The long term objectives of this project are to understand at the molecular level some of the genetic and epigenetic factors responsible for cell differentiation and pattern formation in the mammalian Central Nervous System. With this knowledge it will be possible to understand more fully a wide range of developmental disorders and degenerative diseases that affect the nervous system. Much of this proposal utilizes the retina as a model for CNS development. Previous work from this laboratory has defined the developmental appearance and some aspects of the cellular regulation of a wide array of retinal molecules. One of the major aims of the current proposal is to further characterize nuclear proteins essential for the transcriptional regulation of one photoreceptor molecule, the visual pigment protein opsin. Gel retardation and DNase I protection assays will be used to identify tissue- specific binding proteins and their binding sites. Transient transfection assays using the opsin promoter and beta-galactosidase or luciferase reporter genes will be used to determine functionally important sites. Developmental studies will test the hypothesis that a change in level or activity of some of these proteins is important for the onset of opsin transcription. This analysis will be extended to other photoreceptor proteins whose appearance follows a very similar time course to that of opsin. In this project we will focus on the cGMP-gated ion channel of outer segments that is primarily responsible for the light dependent changes in membrane conductance. This analysis will determine whether promoter regions of this gene bind any proteins that interact with the opsin promoter to provide information about common regulatory mechanisms during neural development. To study the role of diffusible factors in CNS development, known factors and second messengers will be tested for their effects in culture on cell proliferation, cell differentiation and specific transcription of defined retinal genes. The presence of a number of embryonic growth factors will be measured in developing retina using PCR amplification with primers chosen from published sequences. A combination of these approaches will allow testing of the hypothesis that commitment to become a neuronal cell type and the onset of differentiation are regulated by changes in growth factor levels or responses. Monoclonal antibodies will be prepared against partially purified nuclear proteins. These antibodies will allow the testing of hypotheses concerning the generation of cell type specificity of transcription by either unique combinations of ubiquitous nuclear proteins or cell-type specific nuclear proteins. Together, the specific aims in this proposal represent a focused effort to define and understand the cellular and molecular processes governing the formation of neural cell types and transcription of cell-type specific genes of known function.
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