The proposed experiments are designed to utilize a vertebrate genetic system capable of identifying dominant mutations affecting the zebrafish retina, with particular emphasis on mutations involved in rhodopsin trafficking. The long-term objectives of the proposed research include the identification of genes associated with blindness caused by age-related retinal degeneration and the characterization of the cellular and molecular mechanisms underlying retinal degeneration. Mutations causing dominantly inherited night blindness will be isolated by a behavioral assay and confirmed by histological and physiological methods. Proper rhodopsin localization and transport will be evaluated by breeding mutant zebrafish with transgenic zebrafish that express a transgene encoding a rhodopsin-green fluorescent protein (GFP) fusion protein and examining the progeny by confocal fluorescent microscopy. Additional characterization of other transport systems within the photoreceptors of mutant fish will elucidate possible interactions between cellular transport mechanisms. Finally, identification of the genes responsible for these mutations will be achieved by positional cloning strategies. The proposed work will provide insight to the mechanisms underlying photoreceptor cell death that ultimately lead to retinal degeneration and blindness associated with aging.
