Genes controlling zebrafish development : roles for Rabs and Glis
This thesis is divided in two main sections linked by a common question concerning notochord formation and signalling. The first section describes a loss-of-function screen of zebrafish Rab proteins using antisense morpholino oligonucleotides (MO). Rab proteins are small GTPases, which constitute the largest subfamily of the Ras superfamily. There are at least 60 human Rab proteins and they are thought to be involved in every step of vesicular traffic within eukaryotic cells. I identified 63 zebrafish rab genes, but there are probably more as for fourteen human RAB genes the zebrafish counterpart was not identified in this work. The mRNA in situ localization of 20 zebrafish rabs was assessed. All 20 genes were expressed during embryonic development and 6 showed enhanced expression in certain embryonic structures, such as chordamesoderm and polster (rab1b, rab2a2 and rab18), neurons subsets (rab3c1 and rab5a1), or specific brain regions (rab5c). MO injections targeting 20 different rab proteins were carried out and 14 resulted in embryos with obvious phenotypic defects. Several of the Rab morpholino-induced phenotypes are analysed and discussed. The phenotypes observed range from defects in very early developmental processes, such as epiboly (Rab5a2), to defects in highly specialized developmental processes, such as melanophore morphology (Rab1b). Knocking down several of these proteins resulted in embryos with defects in structures with high trafficking activity, such as brain, notochord or blood (Rab7b, Rab9a, Rab14a, Rab15). The second section of the thesis describes efforts to understand the role of Gli3 protein in zebrafish development. Gli proteins are the transcription factors of the Hh signalling pathway and play a variety of roles in processes involving Hh signalling activity such as development of ventral spinal cord and limb. In the zebrafish, mutations in Gli1 and Gli2 proteins are known but no Gli3 homolog had been reported. I cloned the zebrafish gli3 gene and mapped it to linkage group 24. To analyse the loss-of-function phenotype of zebrafish Gli3 in the ventral neural tube a brief analysis of spinal cord markers was done. Using these spinal cord mariners, no obvious mispatterning phenotype was observed in zebrafish embryos injected with a MO targeting gli3. Disrupting Gli3 function in zebrafish embryos, however, resulted in a strong early phenotype. At 40% epiboly Nodal signalling is up-regulated and later in development the embryos show convergence-extension defects. Interestingly, the convergence-extension phenotype can be rescued by a short treatment with cyclopamine, a chemical inhibitor of Hh signalling.