outgrowth.<br />
256
RIC-7 ENCODES A NOVEL PRESYNAPTIC PROTEIN<br />
REQUIRED FOR NEUROTRANSMISSION<br />
Robby M. Weimer, Erik M. Jorgensen<br />
University of Utah, Salt Lake City, UT 84112<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Regulated release of neurotransmitter from presynaptic terminals requires the coordinated function of<br />
several presynaptic proteins. Our current knowledge of this process has come primarily from biochemical<br />
analysis of synaptic proteins. We have taken a genetic approach in <strong>Caenorhabditis</strong> <strong>elegans</strong> to<br />
complement these studies. Previously, we reported the identification of a novel protein, RIC-7, identified<br />
genetically by screens for neurotransmission mutants. We now report that RIC-7 functions in neurons<br />
and is localized to presynaptic terminals.<br />
Mutations in ric-7 result in a pleiotropic neurotransmission phenotype. ric-7 animals lack enteric muscle<br />
contractions during defecation and display a weak shrinker phenotype, indicating of a loss of GABAergic<br />
function. In addition, ric-7 animals are resistant to the acetylcholinesterase inhibitor aldicarb, although<br />
they remain sensitive to the acetylcholine receptor agonist levamisole indicating a presynaptic cholinergic<br />
defect. These phenotypes are not due to defects in neuronal developmental since the overall structure of<br />
the nervous system appears normal. This suggests that mutations in ric-7 disrupt a common presynaptic<br />
step in chemical neurotransmission.<br />
The ric-7 phenotype is rescued by an 18kb PCR fragment that contains a single open reading frame<br />
encoding a predicted 694 amino acid protein with no similarities to known proteins or functional motifs.<br />
Expression of the reporter gene green fluorescent protein (GFP) from the ric-7 promoter identifies a<br />
predominantly neuronal expression pattern. RIC-7 GFP fusion protein is localized to synaptic terminals<br />
independent of UNC-104 function. Furthermore, expression of RIC-7 solely in GABAergic neurons<br />
rescues all GABA-specific phenotypes.<br />
Together, these data suggest that ric-7 encodes a neuronal protein that is localized to and functions at<br />
presynaptic terminals. In addition, ultrastructural data indicate that synaptic vesicle biogenesis is not<br />
affected in a ric-7 mutant background. Thus, RIC-7 is likely to play a role in synaptic vesicle exocytosis.<br />
Ongoing experiments are designed to identify RIC-7 interacting proteins and to determine the molecular<br />
function of RIC-7 in neurotransmission.<br />
257
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
THE REQUIREMENT OF SYNAPTIC VESICLE LOADING FOR<br />
SYNAPTIC VESICLE EXOCYTOSIS<br />
Robby M. Weimer, Janet E. Richmond, Erik M. Jorgensen<br />
Department of Biology, University of Utah, Salt Lake City, UT 84112-0840<br />
Neurotransmitters are released from presynaptic terminals by the fusion of synaptic vesicles with the<br />
plasma membrane. The amount of neurotransmitter in a vesicle causes a consistent amount of current,<br />
called a quanta, in the postsynaptic cell. Studies have shown that quanta are invariant within a synapse.<br />
This observation raises an interesting question. Does synaptic vesicle fusion require the proper loading<br />
of neurotransmitter into the vesicle? We have characterized a mutant in C. <strong>elegans</strong> that will allow us to<br />
address this question.<br />
Loading of neurotransmitter into synaptic vesicles requires a vesicular neurotransmitter-specific<br />
transporter and a driving force. Several studies have shown that the vacuolar H+ ATPase (vATPase)<br />
generates the required driving force for loading. Previously, we reported the cloning of the C. <strong>elegans</strong><br />
homolog of the vacuolar ATPase B subunit, which we now refer to as vha-12 (vacuolar-type H+ ATPase).<br />
A hypomorphic allele of vha-12 results in a pleiotropic neurotransmission phenotype which is consistent<br />
with the know function of the vATPase in neurotransmission. However, animals homozygous for the<br />
hypomorphic allele are not paralyzed. This suggests that neurotransmitter is still released from<br />
presynaptic terminals in a vha-12 animal.<br />
Two models could account for this observation (1) synaptic vesicles are fusing with the plasma membrane<br />
with incomplete loading of neurotransmitter or (2) only completely loaded vesicles are competent to fuse<br />
and the proportion of competent vesicles are decreased in a vha-12 mutant background. In order to<br />
distinguish between these two models we are characterizing neurotransmitter release events in a vha-12<br />
background by electrophysiologically recording from neuromuscular junctions.<br />
258
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
UNRAVELING THE BIOLOGICAL ROLE OF DMWD, A GENE<br />
CLOSE TO THE UNSTABLE CTG-REPEAT IN THE MYOTONIC<br />
DYSTROPHY LOCUS.<br />
J. <strong>West</strong>erlaken 1 , B. Wieringa 2 , P.E. Mains 1<br />
1Department of Biochemistry & Molecular Biology, University of Calgary, Canada<br />
