To examine in more detail the developmental processes in the vertebrate retina, new screening methods will be used in zebrafish. Specifically, transgenic zebrafish expressing the green fluorescent protein (GFP) under a rod photoreceptor specific promoter will be used in two ways: A) A forward genetic screen to identify recessive mutations affecting rod development. Homozygous male transgenic zebrafish will be mutagenized by exposure to ethylnitrosourea. Mutagenized zebrafish will be bred to homozygous transgenic females to produce F2 generation families that are homozygous for the transgene. To identify recessive mutations, F3 generation fish will be examined by light and fluorescence microscopy for changes in GFP expression, indicating an alteration in rod photoreceptor number and/or retinal differentiation. B) Chemical genetic screens to identify molecles that disrupt pathways involved in proper retinal development and rod differentiation. Transgenic embryos will be arrayed at a density of 3 embryos/well of a 96-well plate and exposed to small molecules from libraries of known bioactive substances. The small molecule libraries consist of 2489 compounds and were assembled to contain molecules with known biological activity and with previously identified protein targets. Embryos will be exposed to small molecules at three timepoints significant in eye development: 9 hours post fertilization (hpf), when optic cup formation begins; 24 hpf, a time of retinoblast proliferation but prior to cellular differentiation; 48 hpf, the onset of rod photoreceptor differentiation. Embryos will be examined by standard light microscopy to screen for general morphological defects, differential interference contrast (DIC) microscopy to screen for defects in retinal lamination, and fluorescence microscopy to screen for defects in rod photoreceptor differentiation. Upon identification of specific mutations or small molecules, the phenotypes will be characterized by histology, immunohistochemistry, in situ hybridization, and mosaic analysis, when necessary. The goal of these studies will be to identify pathways in retinal development that could be missed in routine forward genetic screens. ? ?
| Song, Philip I; Matsui, Jonathan I; Dowling, John E (2008) Morphological types and connectivity of horizontal cells found in the adult zebrafish (Danio rerio) retina. J Comp Neurol 506:328-38 |
| Kojima, Daisuke; Torii, Masaki; Fukada, Yoshitaka et al. (2008) Differential expression of duplicated VAL-opsin genes in the developing zebrafish. J Neurochem 104:1364-71 |
| Matsui, Jonathan I; Egana, Ana L; Sponholtz, Todd R et al. (2006) Effects of ethanol on photoreceptors and visual function in developing zebrafish. Invest Ophthalmol Vis Sci 47:4589-97 |
