The long range objectives of this project are to determine the role of cellular interactions in regulating neuronal commitment and differentiation during development and regeneration of the teleost neural retina, with special emphasis on photoreceptors. The new focus of the research plan is on specialized cell-cell junctions as mediators of inductive signalling events. The specific goals for the next project period are: I. Characterize the expression and developmental regulation of cadherins/adherens junctions in teleost retina by electron microsopic and immunocytochemical analyses. II. Develop monospecific antibodies and riboprobes for teleost N-cadherin and R-cadherin by using zebrafish N-cadherin cDNA to clone and sequence cDNAs for zebrafish R-cadherin, and produce synthetic peptides and fusion proteins for generating antibodies that block cadherin-mediated cell-cell adhesion. III. Test the hypothesis that apical junctional complexes are associated with commitment to the cone but not the rod photoreceptor phenotype by examining the profile of expression of cadherin mRNA and protein at the onset of cone differentiation, on rod precursors and differentiating rods, and following lesions that induce regeneration of canes in adult fish. IV. Test the hypothesis that cadherins/adherens junctions mediate choice of photoreceptor fate in an in vitro model system (organ culture of whole embryonic/larval fish eyes). Block N- and R-cadherin-mediated cell-cell adhesion with antibodies, fusion proteins, N-terminal fragments of the molecules, or synthetic peptides containing the specific binding sequence HAV, or indirectly by low Ca+ +, calcium channel antagonists, and the phosphotyrosine phosphatase inhibitor vanadate. Monitor production of photoreceptors with rod-and cone-specific monoclonal antibodies and with in situ hybridization with isotype-specific opsin probes. The studies outlined here, designed to uncover mechanisms involved in photoreceptor development and the regenerative replacement of photoreceptors in the fish retina, may lead to a better understanding of the cellular and molecular factors important for retinal cell commitment and differentiation. This information could provide insights into replacement therapies for damaged retinal tissue.
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