2Department of Cell biology University of Nijmegen, the Netherlands<br />
Myotonic Dystrophy (DM) is a frequent autosomal dominant disorder, caused by the expansion of an<br />
unstable (CTG)n-repeat. The expanding repeat is located in the 3’-untranslated region of the DM protein<br />
kinase gene (DMPK). The DMPKgene is located in a gene dense area, therefore it is unclear if it is<br />
theonly gene involved in disease manifestation. Another possible candidate is the DMWD gene (formally<br />
known as DMR-N9 in mouse, or gene 59 in human), which is located closely upstream of the DMPK<br />
gene. The DMWD gene is mainly expressed in brain, testis and some smooth muscle tissues. In brain the<br />
protein is located in many areas, but most prominently in places where there are synaptic glomeruli. In<br />
testis the protein appears to be located in several distinct stages of spermatogenesis.<br />
The DMWD gene is conserved during evolution and has homologs in plant, yeast and nematodes. To<br />
futher understand the biological role of DMWD we started to investigate the C. <strong>elegans</strong> DMWD gene. In<br />
C. <strong>elegans</strong> DMWD RNA is expressed in embryos and gravid adults. Preliminary results from RNAi studies<br />
suggest that some progeny of the injected worms are sterile. We are further planning to do RNA in situ<br />
hybridisation, GFP tagging of the protein and antibody staining experiments.<br />
259
CALCIUM IMAGING OF THE DEFECATION RHYTHM IN C.<br />
ELEGANS<br />
Jeanna M. Wheeler, James H. Thomas<br />
Department of Genetics, University of Washington, Seattle, WA 98195<br />
Ultradian rhythms (those with a period shorter than 24 hours) have been shown to regulate a number of<br />
biological processes, such as the vertebrate heartbeat and invertebrate swimming behavior. The<br />
defecation cycle in C. <strong>elegans</strong> is an ultradian rhythm regulated by a clock-like mechanism (1). The cycle<br />
has a 45 to 50 second periodicity that is insensitive to changes in temperature between 18° and 30° C. In<br />
addition, the clock mechanism can keep time in the absence of the defecation motor program (when<br />
animals leave their food source), indicating that the clock is distinct from the motor program. It has been<br />
shown that the inositol trisphosphate (IP 3 ) receptor, a calcium channel that regulates cytoplasmic Ca 2+<br />
concentration by release from intracellular stores, is required in the intestine for the defecation cycle (2).<br />
Furthermore, calcium imaging with fura-2 revealed that periodic calcium spikes occur in the most<br />
posterior intestinal cell, just preceding the initiation of each posterior body-wall muscle contraction (pBoc).<br />
The pBoc begins at the posterior of the animal and proceeds anteriorly in a wave-like motion. There are<br />
two simple possibilities for how the calcium spike signals to the posterior body-wall muscles: 1) Ca 2+ may<br />
initiate secretion of a muscle activator only in the posterior intestine, which then propagates anteriorly<br />
through the pseudocoelomic space or by muscle-to-muscle signaling, or 2) the Ca 2+ spike may be<br />
propagated anteriorly through the intestinal cells themselves, initiating signaling to adjacent muscle cells<br />
along the way. In order to distinguish between these possibilities, I am constructing a strain that<br />
expresses fluorescent indicators for Ca 2+ ("cameleons") in the intestine. This strain will be used to<br />
characterize the defecation cycle in wild type worms, as well as in various mutant strains with cycle<br />
defects.<br />
1. D.W. Liu and J.H. Thomas, 1994. J Neurosci 14: 1953-1962.<br />
2. P. DalSanto et al, 1999. Cell 98: 757-767.<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
260
ESTABLISHING THE LEFT/RIGHT ASYMMETRY OF Q<br />
NEUROBLAST POLARISATION AND MIGRATION IN C.<br />
ELEGANS<br />
Lisa Williams, Lee Honigberg, Cynthia Kenyon<br />
University of California, San Francisco<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
The QL and QR neuroblasts are born in bilaterally symmetric positions along the A-P body axis in C.<br />
<strong>elegans</strong> (QL on the left and QR on the right). Shortly after hatching, the two cells undergo characteristic,<br />
stereotypical L/R asymmetric migrations. QL extends a long process to the posterior and its nucleus then<br />
migrates into this projection. Following this migration, the Hox gene mab-5 is expressed in QL and its<br />
descendants remain in the posterior. mab-5 expression is both necessary and sufficient for Q<br />
descendants to remain in the posterior. Conversely, QR extends a process to the anterior and<br />
subsequently migrates anteriorly. mab-5 is not expressed in QR, hence its descendants continue to<br />
migrate anteriorly. Each cell moves to a well-defined final position.<br />
In unc-40 or dpy-19 mutants, polarisation of both Q cells is randomised and the Q nuclei fail to undergo<br />
their migrations. This randomisation of Q polarisation is coupled with a randomisation of mab-5<br />
expression. Either cell, QL or QR, can express mab-5, and this expression then determines the fate of<br />
