The identification of new genes in genomic and cDNA sequencing projects is far outpacing the discovery of their functions. The goal of this project is to close this gap using genomic analysis in zebrafish. A systematic analysis of the expression patterns and map positions of transcribed genes will be used to link cloned genes to mutant phenotypes. This project will 1) facilitate the cloning of zebrafish mutations, and 2) yield functional information about orthologous genes in other vertebrates. The successful analysis of the floating head (flh) mutation illustrates the potential of this approach. The combination of the expression pattern and map position of the zebrafish Not-1 gene led to its identification as a candidate for the flh mutation, which disrupts pattern formation in the embryo. Subsequent analysis confirmed that the Not-1 gene is inactivated in flh mutants. Not-1 homologs have been identified in other vertebrates, and these genes are likely to have the same function in pattern formation as defined by the flh mutation in zebrafish. This project will use expression pattern and map position to identify candidate genes for the large number of zebrafish mutations that have recently been isolated in genetic screens. This proposal has three main goals: 1) Genetic mapping of 250 mutations that define essential gene functions to facilitate the cloning of these genes. These mutations will be mapping by centromere-linkage analysis, an efficient mapping strategy that allows most loci to be localized to a linkage group with approximately 400 PCR assays. 2) Candidate genes for mutations with tissue specific phenotypes will be identified in a systematic screen of the expression patterns of transcribed genes. 1500 novel zebrafish genes will be identified by partial sequence analysis of cDNA clones (production of expressed sequence tags, EST), and the expression patterns of these genes will be determined by wholemount in situ hybridization. 3) Positional candidate genes for the mutations localized in Part 1 will be identified by mapping genes discovered in Part 2 and in other studies. Genes will be mapped by following the segregation of PCR-based gene markers in zebrafish-mouse cell hybrids and in genetic mapping crosses. Since these mutations disrupt many diverse aspects of development and physiology -- and in many cases mimic human diseases -- the gene functions revealed by genomic analysis in zebrafish will be broadly relevant to vertebrate biology and medicine.
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