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Comparative genetic analysis of posterior axis formation in teleosts

Reference	BB/C51454X/1
Principal Investigator / Supervisor	Professor Stephen Wilson
Co-Investigators / Co-Supervisors	Dr Matthias Carl
Institution	University College London
Department	Cell and Developmental Biology
Funding type	Research
Value (£)	320,187
Status	Completed
Type	Research Grant
Start date	01/03/2005
End date	29/02/2008
Duration	36 months

Abstract

Zebrafish and medaka are teleost model systems well-suited for comparative developmental and evolutionary studies of conserved as well as species specific genetic and molecular mechanisms that underlie vertebrate development. In this project we will combine the advantages of working with both fish model organisms to undertake a comparative genetic analysis of posterior axis formation in teleosts. We have identified a group of four non-allelic zygotic, recessive lethal mutations in medaka that result in truncations of posterior structures of differing severity. Remarkably, no mutant phenotype comparable to two of the mutants has yet been described in any other vertebrate. Thus in koepke (kpk) mutants all structures posterior to the hindbrain are absent. Phenotypic analysis of all four mutants using standard approaches will determine whether the mutant phenotypes arise due to defects in fate determination, morphogenesis or both. These studies will provide the basic phenotypic information required for us both to compare between medaka mutants and to compare to zebrafish mutants with gastrulation phenotypes. For a comparative approach, we need to assess whether conserved signalling pathways have equivalent roles in medaka and zebrafish. In zebrafish, several signalling pathways and transcription factors are known to mediate axis formation and extension during gastrulation. We will interfere with these genes pathways in medaka, using morpholino antisense oligonucleotides, wild type or interfering forms of proteins acting in the pathways and or pharmacological reagents. Analysis of the resulting phenotypes will allow us to define the roles, in medaka, of the major signalling pathways and genes known to affect gastrulation in zebrafish. In comparing gene function during gastrulation, we need to know how similar are the morphogenetic movements and the gastrula fate map of posterior structures in medaka and zebrafish. To fate map cells we will use laser activation of caged fluorescent dyes and transplantation of labelled cells into unlabelled hosts. Moreover, we will generate use transgenic lines expressing the UV inducible Kaede protein under the control of regulatory DNA that drives expression in different tissues of the gastrula. These experiments will allow us to determine the origin and behaviour of cells that contribute to posterior structures in medaka and to compare morphogenesis during gastrulation in medaka and zebrafish. To address the question of how morphogenetic movements are affected in the four medaka mutants we will use cell transplantation techniques to determine whether the mutation acts cell autonomously. In parallel, we will determine the nature of the morphogenetic defects by labelling cells using membrane, cytoplasmic and nuclear fluorescent proteins coupled with 4D imaging of cell behaviour and morphology. These experiments will elucidate the cellular basis of the mutant phenotypes and will give important clues regarding the roles of the genes affected by the mutations. To fully understand the role of the mutant loci, we will identify the affected genes. In the course of positional cloning we have already mapped 3 of the 4 mutant loci and are within less than 0,4 cM (appr. 200kb) of the kpk locus. Standard procedures will be used to fine map and clone affected loci. Morpholino based phenocopy of the mutant phenotypes together with rescue experiments and sequencing of the mutated loci will confirm the identities of the mutated genes. Once we have identified the mutated medaka genes, we will compare expression, gene structure and function with the zebrafish orthologues. For the functional analysis, we will employ both the morpholino gene knock down technique and through collaboration, we will derive genetic mutations in zebrafish orthologues. These studies will identify conserved as well as species-specific genetic and molecular mechanisms that underlie development and evolution of posterior axis formation in vertebrates

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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