that cell’s descendants.<br />
In order to gain a more complete picture of how the initial L/R asymmetry of the Q cells is established, we<br />
are studying 11 new mutants from an extensive Q cell migration screen (Queelim Ch’ng and Mary Sym,<br />
unpublished results). These mutants fall into 4 complementation groups and have similar phenotypes to<br />
unc-40 and dpy-19, and are therefore good candidates for new genes in this pathway. Work is underway<br />
to characterise, map and clone these new genes. qid --1 (Q Is Defective, 8 alleles) maps to a 180kb<br />
region on the left arm of chromosome III, and is rescued by injection of a cosmid pool covering this<br />
region. qid-2 (1 allele) maps to the right arm of chromosome X, and further mapping is underway.<br />
Mapping and further characterisation are also planned for qid-3 (1 allele) and qid-4 (1 allele), although<br />
these mutants have less penetrant Q phenotypes and appear to be more pleiotropic.<br />
261
A SCREEN FOR CELL MIGRATION AND AXON OUTGROWTH<br />
MUTANTS<br />
Jim Withee, Gian Garriga<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Molecular and Cellular Biology, University of California, Berkeley<br />
Proper development of the nervous system requires that individual neurons and their processes arrive at<br />
precise coordinates to form the correct pattern of connectivity. Directed migration of cells and growth<br />
cones involves recognition of environmental cues by receptor proteins and the translation of these cues<br />
into growth or movement to precise destinations. Thus, a primary goal of developmental neurobiology is<br />
the identification and characterization of directional cues, their cognate receptors and the intracellular<br />
proteins that interpret these cues.<br />
The left and right CAN cells are born in the head and migrate posterior to the gonad primordium. After<br />
migration is complete, the CAN cells each extend a single, unbranched axon anterior to the base of the<br />
nerve ring and posterior to the lumbar ganglia. <strong>Worm</strong>s expressing GFP from the ceh-23 promoter exhibit<br />
fluorescence from a subset of neurons including the CAN cell (Wang et al, 1993, Cell 74). We are utilizing<br />
a ceh-23::GFP expressing strain and fluorescence microscopy to screen directly for mutations that disrupt<br />
proper migration and axon outgrowth of the CANs. To date, we have identified at least five<br />
complementation groups that display defective cell migration and axon outgrowth. The mutants exhibit a<br />
wide variety of CAN axon defects ranging from simple truncation to extensive ectopic outgrowth and<br />
branching. We are in the process of mapping and characterizing the mutations as we screen for additional<br />
mutants. Because a large portion of cell and axon guidance mechanisms appear to be conserved from C.<br />
<strong>elegans</strong> to vertebrates, it is likely that some of the genes identified in this screen will play a conserved role<br />
in nervous system development.<br />
262
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MAPPING AND CHARACTERIZATION OF HAD-1, AN HSN<br />
AXON GUIDANCE GENE<br />
Lianna Wong, Jim Rader, Gian Garriga<br />
Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204<br />
To understand how growth cones are guided to their synaptic targets, we are studying the outgrowth of<br />
the HSN axons. The HSNs are a bilaterally symmetric pair of serotonergic motor neurons that innervate<br />
the vulval muscles and stimulate egg laying in C. <strong>elegans</strong>. The mature HSN axon morphology can be<br />
visualized using gmIs1, an integrated arrestin::GFP reporter that illuminates the HSN cell bodies and<br />
axons. In wild-type animals, each HSN axon extends ventrally to the ipsilateral ventral nerve cord (VNC)<br />
tract, turns anteriorly along it, then skirts dorsolaterally around the vulva, elaborating varicosities and<br />
small branches that innervate the vulval muscles. The left and right HSN axons then continue their<br />
anteriorly-directed extensions in their respective, parallel axon tracts until they reach the nerve ring in the<br />
head.<br />
Our lab performed a genetic screen for mutants defective in HSN axon guidance. Two of the mutants<br />
isolated, gm188 and gm204, display grossly abnormal HSN axon outgrowth patterns, consisting of circling<br />
and looping of the axons around the vulva, elaboration of excessively long branches, and multiple<br />
crossovers of the axons between the left and right VNC tracts. In some mutant animals, the HSN axons<br />
stop before reaching the nerve ring; in others, the HSN axons turn posteriorly and migrate back to or<br />
beyond the vulva. The gm188 and gm204 mutations both map to LG V and fail to complement one<br />
another. We have tentatively named the gene defined by these mutations as had-1 (HSN axon defective).<br />
Three-factor mapping places had-1 between sma-1 and him-5. There are a number of overlapping<br />
deficiencies in the region; nDf42 and yDf12 both uncover had-1, while arDf1 does not, placing had-1<br />
