(Candidate's Abstract) The experiments described in this proposal are aimed at achieving a better understanding of the mechanisms that underlie degenerative diseases of the retina. Studies in recent years have emphasized the value of using the Drosophila eye as a model system for identifying signaling pathways that regulate the development of the vertebrate eye. Several mutations that lead to retinal degeneration in Drosophila are due to mutations in genes that are conserved between Drosophila and humans. Previous screens for mutations in Drosophila that caused retinal degeneration have required that flies develop to the adult stage. This would make it difficult to identify mutations in genes that are also required in other tissues at earlier stages of development. One way to circumvent this problem is to use mitotic recombination to generate clones of homozygous mutant tissue in the eyes of flies that are heterozygous for lethal mutations. Using a genetic screen where FLP/FRT-mediated mitotic recombination is restricted to the eye, the mentor's laboratory has identified mutations in two loci, burned and pepper which result in retinal degeneration in clones of mutant tissue. Both mutations are homozygous lethal, indicating that these genes have an essential function in the development of the fly.
Specific Aims I and 2 of this proposal are aimed at achieving a phenotypic and molecular characterization of the burned and pepper loci. Immunohistochemical techniques will be used to define the abnormalities in the mutant tissue that eventually lead to degeneration. Mapping strategies and positional cloning will be used to identify the transcription unit that is mutated. Once the genes have been cloned, mammalian hornologues will be searched for using sequence alignment methodologies. The mentor's laboratory has screened the autosomes for mutations of this nature.
Specific Aim 3 describes a genetic screen of the Xchromosome for mutations that cause retinal degeneration in clones. Since the X-chromosome represents 20% of the genome, it is likely that additional loci will be identified that result in degenerative changes in the developing retina. Mutations identified in the screen of the X-chromosome will be characterized biologically, mapped and eventually cloned. It is anticipated that the genes identified and characterized in this screen will contribute to our understanding of retinal degeneration in humans. This project will also train The candidate to function as an independent scientist who can combine clinical neuropathology with basic research.
| Davis, David A; Wilson, Meredith H; Giraud, Jodel et al. (2009) Capzb2 interacts with beta-tubulin to regulate growth cone morphology and neurite outgrowth. PLoS Biol 7:e1000208 |
| Delalle, Ivana; Pfleger, Cathie M; Buff, Eugene et al. (2005) Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration. Genetics 171:1757-65 |
