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.

Basal cells are multipotent airway progenitors that generate distinct epithelial cell phenotypes crucial for homeostasis and repair of the conducting airways. Little is known about how these progenitor cells expand and transition to differentiation to form the pseudostratified airway epithelium in the developing and adult lung. Here, we show by genetic and pharmacological approaches that endogenous activation of Notch3 signaling selectively controls the pool of undifferentiated progenitors of upper airways available for differentiation. This mechanism depends on the availability of Jag1 and Jag2, and is key to generating a population of parabasal cells that later activates Notch1 and Notch2 for secretory-multiciliated cell fate selection. Disruption of this mechanism resulted in aberrant expansion of basal cells and altered pseudostratification. Analysis of human lungs showing similar abnormalities and decreased NOTCH3 expression in subjects with chronic obstructive pulmonary disease suggests an involvement of NOTCH3-dependent events in the pathogenesis of this condition.

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.

The remarkable capacity of the adult olfactory epithelium (OE) to regenerate fully both neurosensory and nonneuronal cell types after severe epithelial injury depends on life-long persistence of two stem cell populations: the horizontal basal cells (HBCs), which are quiescent and held in reserve, and mitotically active globose basal cells. It has recently been demonstrated that down-regulation of the ΔN form of the transcription factor p63 is both necessary and sufficient to release HBCs from dormancy. However, the mechanisms by which p63 is down-regulated after acute OE injury remain unknown. To identify the cellular source of potential signaling mechanisms, we assessed HBC activation after neuron-only and sustentacular cell death. We found that ablation of sustentacular cells is sufficient for HBC activation to multipotency. By expression analysis, nextgeneration sequencing, and immunohistochemical examination, down-regulation of Notch pathway signaling is coincident with HBC activation. Therefore, using HBC-specific conditional knockout of Notch receptors and overexpression of N1ICD, we show that Notch signaling maintains p63 levels and HBC dormancy, in contrast to its suppression of p63 expression in other tissues. Additionally, Notch1, but not Notch2, is required to maintain HBC dormancy after selective neuronal degeneration. Taken together, our data indicate that the activation of HBCs observed after tissue injury or sustentacular cell ablation is caused by the reduction/elimination of Notch signaling on HBCs; elimination of Jagged1 expressed by sustentacular cells may be the ligand responsible.Notch | olfactory epithelium | reserve stem cell | trp63

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.