The capacity of the olfactory epithelium (OE) for lifelong neurogenesis and regeneration depends on the persistence of neurocompetent stem cells, which self-renew as well as generating all of the cell types found within the nasal epithelium. This Review focuses on the types of stem and progenitor cells in the epithelium and their regulation. Both horizontal basal cells (HBCs) and some among the population of globose basal cells (GBCs) are stem cells, but the two types plays vastly different roles. The GBC population includes the basal cells that proliferate in the uninjured OE and is heterogeneous with respect to transcription factor expression. From upstream in the hierarchy to downstream, GBCs encompass 1) Sox2+/Pax6+ stem-like cells that are totipotent and self-renew over the long term, 2) Ascl1+ transit-amplifying progenitors with a limited capacity for expansive proliferation, and 3) Neurog1+/NeuroD1+ immediate precursor cells that make neurons directly. In contrast, the normally quiescent HBCs are activated to multipotency and proliferate when sustentacular cells are killed, but not when only OSNs die, indicating that HBCs are reserve stem cells that respond to severe epithelial injury. The master regulator of HBC activation is the ΔN isoform of the transcription factor p63; eliminating ΔNp63 unleashes HBC multipotency. Notch signaling, via Jagged1 ligand on Sus cells and Notch1 and Notch2 receptors on HBCs, is likely to play a major role in setting the level of p63 expression. Thus, ΔNp63 becomes a potential therapeutic target for reversing the neurogenic exhaustion characteristic of the aged OE.

Adult tissue stem cells can serve two broad functions: to participate actively in the maintenance and regeneration of a tissue or to wait in reserve and participate only when activated from a dormant state. The adult olfactory epithelium, a site for ongoing, life-long, robust neurogenesis, contains both of these functional stem cell types. Globose basal cells (GBCs) act as the active stem cell population and can give rise to all the differentiated cells found in the normal tissue. Horizontal basal cells (HBCs) act as reserve stem cells and remain dormant unless activated by tissue injury. Here we show that HBC activation following injury by the olfactotoxic gas methyl bromide is coincident with the down-regulation of protein 63 (p63) but anticipates HBC proliferation. Gain- and loss-of-function studies show that this down-regulation of p63 is necessary and sufficient for HBC activation. Moreover, activated HBCs give rise to GBCs that persist for months and continue to act as bona fide stem cells by participating in tissue maintenance and regeneration over the long term. Our analysis provides mechanistic insight into the dynamics between tissue stem cell subtypes and demonstrates that p63 regulates the reserve state but not the stem cell status of HBCs.

Summary Adult neurogenesis in the olfactory epithelium is often depicted as a unidirectional pathway during homeostasis and repair. We challenge the unidirectionality of this model by showing that epithelial injury unlocks the potential for Ascl1+ progenitors and Neurog1+ specified neuronal precursors to dedifferentiate into multipotent stem/progenitor cells that contribute significantly to tissue regeneration in the murine olfactory epithelium (OE). We characterize these dedifferentiating cells using several lineage tracing strains and single-cell mRNA-seq, and we show that Sox2 is required for initiating dedifferentiation and that inhibition of Ezh2 promotes multipotent progenitor expansion. These results suggest that the apparent hierarchy of neuronal differentiation is not irreversible, and that lineage commitment can be overridden following severe tissue injury. We elucidate a previously unappreciated pathway for endogenous tissue repair by a highly regenerative neuroepithelium and introduce a system to study the mechanisms underlying plasticity in the OE that can be adapted for other tissues.

Summary The olfactory epithelium (OE) regenerates after injury via two types of tissue stem cells: active globose cells (GBCs) and dormant horizontal basal cells (HBCs). HBCs are roused to activated status by OE injury when P63 levels fall. However, an in-depth understanding of activation requires a system for culturing them that maintains both their self-renewal and multipotency while preventing spontaneous differentiation. Here, we demonstrate that mouse, rat, and human HBCs can be cultured and passaged as P63+ multipotent cells. HBCs in vitro closely resemble HBCs in vivo based on immunocytochemical and transcriptomic comparisons. Genetic lineage analysis demonstrates that HBCs in culture arise from both tissue-derived HBCs and multipotent GBCs. Treatment with retinoic acid induces neuronal and non-neuronal differentiation and primes cultured HBCs for transplantation into the lesioned OE. Engrafted HBCs generate all OE cell types, including olfactory sensory neurons, confirming that HBC multipotency and neurocompetency are maintained in culture.

Neurons in the murine olfactory epithelium (OE) differ by the olfactory receptor they express as well as other molecular phenotypes that are regionally restricted. These patterns can be precisely regenerated following epithelial injury, suggesting that spatial cues within the tissue can direct neuronal diversification. Nonetheless, the permanency and mechanism of this spatial patterning remain subject to debate. Via transplantation of stem and progenitor cells from dorsal OE into ventral OE, we demonstrate that, in mice of both sexes, nonautonomous spatial cues can direct the spatially circumscribed differentiation of olfactory sensory neurons. The vast majority of dorsal transplant-derived neurons express the ventral marker OCAM (NCAM2) and lose expression of NQO1 to match their new location. Single-cell analysis also demonstrates that OSNs adopt a fate defined by their new position following progenitor cell transplant, such that a ventral olfactory receptor is expressed after stem and progenitor cell engraftment. Thus, spatially constrained differentiation of olfactory sensory neurons is plastic, and any bias toward an epigenetic memory of place can be overcome.