Coutinho, Pedro João de Carvalho E. Saraiva; (2001) Molecular characterisation of sneezy, a notochord mutation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutant alleles of sneezy were identified during the first Tubingen and Boston large-scale systematic screens for recessive-zygotic mutations affecting embryogenesis in zebrafish. It affects differentiation of the notochord, pigmentation, fin formation and leads to widespread degeneration of the embryo in the mid hatching period at about 60 hpf. Using a positional cloning approach, I have identified coatomer subunit α (copa) as the gene mutated in sneezy. The coatomer complex, together with the small GTPase ARF1, constitutes the protein coat of COPI vesicles, an essential component of the early secretory pathway. In zebrafish, copa is expressed maternally and during the first 24 hpf shows ubiquitous zygotic expression. This maternal wild-type component is responsible for absence of defects prior to ±24 hpf By tissue transplantation, I show that α-COP function is required within the shield derivatives for normal notochord differentiation. In addition, we find that α-COP activity is required within the neural tube for normal melanophore development. At 24 hpf sneezy mutant embryos display an abnormal maintenance of early chordamesoderm marker gene expression. This correlates with a failure of the chordamesoderm to differentiate into notochord. EM studies of notochord cells in sneezy mutants and wild-type siblings show that the early secretory pathway is blocked in sneezy. This results in disruption of formation or maintenance of the perinotochordal basal lamina. This general block in transport, which may affect the elaboration of integral membrane receptors, leads to a failure in notochord differentiation and subsequent apoptosis. In addition, abnormally high levels of apoptosis occur in the floorplate and posterior dorsal neural tube. Apoptosis in the posterior dorsal neural tube correlates with the lack of pigmentation, in the posterior trunk of mutant embryos. At more anterior levels, where melanophores may survive, the failure to become pigmented probably arises from a failure of the Golgi apparatus, which normally generates the melanosomes.

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