The mammalian olfactory epithelium (OE) is able to regenerate its cellular components (neurons, supporting cells, and duct/gland cells) throughout adult life under normal, maintenance conditions, as well as regenerative conditions after destruction of all differentiated cell types by exposure to the gas methyl bromide (MeBr). This ability together with its peripheral and therefore accessible location makes the OE an attractive system to utilize for purposes of regenerative medicine. Before such a potential can be realized one must first perform a complete structural, biochemical, and functional characterization of the cellular dynamics of the OE. At least two distinct populations of putative stem or progenitor cells have been identified among the basal cells of the epithelium that may be responsible for its regenerative capacity. Globose Basal Cells (GBCs) constitute a heterogeneous population of lineage committed and uncommitted progenitors situated below (basal to) the neuronal layer of the epithelium. Horizontal Basal Cells (HBCs) lie in the basalmost layer of the OE and correspond to an apparently homogeneous, layer of cells in tight contact with the basement membrane of the OE. Both of these basal cell types have been shown by various methods to have the ability to generate all the differentiated cell types of the OE, however HBCs are not present until late in embryonic development whereas GBCs are the progenitor cells involved in the embryonic assembly of the tissue. The transcription factor p63, a member of the p53 family, is necessary for the embryonic appearance of HBCs and loss of p63 expression in the adult regenerative environment appears to be correlated with the ability of HBCs to give rise to differentiated OE cell types. The goal of the proposed research is to exhaustively characterize the functional role of p63 in HBC generation, quiescence, activation, and transdifferentiation. First, nascent HBCs that express p63 but have not yet differentiated toward the HBC phenotype will be characterized based on expression of other markers previously identified in the lab as OE progenitor cell markers. Next, the sufficiency of p63 expression in HBC generation will be probed by forcing expression of p63 in the regenerative environment via retroviral infection. Finally the role of p63 loss will be assessed in the adult normal and post-lesion settings, via a conditional knock-out genetic approach. These studies will go a long way toward elucidating the nature of context dependent plasticity present in the OE specifically and adult stem cell systems in general, as well as provide a mechanistic framework in which to approach the therapeutic application of these cells.
In the course of human ageing, HBCs are one of the last cell types remaining in the olfactory epithelium even in patients with anosmia (loss of the sense of smell). The experiments proposed here will elucidate the mechanisms of HBC plasticity which could be utilized in the clinic either to activate these cells directly for regenerative medicine applications, or to obtain a patient specific activatable stem cell population for disease modeling and drug screening.
