Although the human genome contains about 20,000 genes, the function of only a small fraction have been examined in a vertebrate animal through loss of function analysis. Additional genes have been examined functionally in mammalian cell culture systems. However, the ultimate test of gene function is an animal model knockout where gene function can be surprisingly distinct from that found in cell culture. The zebrafish has provided a valuable vertebrate model for human development and disease. Forward genetic screens in the zebrafish based on mutagenesis of the genome and phenotype-based identification of gene mutations have been a powerful method of gene discovery and gene function analysis. The power of this approach requires that mutant genes be molecularly identified, typically through positional cloning, which requires substantial effort. Furthermore, this approach will not identify functions for many genes in the genome, because forward screens are by design biased by a specific phenotypic assay and are not broad based methods of analysis of all gene functions. A far more rapid and cost efficient method of assessing all gene functions in a vertebrate is a broad-based phenotypic analysis of gene mutations generated by reverse genetic approaches. Several laboratories are currently funded to generate >10,000 molecularly-defined gene mutations in the zebrafish. These labs are examining embryonic/early larval phenotypes for each line and have found that 75% are viable as larvae. Gene function, however, does not end in early larvae. Importantly, human disease typically manifests itself in juveniles and adults. This proposal postulates that a significant fraction of the mutant genes viable as early larvae will display functions during late larval to adult stages. To investigate their functions, 1500 such mutant genes will be screened for late larval and adult phenotypes, expanding the knowledge base of gene function for the ~75% of genes with no evident functions during embryogenesis. Homozygous mutant adults will be examined morphologically, by X-ray analysis to investigate the skeleton, as well as for fertility phenotypes. For mutant genes that cause an adult phenotype, histological analysis will be performed on entire animal sections to investigate internal organ and tissue defects at the cellular level. For mutations lethal prior to adulthood but following early larval stages, the timeframe of lethality will be determined and mutant larvae or juveniles examined morphologically, by X-ray of skeleton and histological analysis to investigate the nature of the defect. The proposed studies will provide a baseline of information to the scientific community for 1500 genes with no apparent function during early development. The resource of phenotypic data would then provide a starting point for future in-depth studies of gene function by individual investigators. This study will decipher the adult functions of a large number of genes in vertebrates, which will provide models for many human disease genes in the zebrafish. Genes acting in organ function and development, bone and skeletal formation, and fertility are among the many juvenile and adult onset disease genes expected.
Human disease typically manifests itself in juveniles and adults. This study will decipher the adult functions of a large number of genes in vertebrates, which will provide models for many human disease genes in the zebrafish. Genes acting in organ function and development, bone and skeletal formation, and fertility are among the many juvenile and adult onset disease genes expected.
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