The long-range goal of this project is to understand the mechanisms controlling the lineage commitment and differentiation of multipotent neural progenitor cells, using the neural crest as a model system. The focus of this proposal is to understand the function and regulation of NRSF, a transcriptional repressor of neuronal genes previously identified in this laboratory. NRSF is expressed in most non-neuronal tissues, as well as in undifferentiated neuroepithelium; however it is not expressed in most neurons. This suggests that NRSF may silence neuronal genes in non-neuronal cells, and/or may transiently repress such genes in neural progenitor cells prior to neuronal differentiation. The latter function would in turn require a mechanism to repress NRSF during neurogenesis. Several parallel and complementary approaches will be undertaken to address these questions. Structure-function analysis, using in vitro DNA binding assays and in vivo silencing assays in transfected mammalian cells, will dissect domains of NRSF important in repression. Further light can be shed on the mechanism of repression by identifying other proteins with which these domains interact, using the yeast 2-hybrid system screen. NRSF domains will also be used to design dominant-negative forms that can be used to perturb NRSF function in various experimental systems, including cultured neural crest cells and Xenopus embryos. Targeted mutations in the NRSF gene will be generated in mice and the phenotype of these mutants characterized using a variety of molecular markers, both in vivo and in cultures of neural crest cells established using a variety of molecular markers, both in vivo and in cultures of neural crest cells established from mutant embryos. The regulation of environmental factors will be examined in primary cultures of neural crest cells grown under conditions that promote commitment to different lineages. The interaction of NRSF with the regulatory circuits that control neuronal differentiation in neural crest cells will be investigated by constitutively expressing intact or mutant NRSF from retroviral vectors in these cells, and examining the response to factors such as BMP2 that promote neurogenesis as well as the expression of positive transcriptional regulators of neurogenesis such as MASH-1. An understanding of the molecular mechanisms that control the commitment and differentiation of multipotential neural progenitors to different lineages will be valuable for human health, both in developing potential cell-replacement therapies for neurologic and neurodegenerative diseases and for developing anti-tumor therapies for cancers of the neural crest such as neuroblastoma.
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