Using forward genetics, and a visual screen for morphological defects in zebrafish embryos, one can isolate mutants with defects in many aspects of early vertebrate development. Roughly 2400 genes can be identified by this approach. About a third are required for the development of multicellular structures, including the embryonic organs. About two thirds encode proteins that are probably required for cell viability and growth. The majority of such embryonic mutants are lethal. To rapidly clone the embryonic lethal genes of the zebrafish, our lab developed and applied a method of insertional mutagenesis. In a large screen we will isolate approximately 620 mutants representing 450-500 different genes, roughly 20 percent of the embryonic lethals of the fish. To date we have cloned candidate genes responsible for about 260 mutants. The genes include many not previously suspected of a role in development. About 20 percent of the genes are novel. All the genes have homologues or related sequences in human, and some are known to be mutated in human diseases. Here we request funding to complete this project. This will take three years and will involve 1) Confirmation of the mutagenic inserts for all mutants and cloning of genes mutated in an additional 200-230 mutants; 2) Phenotypic characterization of all mutants including specifically identification of mutants with defects in development of the jaw, arches, cartilage, kidney, pancreas, liver, vasculature, brain, blood, and pigmentation; 3) Transfer of the mutant lines to the University of Oregon stock center in Eugene, Oregon to allow their unrestricted use by the community; and 4) Freezing sperm from about 250 lines.