between the right breakpoint of arDf1 and him-5, a region of less than one map unit. We have injected<br />
had-1 mutants with cosmids in this region, but thus far have not achieved rescue of the HSN phenotype.<br />
To more precisely define the position of had-1 in this interval, we are presently mapping had-1 relative to<br />
cloned genes and Tc1 polymorphisms.<br />
In addition to mapping and cloning had-1, we are characterizing the HSN axon defects of had-1 mutants<br />
in animals lacking vulval cells and sex muscles to ascertain what roles these tissues play in the Had-1<br />
HSN axon phenotype.<br />
263
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
RECOGNITION OF X-CHROMOSOME-ENRICHED DNA<br />
ELEMENTS BY DOSAGE COMPENSATION PROTEINS<br />
Tammy F. Wu 1 , Jason D. Lieb 2 , Barbara J. Meyer 1<br />
1Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,<br />
Berkeley, CA, 94720<br />
2Howard Hughes Medical Institute and Department of Biochemistry, Stanford University Medical Center,<br />
Stanford, CA 94305<br />
In C. <strong>elegans</strong> X-chromosome dosage compensation is accomplished by repressing gene expression from<br />
both hermaphrodite X chromosomes to equal expression from the single male X. This essential process is<br />
carried out through the action of a large protein complex that localizes to X starting around the 50-cell<br />
stage in XX, but not XO, embryos.<br />
To study how dosage compensation proteins come to be localized to X chromosomes, we are<br />
investigating whether dosage compensation proteins recognize specific cis-acting elements. Previously,<br />
dosage compensation proteins were shown to localize to extrachromosomal arrays bearing specific<br />
regions of the promoter of the autosomal her-1 gene, whose expression is required for male development.<br />
Thus, one approach to the problem of X recognition is to test candidate elements for their ability to be<br />
recognized by dosage compensation proteins. To find such candidates, we have searched the genome<br />
for sequence elements that occur more often on X than on autosomes. Sixteen such elements were<br />
identified, and were found to occur in a wide variety of distributions throughout the X chromosome. These<br />
candidates are being tested for recognition by dosage compensation proteins on arrays in vivo, as<br />
previously described (Carmi et al., Nature 1998, and Dawes et al., Science 1999). Because the sequence<br />
context of these elements may be important for recognition, we are using PCR to amplify products<br />
containing unique surrounding sequence, as well as the candidate element itself.<br />
Two PCR products tested have yielded promising results. Interestingly, the sequences tested have a<br />
roughly 250 base pair region in common, which is itself enriched on the X chromosome. We are currently<br />
investigating the significance of these sequences.<br />
264
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
RAC-LIKE GTPASES AND CELL MIGRATION<br />
Yi-Chun Wu, Li-Chun Cheng, Nei-Yin Weng, Ting-Wen Cheng<br />
Zoology Department, National Taiwan University, No 1 Roosevelt Road Sec4, Taipei 10617, Taiwan,<br />
Republic of China<br />
Cytoskeletal rearrangement and cell polarization are important for fundamental cellular processes, such<br />
as cell migration and the phagocytosis of apoptotic cells. We are interested in how migration cues and<br />
apoptotic signals are interpreted by the receiving cells and subsequently affect their cytoskeletal<br />
rearrangement during cell migration and cell-corpse-phagocytosis in <strong>Caenorhabditis</strong> <strong>elegans</strong>.<br />
At least two C. <strong>elegans</strong> Rac-like GTPases have been shown to be important for cell migration. MIG-2 is<br />
necessary for the migration of Q cells, Q cell descendents and coelomocytes (1). CED-10 is important for<br />
the migration of distal tip cells as well as the engulfment of apoptotic cells (2). To test if these two<br />
GTPases may function redundantly in the migration of other cells, we have constructed ced-10;mig-2<br />
double mutants and are currently analyzing the mutant phenotype. It has been proposed that ced-10 may<br />
function downstream of ced-2 and ced-5 during cell-corpse-engulfment (2). We have also constructed<br />
ced-2; mig-2 and ced-5; mig-2 double mutants. Results will be presented at the meeting.<br />
1. Zipkin et al., Cell, 90, 883-894 (1997)<br />
2. Reddien and Horvitz, Nature Cell Biology, 2, 131-136 (2000)<br />
265
TWO NEW GENES REGULATING NEUROBLAST MIGRATION<br />
IN C. ELEGANS<br />
Lucie Yang, Mary Sym, Queelim Ch’ng, Cynthia Kenyon<br />
Box 0448, 513 Parnassus, Dept. of Biochemistry, San Francisco, CA 94143-0448<br />
In C. <strong>elegans</strong>, the Q cells are bilaterally symmetric neuroblasts present in the posterior body region of the<br />
worm at hatching. During the first larval stage, the Q cells divide and migrate. QR and its descendents<br />
migrate anteriorly whereas QL and its descendents migrate posteriorly. Several genes that regulate the<br />
anterior migrations of QR and its descendents have been identified. These include: 1) lin-39, a homeobox<br />
