FISH and Chips: Applying Microarray Technology and in Situ Hybridization to Understanding Light-Adaptation in Zebrafish

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A major breakthrough in biological research is the development of DNA microarrays in which thousands of different gene segments can be "printed" onto a small slide, or chip. Such chips show differential gene expression under specific physiological conditions. To this point, it has not been possible to assess changes in gene expression in the retina of fish exposed to different lighting regimens or experiencing different circadian phases. This proposal will use microarray technology to identify differences in genes expressed in light- versus dark-adapted zebrafish retinas. Circadian or light-driven oscillations occur in the vertebrate retina for rhodopsin, for various genes involved in regulation of circadian rhythms, for melatonin and the enzymes involved in its biosynthesis, for dopamine and GABA, and for pH. Earlier NSF-funded studies show that acetylcholine stimulates light-adaptive pigment granule dispersion in retinal pigment epithelium (RPE) isolated from the eye of bluegill, and that it acts upon G-protein-coupled muscarinic receptors to do so. Therefore, genes required for synthesis of both upstream and downstream effectors of the muscarinic receptor signaling pathway are of special interest. For this study, two approaches are proposed: (1) comparing mRNA levels between retinas isolated at midday and midnight from fish acclimated to a 12/12 light/dark cycle, and (2) performing in situ hybridization studies to localize expression of genes of interest. Microarray analysis of diurnal regulation of gene expression in the retina will lay the foundation for examining correlations between expression of genes involved in cell signaling, neurotransmitter biosynthesis, and neurotransmitter receptors. Results will be used to relate the phase of expression to the affinity of receptors for ligand, and to better understand the physiological implications of the rhythmic expression. In situ hybridization studies will permit the expressed genes to be placed in the cellular context. The microarray study add to the research skill set for those involved. The research will provide a windfall of data that will likely reveal genes not related to the signaling pathways, but that regulate pigment granule movement. Therefore, students and other researchers will be able to use those data to launch their own research careers. In addition, this proposal will support the thesis research of at least one master's student and at least one Ph.D. student.