The long-term goal of this project is to define basic molecular mechanisms that underlie circadian rhythmicity in vertebrates. Circadian clocks regulate a multitude of biochemical and physiological processes, resulting in coordinated daily rhythms of gene expression, metabolism and behavior. Disruption of the human circadian system due to disease, aging or voluntary disruption of sleep-wake cycles can contribute to diminished sensory and motor performance, sleep disorders or affective disorders. An understanding of basic circadian clock mechanisms may help to define the etiology of these disorders, and may help in the design of treatments. To approach the long-term goal, vertebrate circadian clock genes will be identified and studied by mutagenesis and antisense knockdown analyses in zebrafish. Accelerating advances in zebrafish genetics and genomics and a new method for high-throughput measurement of zebrafish circadian rhythms make this an efficient model system for these approaches.
The specific aims are to: 1) Screen 1500-3000 mutagenized genomes per year for mutations that affect circadian rhythmicity, map each mutation to a chromosome arm, and make all mutants available to other researchers. 2) For selected mutants, clone the mutated gene, characterize phenotypes at the molecular, cellular and behavioral levels, and define the genes' roles in circadian rhythmicity. 3) Screen candidate clock genes for circadian function by knocking down their expression levels with antisense morpholino oligonucleotides. These mutant and morphant screens will exploit new transgenic zebrafish lines in which firefly luciferase expression is driven by the clock. Live larval fish of these lines glow rhythmically when incubated in luciferin, the substrate of luciferase. With available technology, bioluminescence rhythms can be measured simultaneously from 2,000 larvae, making a high-throughput screen for vertebrate clock genes feasible. """"""""Early pressure"""""""" parthenogenesis will be used to reveal recessive mutations in a single generation and to produce mapping panels for centromere linkage analysis. This project is expected to identify novel vertebrate circadian clock genes, and to generate new hypotheses about the functional roles and mechanisms of action of both novel and previously identified clock gene products.
