The overall goal of this project is to understand the mechanisms that generate different classes of neurons during development. We are using the neural crest, which generates the peripheral nervous system, as a model system. Our previous work has focused on the mechanisms that generate autonomic neurons. In this proposal we shall investigate the control of sensory neurogenesis, which has been relatively understudied due to lack of adequate markers. We will carry out these studies at both the cellular and molecular level. Specifically, we will generate and analyze mice containing targeted mutations in neurogenin-1 (ngn-1) and neurogenin-2 (ngn-2), two genes encoding NeuroD-related basic-helix-loop-helix transcriptional regulators that are specifically expressed early in sensory gangliogenesis, as well as in the basal layer of the olfactory epithelium. Using a recently-developed primary explant system for sensory neurogenesis, we will examine the regulation and function of neurogenin-1 and neurogenin-2 in vitro, to complement the in vivo analysis of mutant mice. We will also use this explant system as an assay for putative sensory neuron progenitors, that we propose to isolate by fluorescence-activated cell sorting from heterozygous mice in which the green lantern protein (GLP) has been inserted into the ngn-1 locus by homologous recombination. These isolated sensory neuron progenitors will be immortalized using a recently-developed conditional system, to generate cell lines that can provide large numbers of cells for further study of sensory neuron development and function. In parallel, a similar approach will be undertaken to isolate and immortalize olfactory neuron stem cells from the olfactory epithelium, based on their expression of GLP in the ngn-I locus. Studies of olfactory receptor expression and function in these cell lines will be carried out in collaboration with K. Zinn.
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