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Genetic screen for modifiers of muscular dystrophy in Drosophila melanogaster Muscular dystrophy (MD) is a general term that describes a group of inherited and gradually progressing myogenic disorders that are frequently associated with cardiomyopathy and brain disorders. Genetically the pattern of inheritance can be X-linked recessive as in Duchenne or Becker muscular dystrophy (DMD/BMD), autosomal dominant as in limb-girdle muscular dystrophy type 1 (LGMD type 1), or autosomal recessive as in limb-girdle muscular dystrophy type 2 (LGMD type 2) (Campbell, 1995; Straub, Campbell, 1997). DMD is a severe progressive muscle-wasting disease affecting approximately 1 out of 3500 males (Blake et al, 2002). One of the most important advances in understanding the molecular genetics of neuromuscular diseases has been the cloning of the gene encoding dystrophin, the protein absent in muscle of DMD patients. In the last few years the role of dystrophin in skeletal muscle has been studied, and several dystrophin-associated proteins that form a Dystrophin-Glyco- protein Complex (DGC) have been identified. The DGC consists of dystrophin, the dystroglycans, the sarcoglycans, sarcospan, the syntrophins and dystrobrevin (Durbeej and Campbell, 2002). The DGC components are now being characterized and evidence is beginning to indicate that proteins of this complex may be responsible for other forms of muscular dystrophy (Campbell, 1995). Unfortunately all known cases of MD belong to the category of incurable, therefore further studying of the DGC function is very important. A number of animal models have been established for DMD, but severe muscular dystrophy in the absence of dystrophin alone has only been observed in dogs (reviewed in Collins and Morgan, 2003). Mice and C.elegans exhibit muscle degeneration in the absence of dystrophin when also lacking myoD (Gieseler et al., 2000; Megeney et al., 1996), a gene required for muscle regeneration. This fact makes it difficult to study the mechanisms of MD utilizing such models, Doktorandenseminar - Seminar for PhD students Organizing team: W. Fischle, G. Groenhof, C. Höbartner, S. Jakobs, A. Lange Summary of the “Seminar for PhD Students“ by Mariya Kucherenko, Max Planck Research Group of Gene expression and signaling, 12.8.2009 hence development of a new remarkably good model for genetic manipulations remains an open task. Drosophila melanogaster was used as a model for human diseases previously. Characterization of the fly DGC shows that the fruit fly retains all essential components of the DGC, but with substantially less diversity (Greener and Roberts, 2000). Furthermore, regions and domains known to mediate interactions between members of the complex are highly conserved between human and fly, suggesting that the overall structure of the complex is identical (Neuman et al, 2005). Recently, a model for muscular dystrophy caused by Dystrophin (Dys) and Dystroglycan (Dg) deficiency was established in Drosophila melanogaster and phenotypes similar with human neuromuscular diseases were described (Shcherbata et al, 2007). Mutations in Drosophila Dystrophin and Dystroglycan genes cause shortened lifespan and reduced mobility as a result of age dependent muscle degeneration. In addition, the (+) inretaction; (-) no interaction; (En) enhancement. Seite 20 reduction of Dys and Dg leads to defects in eye development as manifested by altered photoreceptor axon path-finding. To review new components that interact with the DGC or regulate its function we used a Drosophila melanogaster model for muscular dystrophy and performed genetic interaction screens to identify dominant modifiers of Dg and Dys related mutant phenotypes. In the primary large scale screen we looked for modifications of an easily score-able phenotype such as alterations in the posterior crossvein. Using this screening strategy we have found 38 modifiers that belong to different functional groups: genes involved in muscle function, neuronal/cell migration and motor function as well as cytoskeletal components and components of the TGFbeta, EGFR and Notch signaling pathways (Kucherenko et al, 2008). In order to shed light on the mechanisms of muscle degeneration caused by Dys and Dg down regulation a secondary screen in muscle tissue was performed (Figure Table 1. Modifiers of muscle degeneration phenotype caused by reduced level of Dys and Dg.
Doktorandenseminar - Seminar for PhD students Organizing team: W. Fischle, G. Groenhof, C. Höbartner, S. Jakobs, A. Lange 1). In this genetic screen the Dys and Dg loss-of-function alleles (Dg 323 , Dg O86 and DysDf) were used to identify heterozygous interactions. To find suppressors and/ or enhancers of muscle degeneration we used both Dg/Dys and RNAi mutants (Dys RNAi :act-Gal4 and Dg RNAi :tub-Gal4), which show a moderate degeneration phenotype (Figure 1, C). The pre-selected primary screen modifiers were crossed to Dys and Dg mutants and the muscle structure of F1 progeny was analyzed. The paraffin sections of Drosophila indirect flight muscles were stained with hematoxyline and eosine and analyzed utilizing light microscopy. As a result of the secondary screen, 11 modifiers that enhanced the muscle degeneration phenotype were identified (Figure 1, Table 1). Further studies of identified components showed their requirement in either muscle or nervous tissue, where specific interaction with the DGC components may occur. The novel components that contribute to DGC dependent muscle maintenance are being analyzed. References: Blake, DJ., Weir, A., Newey, SE., Davies, KE. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 82: 291–329 Campbell, KP. Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell. 1995 Mar 10;80(5):675–679 Collins, CA., Morgan, JE. Duchenne‘s muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies. Int J Exp Pathol 84: 165–172 Durbeej, M. and Campbell, K.P. Muscular Dystrophies Involving the Dystrophin-Glycoprotein Complex: and overview of Current Mouse Models. Current Opinion in Genetics & Development 12:3:349-361, 2002 Fig. 1. Enhancement of muscle degeneration phenotype caused by reduced levels of Dys and Dg. (A) Location of transverse section of Drosophila indirect flight muscles (IFM). (B) The muscle section of a wild type fly. (C) The muscle section of Dystrophin RNAi mutant. (D) Enhancement of Dystrophin phenotype by reduction of one copy of mbl. (E) Enhancement of Dystroglycan phenotype by reduction of one copy of chif. Gieseler, K., Grisoni, K., Segalat, L. Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans. Curr Biol 10: 1092–1097 Greener, MJ., Roberts, RG. Conservation of components of the dystrophin complex in Drosophila. FEBS letters 2000; 482(1-2):13-8 Kucherenko, MM., Pantoja, M., Yatsenko, AS., Shcherbata, HR., Fischer, KA., Maksymiv, DV., Chernyk, YI., Ruohola-Baker H. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex. PLoS One. 2008 Jun 11; 3(6): e2418 Megeney, L., Rudnicki, MA. Determination versus differentiation and the MyoD family of transcription factors. Biochem. Cell Biol. 1995;73:723–732 Seite 21 Neuman, S., Kovalio, M., Yaffe, D., Nudel, U. The Drosophila homologue of the dystrophin gene - introns containing promoters are major contributors to the large size of the gene. FEBS Lett 579: 5365–5371 Shcherbata, HR., Yatsenko, AS., Patterson, L., Sood, VD., Nudel, U., Yaffe, D., Baker, D., Ruohola-Baker, H. Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 2007 Jan 24;26(2):481-93 Straub, V., and Campbell, K.P. 1997. Muscular dystrophies and the dystrophin-glycoprotein complex. Curr. Opin. Neurol. 10: 168-175
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