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The long-term objectives of this proposal are to elucidate the molecules and the mechanisms which direct, regulate and maintain structural and functional differentiation of the retinal pigment epithelium (RPE). By screening a panel of monoclonal antibodies produced against RPE cells, two antibodies were identified to use as differentiation markers in these studies, 3C4 and F22. The 3C4 antigen is a transmembrane protein which is expressed only in RPE cells. During development, 3C4 becomes polarized to the basal-lateral surface of mature chick RPE where it increases its association with the submembranous cytoskeleton. This linkage appears to be mediated directly through actin. To further characterize the structure and function of this unique protein, the 3C4 antigen will be cloned from an RPE expression library and sequenced. The clones will be used to look for expression of the 3C4 in mammalian RPE cells by in situ hybridization. Eukaryotic expression technology will be used for functional analysis of the cloned 3C4 gene. The F22 mAb recognizes an extracellular matrix molecule which is deposited in the interphotoreceptor matrix ( IPM), at the vitreoretinal junction and in the cartilaginous plate in the sclera. The distribution of the antigen is consistent with it having a role in ganglion cell differentiation and axonal guidance during ocular development. The localization of F22 in the IPM and the vitreoretinal juncture suggests that it could be important for maintaining retinal attachment to the RPE and vitreous. The structural characterization of the F22 antigen will be obtained by cloning and sequencing the gene from a RPE cDNA library. The functional roles of the F22 antigen will be determined by antibody blocking experiments at critical stages in ocular development.

The role of beta-1 integrin receptors in directing and maintaining RPE cell differentiation will also be examined. Integrins are a family of transmembrane proteins that provide a link between the extracellular matrix and the cytoskeleton. Signals transmitted by integrins from the extracellular matrix to the cytoskeleton effect cell attachment, migration and differentiation. Immunocytochemical techniques will be used to identify and map the expression of specific integrin heterodimers during development. Integrins which are directly involved in linking the RPE cells to Bruch's membrane will be identified using bivalent crossinglinking reagents and differential extraction methods. To determine if reversible phosphorylation of adhesion plaque proteins modulates integrin signalling during development, the association of phosphotyrosine proteins with integrins will be examined using a panel of antibodies produce against phosphotyrosyl adhesion plaque proteins. The expression of specific laminin isoforms will be investigated in the developing chick eye using cytochemical approaches with antibodies and cDNA probes. Their role in the development of a polarized RPE phenotype will be determined.
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