gene required in QR and its descendents for migration (Wang B. et al, 1993; Clark S. et al, 1993); 2)<br />
mig-13, a novel transmembrane protein required outside of QR and its descendents for migration (Sym M<br />
et al, 1999); and 3) egl-20, a Wnt homolog expressed in cells in the tail region (Whangbo J.. and Kenyon<br />
C, 1999). Two mutant screens (Mary Sym and Queelim Ch’ng) were conducted to identify additional<br />
genes that regulate the migration of QR and its descendents.<br />
From these screens, mutations in two new genes were identified that cause certain cells in the QR<br />
lineage to stop migrating prematurely. These two genes seem likely to be involved in guidance rather than<br />
in providing cells with the ability to migrate because, in these mutants, other cells sometimes migrate in<br />
the wrong direction. Current efforts are directed toward cloning these two genes and determining how<br />
these genes regulate the migration of QR and its descendents together with genes previously known to<br />
regulate these migrations.<br />
References:<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Clark SG, Chisholm AD, and Horvitz HR. 1993. Control of Cell Fates in the Central Body Region of C.<br />
<strong>elegans</strong> by the Homeobox Gene lin-39. Cell 74: 43-55.<br />
Sym M, Robinson N, Kenyon C. 1999. mig-13 Positions Migrating Cells Along Anteroposterior Body Axis<br />
of C. <strong>elegans</strong>. Cell 98: 26-36.<br />
Wang BB, Muller-Immergluck MM, Austin, J, Robinson, NT, Chisholm, A, Kenyon, C. A Homeotic Gene<br />
Cluster Patterns the Anteroposterior Body Axis of C.. <strong>elegans</strong>. Cell 74: 29-42.<br />
Whangbo J, Kenyon, C. A Wnt Signaling System that Specifies Two Patterns of Cell Migration in C.<br />
<strong>elegans</strong>. Molecular Cell 4: 851-858.<br />
266
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
IDENTIFICATION AND CHARACTERIZATION OF TELOMERE<br />
BINDING PROTEINS IN THE NEMATODE C. ELEGANS<br />
Su Young Yi, Seunghyun Kim, Junho Lee<br />
Department of Biology, Yonsei University, Seoul 120-749<br />
Telomeres are multifunctional elements that shield chromosome ends from degradation and end-to-end<br />
fusions, prevent activation of DNA damage checkpoints, and modulates the maintenance of telomeric<br />
DNA by telomerase. Telomeric DNA has been found to interact with proteins in many organisms. For<br />
example, hTRF1, a human telomeric-repeat binding factor, is involved in the telomere length regulation.<br />
We wanted to identify and elucidate functions of telomere binding proteins in the nematode C. <strong>elegans</strong>.<br />
Telomeric repeat of C. <strong>elegans</strong> consists of a long stretch of (TTAGGC)n.<br />
1. We have isolated Ceh-37, a novel homeotic protein in C. <strong>elegans</strong>, as double-stranded telomere binding<br />
factor by yeast one-hybrid screening. We found that Ceh-37 specifically binds C. <strong>elegans</strong> telomere in<br />
vitro. We found that the N-terminal domain and the homeotic domain is sufficient for telomere binding. We<br />
also found that Ceh-37 binds the telomere as dimers, and that the N-terminal is required for the<br />
dimerization. Ceh-37 is expressed not in all cells, but in subsets of cells. Over-expression of Ceh-37<br />
caused death in late larval stages.<br />
2. We have identified a protein (CeST-BP) in C. <strong>elegans</strong> embryonic nuclear extract that specifically binds<br />
single-stranded telomere sequences by gel mobility shift assay. We found that the two Ts and Gs of the<br />
telomere sequence were necessary to efficiently bind the C. <strong>elegans</strong> single-stranded telomere binding<br />
protein (CeST-BP). CeST-BP did not efficiently bind with repeated RNA sequences of UUAGGC, thus we<br />
concluded that CeST-BP bound specifically with DNA only. We also found that CeST-BP needs at least<br />
2.5 repeats of (GGCTTA) sequence for binding. CeST-BP was sensitive to salt concentrations, and<br />
insensitive to RNase treatment. The size of CeST-BP was found to be about 40 kD in South-<strong>West</strong>ern<br />
hybridization. We plan to identify CeST-BP by purification using DNA-streptavidin sepharose affinity<br />
chromatography and MALDI-TOF protein sequence search. Subsequently, studies of the functions of<br />
CeST-BP in vivo will follow.<br />
267
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
MOLECULAR ANALYSIS OF THE DOSAGE COMPENSATION<br />
GENE DPY-21<br />
Stephanie Yonker, Edith Cookson, Barbara J. Meyer<br />
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,<br />
Berkeley, CA 94720<br />
Dosage compensation in <strong>Caenorhabditis</strong> <strong>elegans</strong> equalizes X chromosome expression between XO<br />
males and XX hermaphrodites. DPY-21 is one of several proteins required for proper dosage<br />
compensation. However, DPY-21 is unique among known dosage compensation proteins. While other<br />
dosage compensation mutations cause maternal-effect lethality and affect only XX animals, dpy-21<br />
