While the generation of neurons in the peripheral (PNS) and central nervous systems (CNS) is highly reproducible spatially and temporally, little is known of regulatory mechanisms. During the previous award we developed a model PNS sympathetic cell culture system of dividing precursors (neuroblasts) and found that insulin growth factors (IGF's), EGF, and transsynaptic signals were highly specific mitogens. We now hypothesize that a). population-specific epigenetic factors regulate CNS ontogeny including proliferation, differentiation and survival, and b). environmental signals regulate ontogenetic processes independently. Our recent development of a brain neuroblast system, composed of cerebellar granule neurons and precursors, now allows definition of mitogenic and trophic factors, second messengers and interacting cellular programs in CNS neurogenesis.
Our specific aims are to define epigenetic signals regulating brain precursors and to characterize relationships of division to neuronal differentiation in PNS and CNS populations. Second, extending our discovery that VIP is a developmentally regulated mitogen and trophic factor, we plan to characterize peptide production and intracellular transduction mechanisms. Third, we will define the role of Id, a member of the helix-loop-helix transcription factor family, in mediating neuroblast response to epigenetic signals. Our strategy employs pure populations of PNS and CNS neuroblasts in serum-free culture. We examine trophic and mitogenic effects by assaying cell number and [3H]thymidine incorporation, using autoradiography and scintillation spectroscopy. Video-enhanced DIC time-lapse image analysis will be employed to define relationships of mitosis to differentiation and epigenetic regulation. We will define mechanisms of VIP production by assaying peptide and mRNA levels in vivo and in vitro. Second messenger systems mediating VIP effects will be assessed by characterizing peptide-induced phosphorylation of exogenous and endogenous proteins. Finally, the expression and epigenetic regulation of Id will be characterized by in situ hybridization, Northern analysis, and immunocytochemistry in vivo and in culture. By characterizing population-specific epigenetic signals and intrinsic molecular mechanisms regulating neuroblast mitosis, we hope to identify unrecognized loci where disease processes intervene to derange neurogenesis. In turn, we may gain insight into mechanisms underlying congenital diseases such as neuronal systems degeneration, and neural tube dysgenesis and potentially design novel approaches to brain repair.
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