Abbas, Leila; (2001) Rho GTPases and early zebrafish development. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The work in this thesis examines the role of the Rho family of small GTPase proteins in vertebrate embryogenesis, using the zebrafish, Brachydanio rerio, as an experimental model. These proteins have been shown to mediate actin rearrangements underlying cell movement and morphology changes both in vitro and in vivo. Their function has been well-characterised in single cells, but less is known about their role in the movements of cells in whole sheets, such as those occurring in the processes of epiboly and gastrulation in embryogenesis. Firstly, the cloning of two, previously unknown members of the zebrafish Rho GTPase family is described - a full length RhoA homologue and a partial fragment of Racl. The expression patterns of both of these genes is determined over the course of embryogenesis, at several stages from the 4 cell stage embryo to the 72 hour larva. Additionally, the expression patterns of these genes in the adult brain are determined. To address the functioning of the Rho pathway in vivo, synthetic mRNA transcripts encoding differing forms of human RhoA or the Rho-blocking C3 exoenzyme were injected into 2-4 cell stage embryos. The resultant phenotypes are presented, as analysed by in situ hybridisation, immunohistology and examination of embryonic morphology. Injection of constitutively active V14Rho results in a defective epiboly phenotype which is resolved into cyclopia and which phenocopies the silberblick/Wnt11 mutant phenotype. Blocking Rho function with C3 has a dramatic and rapid effect on embryogenesis, affecting cellularisation and axial polarity in the early embryo and resulting ultimately in death. The final chapter deals with the characterisation of a mutant strain of fish arising from an ENU mutagenesis screen. Point mutations chemically induced in male germlines were bred to homozygosity over a three generation scheme and the F3 progeny were screened for the presence of homozygous recessive mutations. One such recovered mutation, gimpy, is described here. Affected embryos are shorter than their sibs, have aberrant brain morphology and lack a differentiated notochord - thus, they resemble the dwarf clsss of mutants recovered in other mutagenesis screens. I report the analysis of this mutant using the observation of embryonic morphology, in situ analysis and histological techniques and discuss the subsequent identification of this mutation as a knock-out of the LamininC gene.

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