Regulation of Stem Cells and Neurogenesis in the Adult Olfactory Epithelium.

The olfactory epithelium (OE) possesses a robust capacity to regenerate neurons and non-neuronal cells, throughout the life of the animal, to maintain the perceptual integrity of olfaction. The stem and progenitor cells responsible for homeostasis and recovery from injury are just beginning to be identified and their mechanisms of action are poorly understood. The transcription factor NeuroD1 plays an important role in progression during neurogenesis in other settings and we hypothesized that it anticipates neuronal differentiation among neuronal progenitors in the OE. By immunochemistry, lineage tracing from the NeuroD1-promoter and targeted knockout mice we show that the NeuroD1-expressing progenitors are characteristically immediate neuronal precursors. In addition, most, if not all, olfactory sensory neurons (OSNs) go through a NeuroD1-expressing stage during their development, however a complete sensory epithelium develops when NeuroD1 is genetically deleted.In other neurogenic matrices, Pax6 and Sox2 are expressed in multipotent progenitors and play roles in maintaining an undifferentiated state. Similarly, OE progenitors also express them, therefore we hypothesized that they function to suppress neurogenesis in the OE. Using retroviral vectors, we have manipulated the levels of Pax6 and Sox2 during regeneration, by over-expressing them separately and in concert, as well as eliminating Sox2 conditionally in floxed-Sox2 mice by retroviral expression of Cre recombinase. We find that Pax6 and Sox2, separately and together can suppress the formation of neurons in clones, though this effect is incomplete. In those clones that contain neurons, Pax6 reduces their numbers, while Sox2 expands them. Moreover, conditional elimination of Sox2 nearly extinguishes the neuronal population, suggesting that Pax6 and Sox2 play counteracting roles to regulate neurogenesis in the OE.Relating to multipotency on a cellular level, rather than a molecular one, we investigated the influence of the regenerating environment on horizontal basal cell (HBC) potency. By way of lineage tracing, HBCs are known to demonstrate multipotency in situ, in response to injury, however upon transplantation derived from normal OE, these cells fail to engraft and do not contribute to the recovery of the tissue. Therefore, we hypothesized that the regenerating environment provides cues that confer a cell-autonomous multipotency, enabling HBCs to actively participate in recovery from injury. We used CD54, an HBC marker in normal OE, to isolate HBCs by FACS and compared donor cells from normal and 2-day post-methyl bromide lesion tissue by transplantation, testing their potency in a colony-forming unit assay. Similar to previous findings, normal HBCs poorly engraft and make small, monotypic clones, whereas 2-day post-lesion, CD54 (+) cells engraft much better and give rise to large, multi-lineage clones, similar to in vivo lineage tracing.Taken together, the data presented in this thesis suggest that in the OE NeuroD1 is a marker for immediate neuronal precursors, Pax6 and Sox2 play counteracting roles to regulate neurogenesis, and that the post-lesion environment is instructive upon resident stem cells, activating their multipotent capacities.