Neurogenesis, the proliferation of neuronal precursor cells and the terminal differentiation of their progeny into neurons, is the primary event that determines neuronal number during nervous system development. It appears that this process is tightly regulated in vivo, but little is known about the mechanisms of its regulation or the molecules involved. The goal of the proposed research is to identify basic mechanisms that regulate neurogenesis in the mammalian embryonic nervous system. To accomplish this, a tissue culture system has been developed for studying neurogenesis in a model neuroepithelium, the olfactory epithelium of the mouse embryo. Unique properties of the olfactory epithelium that make it an ideal system for studying neurogenesis include: It is structurally simple, producing only one type of neuron; it maintains the ability to generate neurons throughout adult life; and there is good evidence that regulatory signals control its production of neurons in vivo. Preliminary studies on explant cultures of olfactory epithelium have demonstrated that neurogenesis can occur efficiently in vitro. Using this system, it has been possible to identify distinct cellular stages in the neurogenic pathway that are likely to be subject to regulation. Significantly, a neuronal precursor cell, which divides to give rise to daughter neurons in defined, serum-free culture, has been identified and named the Immediate Neuronal Precursor (INP). Methods have now been developed to purify INPs and culture them in a rigorously controlled environment. In addition, procedures are being developed for purifying the neuronal progeny of INPs, the olfactory receptor neurons, using monospecific antibodies directed against a unique cell-surface protein expressed by these neurons in transgenic mice. Using these purified populations of cells, experiments will be carried out to determine how the environment of neuronal precursor cells controls their identify and fate. The process of neurogenesis will be analyzed in cultures of purified INPs, to characterize the pattern of cell divisions and the developmental fates of neuronal precursors isolated from other cell types. Experiments to identify cell-cell interactions that regulate neurogenesis will focus on the role of cell contact and cell density and on the possibility that mature olfactory receptor neurons regulate the proliferation of their own precursors. Molecules mediating these cell- cell interactions will be identified in experiments localizing the expression of polypeptide growth factors and their receptors. By providing information about how the production of neurons is controlled, this work will aid in the understanding and treatment of developmental disorders of the nervous system, such as neural tube defects. In addition, by identifying mechanisms that regulate the production of neurons, this work may aid in the development of neuronal replacement therapies for neurodegenerative diseases.
