PhD thesis

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Material and methods 13 Detection of proliferating cells with BrdU (5-bromo-2-deoxyuridine) staining BrdU labeling was carried out, in order to identify possible growth zones in rhynchonelliform brachiopod larvae. BrdU is incorporated into the DNA of proliferating cells during the S-phase of the cell cycle. Staining of BrdU thus allows for visualization of dividing cells and their progenies. Larvae of Terebratalia transversa of the following developmental stages: 6, 11, 24, 35, 48, 60, and 96 hours after fertilization (hpf) were incubated in 0.1mM BrdU (Sigma-Aldrich, St. Louis, MO, USA) in seawater at 11.5ºC for 6 – 48h. In another experiment larvae were cultured in 10mM BrdU in seawater for 30 min and subsequently the larvae were cultured in BrdU free seawater (pulse-chase experiment). After the treatment with BrdU the larvae were fixed in 4% paraformaldehyde in PBS for 1 hour at room temperature and then treated for 10 min at 37ºC in 0.01mg/ ml proteinase K in PBS. After that they were kept for 10 min in 0.1N HCl on ice, 1 hour at 37ºC in 2N HCl, 1 hour in PBS with three changes, and 15 min in PBT (PBS with Tween 20). Then, the larvae were incubated in 1:500 mouseanti-BrdU antibody in PBT over night at 4 ºC, washed for 1 hour in PBS with three changes, 1 hour in 1:200 diluted TRITC, and finally 1 hour in PBS with three changes. Stained larvae were mounted in glycerol and analyzed with a Leica DM RXE 6 TL fluorescence microscope equipped with a TCS SP2 AOBS laserscanning device (Leica Microsystems, Wetzlar, Germany). Gene expression analyses The expression of developmental genes was studied by whole mount in situ hybridization (WMISH). Thereby, target mRNA is visualized with a complementary RNA probe which contains DIG labelled uridine (Digoxigenin- 11-uridine-5’-triphosphate). The digoxigenin is subsequently stained with a Anti-DIG-AP, fab fragments antibody that contains alkaline phosphatase (AP) which in turn is made visible by a reaction with BCIP (5-Bromo-4-chloro-3- indolyl phosphate) and NBT (nitro blue tetrazolium chloride). In this reaction BCIP is dephosphorylated by AP and dimerizes to leucoindigo. This dimer is then oxidized by NBT to an insoluable dark blue 5,5’-dibromo-4,4’ precipitate (Trinh et al. 2007). The precipitate is visible in daylight conditions and also reflects laser light which allows the use of this technique in combination with a confocal laserscanning microscope (Jekely and Arendt 2007). There are several WMISH protocols available which usually have to be adapted to the organism they are intended for. Protocols developed for several species were tested in this study, namely one for the sea urchin Strongylocentrotus
14 Material and methods purpuratus, the cnidarian Nematostella vectensis, and the polychaete Platynereis dumerilii, respectively (Arendt et al. 2001; Long and Rebagliati 2002; Martindale et al. 2004; Venuti et al. 2004). The N. vectensis protocol was found to be the best of the tested protocols for the brachiopod Terebratalia transversa and was used accordingly to investigate the expression patterns of TtrNot and TtrCdx (Chapter IV). Illustrations Illustrations were done with Photoshop CS3 and Illustrator CS3 software (Adobe, San Jose, CA, USA).
Page 1 and 2: Comparative neurogenesis, muscle de
Page 3 and 4: 2 Principal supervisor Assoc. P
Page 5 and 6: 4 Preface This thesis presents
Page 7 and 8: 6 Abstract Brachiopods are a sm
Page 9 and 10: 8 Acknowledgements The endeavou
Page 11 and 12: 10 Introduction Nervous system Micr
Page 13: 12 Material and methods Material an
Page 17 and 18: 16 Results and discussion Results a
Page 19 and 20: 18 Results and discussion posterior
Page 21 and 22: 20 Results and discussion Myogenesi
Page 23 and 24: 22 Results and discussion Wanninger
Page 25 and 26: 24 Results and discussion Growth pa
Page 27 and 28: 26 Results and discussion argument
Page 29 and 30: 28 References References Abdelkhale
Page 31 and 32: 30 References priapulids and protos
Page 33 and 34: 32 References James, M. A., Ansell,
Page 35 and 36: 34 References regionalization, prol
Page 37 and 38: 36 References 211. Williams, A., Ca
Page 39 and 40: 38 Chapter II Frontiers in Zoology
Page 53 and 54: 52 Chapter III Chapter III Altenbur
Page 55 and 56: 54 Chapter III Altenburger and Wann
Page 63 and 64: 62 Chapter IV Chapter IV Altenburge
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64 Submitted manuscript Chapter IV
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PhD thesis

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