The proposal is concerned with studies designed to analyze both in vitro and in vivo, the function of Brn-3b, a POU-domain transcription factor, in RGC development, and to explore underlying mechanisms for RGC degeneration under pathological conditions using the brn-3b gene locus as a molecular tool. First, transcriptional properties and structure/function relationships of Brnb-3b will be analyzed using biochemical approaches including gel mobility shift, methylation interference foot printing, and co-transfection transcription assays. These in vitro studies will provide initial information on how Brn-3b may specifically control its target gene expression in RGC development. Secondly, various histochemical and immunochemical approaches will be utilized to investigated whether Brn-3b is activated in dividing retinoblasts, and to compare cell proliferation and death between Brn-3b (+/+) and (-/-) retinas. These in vivo studies are expected to gain crucial information regarding the timing and mode of Brn-3b function during RGC development. Thirdly, Brb-3b will be replaced by homologous recombination in mice with the alkaline phosphatase reporter to examine the projections of axons, or with bcl-2 gene to study rescue of apoptosis in Brn-3b-expressing RGCs. The resulting data will help in understanding the function of Brn-3b in RGCs, and may also shed light on degeneration of RGCs in pathological conditions. Together, the proposed studies on Brn-3b function will provide fundamental insights into the molecular mechanism that control mammalian retinal development. In the long term, the information should help better understand the mechanisms leading to certain blinding disorders, and also may provide new directions for treatment modalities.
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