mutations cause only modest lethality and affect both XX and XO animals. Furthermore, different assays<br />
of X-linked gene expression have shown that dpy-21 mutations cause both elevation and reduction in<br />
X-linked gene expression in XO animals. We have cloned dpy-21 by injecting single-stranded RNA from<br />
candidate ESTs that map to a YAC close to dpy-21. Two positive ESTs were found to represent the same<br />
gene and were used to assemble the dpy-21 mRNA sequence in conjunction with 5’ and 3’ RACE<br />
products. To verify that we had identified dpy-21, we sequenced six dpy-21 alleles and found DNA lesions<br />
within the predicted coding sequence. Three of the sequenced dpy-21 alleles contain premature STOP<br />
codons. The 5610 nucleotide dpy-21 transcript, which is both SL1 and SL2 spliced, encodes a novel 1651<br />
amino acid protein. While there is no sequence similarity to known proteins, putative homologues are<br />
present in Drosophila melanogaster and human genomes. Preliminary immunoflourescence experiments<br />
with DPY-21 antibodies indicate that DPY-21 localizes to the nucleus.<br />
268
A SEARCH FOR LETHAL SYNAPTIC FUNCTION MUTANTS<br />
USING A SENSITIZED BACKGROUND<br />
Karen Yook, Erik Jorgensen<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Dept. of Biology, University of Utah, Salt Lake City, UT 84112 USA<br />
We are interested in identifying genes essential for neurotransmitter release. However mutations in<br />
genes essential for neurotransmission can result in L1 lethality, as observed with animals homozygous for<br />
null mutations in cha-1, the biosynthetic enzyme for acetylcholine. In the course of characterizing<br />
synaptic function mutants, we noticed that mutations in separate synaptic function loci do not complement<br />
one another when heterozygous, that is, they exhibit nonallelic noncomplementation. In an in-depth<br />
analysis of nonallelic noncomplementation among synaptic function alleles, we demonstrated that<br />
nonallelic noncomplementation occurs between lethal synaptic function alleles and hypomorphic alleles at<br />
other loci (manuscript in preparation). Therefore, in order to uncover synaptic function loci which have<br />
been mutated to lethality, we adopted a nonallelic noncomplementation strategy to screen for mutations<br />
that interact with a hypomorphic allele of unc-13. Specifically, we looked for double heterozygotes (mut/+;<br />
unc-13(n2813)/+) which decrease neurotransmitter release. Although the noncomplementation<br />
interaction between synaptic function loci is robust, we increased the sensitivity of our screening assay<br />
with an additional background mutation in unc-29, a subunit of the acetylcholine receptor. Therefore we<br />
carried out a screen for genes whose functions are effected by mutations in unc-13 and unc-29. Initially,<br />
we screened 2800 EMS mutagenized genomes in this sensitized background and uncovered 117<br />
potential synaptic function loci of which 37 exhibited an uncoordinated phenotype on its own and another<br />
12 exhibited L1 lethality. Characterization of the uncoordinated mutants identified mutant alleles of<br />
unc-26, unc-2, aex-3, ric-4, unc-38 all synaptic function loci known to be involved in neurotransmission.<br />
In addition, we uncovered a novel locus. These results suggest that the sensitized background is a good<br />
strategy for identifying the components of the synaptic transmission machinery. More recently, we used<br />
the ENU mutagen and screened through 3900 genomes. This screen has turned up 134 potential<br />
synaptic function mutations; 22 of these mutations are associated with a lethal phenotype, of which 13<br />
exhibit L1 lethality. We are currently pursuing the identity of the lethal loci.<br />
269
IDENTIFICATION OF DOWNSTREAM TARGET GENES IN<br />
DAF-2 PATHWAY<br />
Hui Yu, Pamela L. Larsen<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
Molecular Biology Program and Division of Biogerontology, University of Southern California, Los<br />
Angeles, CA 90089<br />
In a search for downstream genes in the daf-2 insulin-like receptor signaling pathway, nine genes were<br />
identified by differential display PCR and verified by Northern analysis to be differentially expressed in<br />
daf-2 mutants compared to N2 animals. The genes discovered have been named dao for daf-2 pathway<br />
over- and under-expressed genes. Three dao genes, dao-1, dao-8 and dao-9, are homologs of an<br />
intracellular transducer FK-506 binding protein. These three genes were down-regulated in daf-2(m41)<br />
animals and this effect in part relies upon daf-16. Among the other six genes, dao-2, dao-3 and dao-4<br />
were positively regulated by daf-2 signaling, while the opposite pattern was observed for dao-5, dao-6<br />
and dao-7. dao-2, dao-4 and dao-6 encode novel proteins. By homology, dao-3 is probably a<br />
methylenetetrahydrofolate dehydrogenase. The predicated DAO-5 protein has many phosphorylation site<br />
repeats and showed 38% identity to xNopp180, a nuclear localization sequence-binding protein shuttling<br />
