Analysis of genes controlling notochord development in zebrafish
The notochord is a vital and defining organ in vertebrates. Mutagenesis screening in zebrafish identified seven 'dwarf mutants that lack notochord development grumpy, sleepy, bashful, dopey, happy, sneezy and doc. This thesis is concerned with the identification and positional cloning of the doc locus as well as the confirmation and characterisation of the dopey and happy loci. Previous positional cloning efforts identified the grumpy, sleepy and bashful genes, demonstrating a requirement for the laminin chains pi, yl and al in formation of the notochord basement membrane (Parsons et al., 2002b Pollard, 2002) and the mutant sneezy, which has been shown to encode the COPI subunit a (Coutinho et al., 2004). This thesis establishes that the doc locus lies within a 0.5Mb region on linkage group 18, containing several genes, including a novel gene encoding a predicted protein with 14 WD40 domains. Antisense morpholino (MO) knock-down of doc results in a phenocopy of doc and insitu expression demonstrates that this gene is expressed specifically within the notochord during development. Expression analysis of echidna hedgehog (ehh) demonstrated that MO knock-down of this gene results in a lack of notochord differentiation. I therefore expect this novel gene is doc. Analysis of the mutants dopey and happy has demonstrated that they encode the coatomer subunits COPp' and COPp respectively. Expression of these and other COPI subunits demonstrate that the majority of COPI subunits are up-regulated within the notochord during development and maintained abnormally in COPI deficient embryos. I have investigated the mechanism of coatomer gene regulation and found that loss of coatomer function leads both to up-regulation of coatomer mRNA and activation of the unfolded protein response (UPR). Suggesting that the UPR is the regulator of mRNA expression, functioning to maintain the secretory network during development, though work to provide definitive proof remains.