The identification of new genes is far outpacing the discovery of their functions. The goal of this project is to close this gap using genomic analysis in zebrafish. Using complementary forward and reverse genetic approaches, this project will define loss-of-function phenotypes for hundreds of zebrafish genes and provide functional information about their counterparts in other vertebrates, including human. In the reverse genetic approach, loss-of-function phenotypes will be determined for 250 genes by injection of antisense morpholino oligonucleotides. Many of these 250 will form duplicated gene pairs, which often have overlapping functions that are difficult to analyze with forward genetic approaches. In addition, by systematically analyzing genes in duplicated chromosomal segments, this project will provide critical insight into the mechanisms by which duplicated genes acquire new functions that can drive evolutionary innovation. These reverse genetic experiments will generate an important database of zebrafish gene functions, providing the first loss-of-function study for many essential vertebrate genes. In the forward genetic approach, genetic mapping studies will link cloned genes to mutant phenotypes. Zebrafish genetic screens have identified thousands of mutations that disrupt developmental, behavioral, and physiological traits shared by fish and humans, but only a small number of the affected genes are known. To accelerate molecular analysis of zebrafish mutations, this project will localize 250 mutations with a newly developed mapping strategy in which ~600 single nucleotide polymorphisms covering the genome are scored in parallel in a single hybridization to an oligonucleotide microarray. The SNP microarray method is significantly faster than other available methods, alleviating a key bottleneck in the molecular analysis of zebrafish mutations. Map information will be used to identify positional candidate genes from previously constructed zebrafish gene maps and from the rapidly increasing zebrafish genomic sequence database. Because zebrafish 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. Immediate data release for both morpholino and mapping projects will ensure that the innovative approaches and results from this research will be widely disseminated to the research community.
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