between the cytoplasm and the nucleus. dao-7 contains a zinc finger region similar to mammalian ZFP36<br />
protein and thereby is a potential transcription factor. Distinct expression patterns and molecular identities<br />
of these new downstream targets in the daf-2 pathway indicates that daf-2 signaling is a complicated<br />
signaling network involved in multiple cellular processes.<br />
270
FATE SPECIFICATION IN MALE P(9-11).P EQUIVALENCE<br />
GROUP<br />
Hui Yu, Paul W. Sternberg<br />
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
HHMI and Division of Biology, California Institute of Technology, Pasadena, CA 91125<br />
C. <strong>elegans</strong> P(9-11).p cells in male pre-anal ganglion (PAG) equivalence group have three potential fates,<br />
1° , 2° , and 3° . In wild-type males, P10.p and P11.p adopt the 2° and 1° respectively, divide to generate<br />
distinct subsets of cells. P9.p (3° fate), which is undivided, fuses with hyp7. osm-6::gfp is specifically<br />
expressed in two hook neurons HOA and HOB derived from the 2° lineage. To understand the fate<br />
specification in P(9-11).p cells, we constructed a series of strains carrying the osm-6::gfp marker with<br />
mutations in lin-12, lin-15 and mab-5 genes. Instead of normal one pair of GFP expressing cells, lin-12(gf)<br />
mutants showed 2-3 pairs of GFP expression characteristic of the HOA and HOB neurons, indicating a<br />
transformation to the 2° fate in P9.p and P11.p cells. In lin-15(lf) mutants, P9.p could divide and a second<br />
pair of GFP expression was observed anteriorly sometimes because of a P9.p Æ P10.p fate<br />
transformation. The results suggested that the lin-12 activity is sufficient to promote the 2° fate and lin-15,<br />
a negative regulator of let-23 -ras pathway, is required for the 3° fate. The roles of mab-5 and let-23/ras<br />
are under investigation. A genetic screen for other candidates involved in P(9-11).p fate determination is<br />
also in progress.<br />
271
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
LOSS OF A DYNAMIN RELATED PROTEIN MGM-1 CAUSES<br />
EXCESSIVE MITOCHONDRIAL FRAGMENTATION<br />
Mauro Zappaterra, Alexander van der Bliek<br />
Department of Biological Chemistry, UCLA School of Medicine, P.O. Box 951737, Los Angeles, CA<br />
90095-1737<br />
Our lab studies the functions of dynamin and dynamin related proteins. These proteins form a small family<br />
of large GTP-binding proteins. We have recently shown that the C. <strong>elegans</strong> dynamin related protein<br />
DRP-1, is involved in the scission of the mitochondrial outer membrane (1). Since little is known about<br />
mitochondrial division and mitochondrial morphology in animal cells, we are investigating the mechanisms<br />
that regulate these processes.<br />
MGM-1 is another member of the dynamin family present in animal cells. As of now we know that this<br />
protein is present in yeast, C. <strong>elegans</strong>, and humans. Unlike other members of the dynamin family, MGM-1<br />
has an N-terminal mitochondrial leader sequence. MGM-1 is also more closely related to bacterial<br />
dynamin-like proteins than it is to dynamin or DRP-1, suggesting that this protein has followed a different<br />
evolutionary path. We speculate that MGM-1 was introduced into eukaryotic cells by the progenitors of<br />
mitochondria. Expression studies using ß-galactosidase under the control of the mgm-1 promoter show<br />
high levels of expression in intestines, body wall muscles, and neurons. These high levels might reflect<br />
high metabolic rates in those tissues, because the MGM-1 expression pattern is similar to that of another<br />
dynamin-related protein, DRP-1, which is also important for mitochondrial maintenance. Mgm-1 RNAi<br />
drastically slows the growth rate and approximately doubles the life span of C. <strong>elegans</strong>. We show that<br />
excessive fragmentation of mitochondria is induced by RNAi and mgm-1 antisense cDNA. We present<br />
evidence that MGM-1 is localized to the mitochondrial matrix where it might regulate the morphology of<br />
the mitochondrial inner membrane.<br />
1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular Cell 4: 815-826.<br />
1999.<br />
272
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
ISOLATION AND PHENOTYPIC ANALYSIS OF SYD-7<br />
Mei Zhen 1,2 , Nikki Alvarez 1 , Yishi Jin 1<br />
1Department of Biology, 327 Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064<br />
2zhen@darwin.ucsc.edu<br />
Using a synaptic vesicle-tagged GFP marker, Punc-25-SNB-1::GFP, that is localized to the presynaptic<br />
terminals of VD and DD motoneurons, we isolated two recessive loss-of-function mutations, ju43 and<br />
ju58, that define the syd-7 locus. In wild-type animals, the Punc-25-SNB-1::GFP marker is expressed as<br />
discrete fluorescent puncta spaced evenly along the ventral and dorsal sides of the animal. In syd-7<br />
mutants some of the fluorescent puncta are absent in various regions, while the remaining puncta appear<br />
normal in morphology and spacing. 90% of ju43 and ju58 animals lose SNB-1::GFP puncta in places<br />
