Expression of proneural basic helix-loop-helix (bHLH) transcription factors such as neurogenins (Ngns) within a neuronal progenitor initiates neurogenesis. Ngns suppress expression of SoxB1 proteins that are required for maintenance of a progenitor state. However, the mechanisms underlying this suppression are not understood. The central hypothesis of this proposal is that once the level of Ngn reaches a threshold within a progenitor cell, Ngn transcriptionally suppresses expression of progenitor genes by a feed-forward repression mechanism mediated by its downstream factor, MTGR1, and ensures irreversible progression of differentiation.
The aim of this application is to carry out key experiments to demonstrate the involvement of Ngn and MTGR1 in progenitor gene suppression and to create molecular tools to analyze such regulation. Two experimental systems, Xenopus primary neurons and chick spinal cord, which have distinct experimental advantages, will be used. The focus of the research is the interaction of Xngnr-1, the earliest expressed Ngn member in frogs, or cNgn2, the chick counterpart of Xngnr-1, and Sox2, a member of the SoxB1 family and well-studied marker of neuronal progenitors.
In Specific Aim 1, we will determine whether Xngnr-1 is directly or indirectly involved in suppression of Sox2 mRNA.
In Specific Aim 2, we will determine if XMTGR1, a transcriptional repressor induced by Xngnr-1, is involved in suppression of Sox2 mRNA.
In Specific Aim 3, an in vivo reporter gene assay system using Sox2 enhancers will be established using the chick spinal cord, and the effect of cNgn2 on the reporters will be examined. Based on the results obtained from this proposed project, we will propose to molecularly characterize the nature of progenitor gene suppression in our future R01 proposal. We expect that this line of research will reveal a general mechanism of progenitor gene suppression employed during neuronal differentiation, and will provide a new model of how neuronal differentiation and progenitor maintenance are regulated. The clinical importance of SoxB1 proteins has been demonstrated in several reports. For example, experiments in mouse and chicken embryos show continued expression of SoxB1 proteins inhibits neurogenesis, whereas loss-of-function of the Sox2 gene is correlated with cerebral malformations, epilepsy, degeneration of neurons and the appearance of cytoplasmic protein aggregates in neurons, features seen in diverse human neurodegenerative diseases. Mutations in human Sox2 and Sox3 genes are associated with anophthalmia and X-linked mental retardation, respectively. Therefore, studies proposed here have broad implications for understanding the underlying biology of multiple nervous system disorders and may provide insight into future therapeutic strategies, particularly as they relate to stem cell manipulation. ? ? ?
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