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Chapter IV Submitted manuscript 65 because their valves are not connected to each other by a hinge (Cohen and Weydmann 2005). We investigated the brachiopod Terebratalia transversa, a representative of the rhynchonelliform (articulate) brachiopods, the largest group within recent Brachiopoda. So far, several Hox gene sequences have been characterized for the linguliform brachiopod Lingula anatina (de Rosa et al. 1999). However, no expression data for any Hox or homeobox containing genes are currently available for Brachiopoda. With the investigation of Not and Cdx expression in Terebratalia transversa we aim to shed light on the function of these genes in invertebrate body patterning and thereby contribute to the discussion concerning their ancestral roles in eumetazoan (i.e., placozoan, diploblast, and triploblast) development and evolution. MATERIAL AND METHODS Animal collection, rearing, and fixation Adult animals were dredged in the vicinity of the Friday Harbor Laboratories, Washington, USA, at 48º32’869 N; 122º58’452 W during summer 2008 and spring 2009. The animals were placed in running seawater tables at ambient seawater temperature (approx. 11.5ºC). Embryos were obtained by artificial fertilization. To this end, gonads were dissected from the specimens and stored individually in beaker glasses. The eggs were washed several times with seawater and left in 100ml seawater until germinal vesicle breakdown, which usually occurred within 10-16 hours after dissection. Sperm cells were left until they had acquired a high degree of motility, which usually occurred after 4-14 hours. Sperm remained active until up to 48 hours after dissection. For fertilization, a few drops of the sperm suspension were added to the beaker glasses containing the eggs. Development of embryos and larvae was monitored closely and the beaker glasses were cleaned daily from debris with help of a glass pipette driven by a peristaltic pump. Larvae were fixed at various developmental stages in 4% paraformaldehyde in 0.5M NaCl, 0.1M MOPS (pH 7.5) for 8-10 hours at 4ºC, washed in 50% EtOH for 30 min, and finally stored in 80% EtOH at -20ºC. Cloning and in situ hybridization RNA was extracted from larvae at various developmental stages with a miRCURY RNA Isolation Kit (Exiqon, Vedbaek, Denmark). It was reversely transcribed into cDNA with a RETROscript Kit using oligo(dT) primers (Applied Biosystems/Ambion, Austin, TX, USA). In order to screen for homeobox containing genes, the cDNA was used as template for PCR reactions with the following degenerate primers: HoxF 5’-GCT CTA GAR YTN GAR AAR GAR TT-3’, which recognizes the peptide sequence ELEKEF, and HoxR 5’-GGA ATT CRT TYT GRA ACC ADA TYT T-3’, which recognizes the peptide sequence KIWFQN (Murtha et al. 1991; Balavoine and Telford 1995). PCR was carried out under the following conditions: 3 min 94 °C, followed by 40 cycles of 45s at 94 °C, 45s at 50 °C, and 60s at 72 °C, followed by a final extension step of 10 min at 72 °C. PCR products were purified over column with a QIAquick Gel Extraction Kit (Qiagen, Venlo, The
66 Submitted manuscript Chapter IV Netherlands) and subsequently ligated into a pGEM-T Easy vector (Promega, Madison, WI, USA). Ligation products were transformed into One Shot TOP10 E. coli competent cells (Invitrogen, Carlsbad, CA, USA). Cells were allowed to grow over night; clone DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen). Insert sequences were sequenced at the sequencing facility of the University of Barcelona and identified using the tBLASTx algorithm. Two specific forward primers were subsequently designed from the TtrNot and the TtrCdx PCR sequences: TtrNotF1 5’- GGA GAA GGA GTT CGA AAG GCA ACA A-3’, TtrNotF2 5’-CCG AAT CCC AAG TGA AGA TCT GGT-3’, TtrCdxF1 5’-CCT GGA GCT GGA GAA GGA GTT CTG T-3’, and TtrCdxF2 5’-AAC AAC CTT GTA CTT TCA GAG AGA CAG G-3’. The specific primers were used nested in a 3’RACE-PCR using a SMART RACE kit following the manufacturer’s protocol (Clontech, Mountain View, CA, USA). The sequences of the RACE-PCR products were again checked by BLAST and the positive clones were used for in situ probe production using the DIG RNA Labeling Kit (SP6/T7, Roche, Basel, Switzerland). In situs were done following a standard protocol with a 5 min proteinase K step and at least 48 hours of hybridization time at 40ºC or 45ºC (Martindale et al. 2004; Hejnol and Martindale 2008). For cohorts aged 0-64 hours after fertilization (hpf), in situs were performed on developmental stages that were 2-4 hours apart, for the age group of 64-154 hpf, in situs were done every 5-10 hours, while for later stages longer intervals were chosen. The latest stages investigated were 540 hpf old, which corresponded to 420 hours after settlement/onset of metamorphosis (hps). Sense probes were generated as controls for in situ hybridization. Since they didn’t give any signal they are omitted in the figures. Stained specimens were photographed with a Leica ProgRes C3 digital camera mounted on a Leica MZ 16F stereomicroscope. Schematic illustrations were generated using Adobe Illustrator CS3 and CS4 graphics software (Adobe, San Jose, CA, USA). Analysis of gene sequences and primer design was done with CLC Main Workbench 5 (CLC bio, Aarhus, Denmark). Immunostaining and confocal laserscanning microscopy Larvae were stained with antibodies against serotonin (ImmunoStar, Hudson, WI, USA) and acetylated α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). In addition, cell nuclei were labeled using DAPI (Invitrogen, Eugene, OR, USA). Prior to staining, larvae were washed thrice for 15min each in phosphate buffer (PB) and incubated for 1h in PB containing 0.2% Triton X-100 (Sigma-Aldrich) at room temperature. Thereafter, the larvae were incubated over night at 4ºC in 6% normal goat serum in 0.1M PB and 0.2% Triton X-100 (blocking solution). Then, the larvae were incubated for 24 hours at 4ºC in blocking solution containing a 1:800 dilution of the polyclonal serotonin antibody, 3µg/ml DAPI, and a 1:800 dilution of the monoclonal acetylated
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Comparative neurogenesis, muscle de
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2 Principal supervisor Assoc. P
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4 Preface This thesis presents
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6 Abstract Brachiopods are a sm
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8 Acknowledgements The endeavou
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10 Introduction Nervous system Micr
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12 Material and methods Material an
Page 15 and 16: 14 Material and methods purpuratus,
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
Page 65: 64 Submitted manuscript Chapter IV
Page 69 and 70: 68 Submitted manuscript Chapter IV
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