corresponding to the presynaptic regions of VD1 and VD2 neurons. 50% of ju58 animals also lack<br />
SNB-1::GFP puncta in presynaptic regions of different DD neurons. Despite of the defects in GFP marker,<br />
the locomotion of syd-7 animals is indistinguishable from wild-type animals. Absence of SNB-1::GFP<br />
puncta can be caused by defects in either cell fate or neural differentiation. To distinguish these<br />
possibilities we are examining the axonal morphology of D neurons and the expression of cell-type<br />
specific markers in syd-7 animals.<br />
We mapped syd-7 to chromosome V, in a small region overlapped by yDf12 and arDf1. This tentatively<br />
places syd-7 between eat-6 and him-5. Currently we are further fine-mapping syd-7 and trying to rescue<br />
syd-7 with cosmids in the region. We are also in the progress of testing for the genetic interactions<br />
between syd-7 and genes known to be involved in the development of the nervous system.<br />
273
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A SCREEN TO IDENTIFY GENES THAT REGULATE THE<br />
ACTIVITY OF THE C. ELEGANS GLUTAMATE RECEPTOR<br />
GLR-1.<br />
Yi Zheng, Heng Xie, Pene J. Brockie, Andres V. Maricq<br />
Department of Biology, University of Utah, Salt Lake City, Utah 84112<br />
Efficient synaptic transmission relies on an organized distribution of neurotransmitter receptors.<br />
Activity-dependent changes in synaptic strength are believed to be crucial for information processing and<br />
for the refinement of neuronal network during development. The presence of silent synapses and their<br />
activation indicates rapid and activity-dependent changes in the number of glutamate receptors at<br />
excitatory glutamatergic synapses. However, the molecular mechanisms underlying the regulation of the<br />
density of receptors at synapses are unclear.<br />
We have previously shown that the C. <strong>elegans</strong> glutamate receptor subunit GLR-1 is expressed in the<br />
interneuronal circuitry that is required for normal locomotion. To perturb the function of this circuit, we<br />
engineered into GLR-1 the A/T amino acid change first identified in the d2 glutamate receptor of the<br />
mutant Lurcher mouse. Transgenic worms (akIs9) that express this mutant glutamate receptor show a<br />
striking change in locomotion where they rapidly alternate between forward and backward movement,<br />
presumably secondary to the constitutive depolarization of the command interneurons by the GLR-1(A/T)<br />
channel. In contrast, worms that have a null mutation in GLR-1 (ky176) move about the same as wild-type<br />
animals.<br />
The dramatic difference between ky176 and akIs9 worms provides an opportunity to identify genes that<br />
regulate glutamate receptor density in C.<strong>elegans</strong>. Mutations that decrease the membrane density of<br />
GLR-1(A/T) should suppress the hyper-reversal phenotype of akIs9 worms and make them move more<br />
like ky176 animals. We mutagenized the akIs9 strain and screened ~25,000 haploid genomes for<br />
mutations that suppress the locomotory phenotype. We have isolated 17 candidate mutants that suppress<br />
the hyper-reversal movement and we are in the process of characterizing them. Amongst these mutants,<br />
we expect to find mutations that affect the insertion or stability of glutamate receptors at the synapse.<br />
274
<strong>West</strong> <strong>Coast</strong> <strong>Worm</strong> <strong>Meeting</strong> 2000<br />
A RESOURCE FOR C. ELEGANS MICROARRAYS<br />
Stuart K. Kim, Min Jiang, Kyle Duke<br />
Department of Developmental Biology and Genetics, Stanford University Medical Center, Stanford, CA<br />
We are entering a new age in molecular genetics in which we can use sequence data from genome<br />
projects to dissect cell, developmental and disease pathways more completely and more sensitively than<br />
ever before. One key functional genomics approach is to use DNA microarrays to define changes in gene<br />
expression patterns during development and during the onset of disease. DNA microarrays could be used<br />
to identify genes that are regulated by specific transcription factors, by specific cell signaling pathways, by<br />
expression of homologs of human disease genes in transgenic animals or by addition of various<br />
pharmaceutical drugs.<br />
We are currently producing DNA microarrays that contain every gene in the C. <strong>elegans</strong> genome. Our goal<br />
is to develop C. <strong>elegans</strong> microarray technology, and to make this technology available to all other C.<br />
<strong>elegans</strong> labs. We will provide these microarrays to any C. <strong>elegans</strong> lab by hybridizing their RNA samples<br />
to the full genome microarrays. The results are deposited in the Stanford Microarray Database, and can<br />
be accessed over the web. We have already performed about 200 microarray hybridizations using the full<br />
genome microarrays, in collaboration with 27 different academic C. <strong>elegans</strong> labs. We are also helping to<br />
provide needed tools and reagents so that other labs can establish their own microarraying facilities.<br />
275