The long term goal of this project is to understand at the molecular level the genetic and epigenetic factors responsible for cell determination and differentiation in the mammalian Central Nervous System. This knowledge is essential for understanding and treating many diseases caused by mutation or injury that result in abnormal brain development and for manipulating cell phenotypes in neural regeneration and replacement therapies. This project builds on past findings and has four specific aims. First, a continued analysis using transgenic mice will test the hypothesis that a specific DNA element found in many photoreceptor specific genes, RET 1, is both sufficient and necessary to direct photoreceptor expression of genes. Using specific DNA sequences, consisting of either RET 1 alone or the opsin promoter in which RET 1 has been mutated, linked to a lac Z marker gene, the spatial and temporal expression of the marker will be examined using X-gal histochemistry. If expression is detected in any cells other than photoreceptors, these will be examined for the RET 1 binding protein and for negative regulators that normally block expression of opsin. Second, the protein binding to the RET 1 element has been purified and it is now proposed to clone it and study its structure and activity in more detail. The cDNA sequence will categorize this molecule as a member of a new or an existing family of transcriptional regulators, the genomic sequence will identify promoter elements controlling its expression and antibodies will allow a precise description of the cellular extent of its expression and will provide tools to isolate associated proteins. These experiments will provide both new information about neuronal transcriptional regulators and probes to study molecular events closer to the time of cell birth and commitment. Third, retinoic acid and basic fibroblast growth factor have been shown to affect the binding activity of nuclear proteins interacting with the RET 1 sequence, and it is now proposed to study the ways in which these factors influence binding and thus provide insights into the molecular mechanisms by which they can influence neuronal development. Fourth, the hypothesis that transcription factors expressed during earlier phases of forebrain and retinal development are essential for terminal differentiation of neurons such as photoreceptors, and expression of cell type -specific molecules such as opsin, will be tested using a culture system in which a retina is formed by transdifferentiation of the adjacent retinal pigment epithelium. Together these specific aims represent a focussed effort to define specific and general rules governing the formation of neural cell types and the transcription of cell-type specific genes of known function in the mammalian Central Nervous System.
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