The long-range goal of this proposal is to understand the role that tight junctions play in regulating the outer blood-retinal barrier. Malfunction of the blood-retinal barrier is implicated in many retinopathies. This proposal examines how immature, leaky, tight junctions are converted into an effective barrier, and how this process is regulated by the neural retina. During development, rudimentary tight junctions of the retinal pigment epithelium (RPE) undergo this conversion to form the outer blood-retinal barrier. This laboratory devised an innovative culture model that revealed different stages in the assembly of a continuous network of tight junctions followed by its conversion to a low permeability form. The current proposal focuses on the ability of retinal conditioned medium to reorganize patches of tight junctions into a continuous network, and on the differences in the permeability of tight junctional strands that are obtained in older versus younger embryonic RPE. Differential screening of high density microarrays and more traditional molecular techniques will be used to address two hypotheses: 1) Retinal factors regulate the formation and assembly of junctional strands into a functional network by regulating the expression of tight junctional proteins and the effectors of tight junctional proteins. 2) The permeability properties of individual junctional strands assembled under retinal stimulation depends upon the embryonic age of the RPE because of the differential expression of interchangeable tight junction components. Although studies reported in the literature suggest candidate proteins to fill these roles, the unbiased genomic screen proposed here is expected to reveal novel tight junctional proteins and regulatory pathways. The regulation of the tight junction has proved to be enigmatic. Recent advances in the development of reagents coupled with this novel culture model will advance our understanding of this essential structure of the outer blood-retinal barrier. ? ?
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