This proposal signifies an expansion of my research efforts. My previous work has focused on defining early and late stages in the differentiation of olfactory sensory neurons, the capacity of the primary olfactory projection to recover from injury and the histopathology of human olfactory disorders. All three of these issues are enmeshed with the epithelium's unique capacity for neurogenesis throughout the life of the animal and for regeneration of the neuronal population after injury. This grant focuses on the biology of the basal cells in the olfactory epithelium, which are the neuronal precursor cells underlying the epithelium's neurogenic capacity. However, the regulation of olfactory neurogenesis is poorly understood at either a phenomenological or mechanistic level. The experiments proposed here seek answers to three questions about the basal cells of the olfactory epithelium. (1) Are basal cells functionally heterogenous with respect to their capacity to remain in the mitotic cycle? Their capacity as stem cells can be assayed by combining the sequential administration of 3H-thymidine and bromodeoxyuridine with an immunochemical definition of cellular phenotype. (2) What is the life history of individual basal cells? Genomic incorporation of a replication incompetent retroviral vector will be used to permanently label individual basal cells and their descendants, which will inherit this marker. (3) Can monoclonal antibodies be generated that will selectively mark basal cells and not neurons, and can ones be generated that will distinguish between various categories of basal cells? In contrast with previous attempts to answer this question, the immune response of the animal will be manipulated to focus more specifically on the basal cells, and the immunogen will consist of an enriched preparation of basal cells (partially purified either by """"""""panning"""""""" procedures or fluorescence activated cell sorting based on the incorporation of bromodeoxyuridine by dividing cells). Answers to these questions have implications for identifying how neurogenesis is regulated in the olfactory epithelium, for relating neurogenesis to sensory function, and for understanding those human olfactory diseases that are disorders of neurogenesis. In addition, collaborative arrangements are outlined that will enable me to acquire new training, including experience with molecular biological techniques. this additional training will prepare me for the pursuit of my long-term research objectives related to the cellular and molecular biological mechanisms regulating neurogenesis and neuronal differentiation in the olfactory epithelium.
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