The basic-helix-loop-helix (bHLH) family of transcription factors are key regulators of neuron formation and differentiation in vertebrates. Neural bHLH proteins are required for neuron formation in the mouse, and forced expression of these proteins can initiate cell cycle withdrawal and neuronal differentiation in neural progenitors or uncommitted embryonic carcinoma cells. Our data indicates that the bHLH proteins increase expression of at least one cyclin-dependent-kinase inhibitor (CDKi), and this is likely to contribute to neuronal cell cycle exit. Here I propose to further analyze the role of CDKi proteins in neuronal cell cycle exit, in response to forced expression of bHLH proteins and in differentiating neurons from the mouse cerebral cortex. In addition, we will characterize the mechanism(s) by which CDKi expression is regulated by the neural bHLH proteins. During neuronal differentiation, neural bHLH proteins are expressed in a sequential cascade, with some bHLH proteins expressed in neural progenitors and others expressed during differentiation. It is not known if the bHLH proteins expressed during neuronal differentiation have functions distinct from those bHLH proteins expressed in neural precursors. To address this question, we will determine whether members of the neuroD family of bHLH proteins, which are expressed during neuronal differentiation, are functionally required downstream of neural precursor bHLH proteins such as neurogenin 1 to mediate cell cycle exit and neuronal differentiation. These studies, as well as our studies on CDKi function, will be facilitated by a mammalian expression vector-based method for RNA interference that we have recently developed. This research should lead to a better understanding of the mechanisms that regulate neurogenesis and neuronal differentiation in mammals, including humans. In the long term, such information should contribute to developing strategies for replacement of neurons lost due to injury or neurodegenerative diseases.
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