Horizontal basal cells self-govern their neurogenic potential during injury-induced regeneration of the olfactory epithelium.

Horizontal basal cells (HBCs) residing within severely damaged olfactory epithelium (OE) mediate OE regeneration by differentiating into odorant-detecting olfactory sensory neurons (OSNs) and other tissue supporting non-neuronal cell types. Depending on both tissue type and integrity, the Notch signaling pathway can either positively or negatively regulate resident stem cell activity. Although Notch1 specifies HBC dormancy in the uninjured OE, little is known about how HBCs are influenced by the Notch pathway following OE injury. Here, we show that HBCs depend on a functional inversion of the Notch pathway to appropriately mediate OE regeneration. At 24 h post-injury, HBCs enhance Notch1-mediated signaling. Moreover, at 3 days post-injury when the regenerating OE is composed of multiple cell layers, HBCs enrich both Notch1 and the Notch ligand, Dll1. Notably, HBC-specific Notch1 knockout increases HBC quiescence and impairs HBC differentiation into neuronal progenitors and OSNs. Interestingly, complete HBC knockout of Dll1 only decreases differentiation of HBC-derived OSNs. These data underscore the context-dependent nature of Notch signaling. Furthermore, they reveal that HBCs regulate their own neurogenic potential after OE injury.

Molecular Evidence for Olfactory Neuroblastoma as a Tumor of Malignant Globose Basal Cells.

Olfactory neuroblastoma (ONB, esthesioneuroblastoma) is a sinonasal cancer with an underdeveloped diagnostic toolkit, and is the subject of many incidents of tumor misclassification throughout the literature. Despite its name, connections between the cancer and normal cells of the olfactory epithelium have not been systematically explored and markers of olfactory epithelial cell types are not deployed in clinical practice. Here, we utilize an integrated human-mouse single-cell atlas of the nasal mucosa, including the olfactory epithelium, to identify transcriptomic programs that link ONB to a specific population of stem/progenitor cells known as olfactory epithelial globose basal cells (GBCs). Expression of a GBC transcription factor NEUROD1 distinguishes both low- and high-grade ONB from sinonasal undifferentiated carcinoma, a potential histologic mimic with a distinctly unfavorable prognosis. Furthermore, we identify a reproducible subpopulation of highly proliferative ONB cells expressing the GBC stemness marker EZH2, suggesting that EZH2 inhibition may play a role in the targeted treatment of ONB. Finally, we study the cellular states comprising ONB parenchyma using single-cell transcriptomics and identify evidence of a conserved GBC transcriptional regulatory circuit that governs divergent neuronal-versus-sustentacular differentiation. These results link ONB to a specific cell type for the first time and identify conserved developmental pathways within ONB that inform diagnostic, prognostic, and mechanistic investigation.

Spatial Determination of Neuronal Diversification in the Olfactory Epithelium.

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.SIGNIFICANCE STATEMENT Spatially restricted differentiation of olfactory sensory neurons is both key to normal olfactory function and a challenging example of biological specificity. That the stem cells of the olfactory epithelium reproduce the organization of the olfactory periphery to a very close approximation during lesion-induced regeneration begs the question of whether stem cell-autonomous genomic architecture or environmental cues are responsible. The plasticity demonstrated after transfer to a novel location suggests that cues external to the transplanted stem and progenitor cells confer neuronal identity. Thus, a necessary prerequisite is satisfied for using engraftment of olfactory stem and progenitor cells as a cellular therapeutic intervention to reinvigorate neurogenesis whose exhaustion contributes to the waning of olfaction with age.

Identifying Treatments for Taste and Smell Disorders: Gaps and Opportunities.

The chemical senses of taste and smell play a vital role in conveying information about ourselves and our environment. Tastes and smells can warn against danger and also contribute to the daily enjoyment of food, friends and family, and our surroundings. Over 12% of the US population is estimated to experience taste and smell (chemosensory) dysfunction. Yet, despite this high prevalence, long-term, effective treatments for these disorders have been largely elusive. Clinical successes in other sensory systems, including hearing and vision, have led to new hope for developments in the treatment of chemosensory disorders. To accelerate cures, we convened the "Identifying Treatments for Taste and Smell Disorders" conference, bringing together basic and translational sensory scientists, health care professionals, and patients to identify gaps in our current understanding of chemosensory dysfunction and next steps in a broad-based research strategy. Their suggestions for high-yield next steps were focused in 3 areas: increasing awareness and research capacity (e.g., patient advocacy), developing and enhancing clinical measures of taste and smell, and supporting new avenues of research into cellular and therapeutic approaches (e.g., developing human chemosensory cell lines, stem cells, and gene therapy approaches). These long-term strategies led to specific suggestions for immediate research priorities that focus on expanding our understanding of specific responses of chemosensory cells and developing valuable assays to identify and document cell development, regeneration, and function. Addressing these high-priority areas should accelerate the development of novel and effective treatments for taste and smell disorders.

Notch1 maintains dormancy of olfactory horizontal basal cells, a reserve neural stem cell.

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, next-generation 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.

Canonical Notch Signaling Directs the Fate of Differentiating Neurocompetent Progenitors in the Mammalian Olfactory Epithelium.

The adult olfactory epithelium (OE) has the remarkable capacity to regenerate fully both neurosensory and non-neuronal cell types after severe epithelial injury. Lifelong persistence of two stem cell populations supports OE regeneration when damaged: the horizontal basal cells (HBCs), dormant and held in reserve; and globose basal cells, a heterogeneous population most of which are actively dividing. Both populations regenerate all cell types of the OE after injury, but the mechanisms underlying neuronal versus non-neuronal lineage commitment after recruitment of the stem cell pools remains unknown. We used both retroviral transduction and mouse lines that permit conditional cell-specific genetic manipulation as well as the tracing of progeny to study the role of canonical Notch signaling in the determination of neuronal versus non-neuronal lineages in the regenerating adult OE. Excision of either Notch1 or Notch2 genes alone in HBCs did not alter progenitor fate during recovery from epithelial injury, whereas conditional knock-out of both Notch1 and Notch2 together, retroviral transduction of progenitors with a dominant-negative form of MAML (mastermind-like), or excision of the downstream cofactor RBPJ caused progeny to adopt a neuronal fate exclusively. Conversely, we show that overexpressing the Notch1-intracellular domain (N1ICD) either genetically or by transduction blocks neuronal differentiation completely. However, N1ICD overexpression requires both alleles of the canonical cofactor RBPJ to specify downstream lineage. Together, our results suggest that canonical RBPJ-dependent Notch signaling through redundant Notch1 and Notch2 receptors is both necessary and sufficient for determining neuronal versus non-neuronal differentiation in the regenerating adult OE.SIGNIFICANCE STATEMENT Despite the substantial reconstitution of the olfactory epithelium and its population of sensory neurons after injury, disruption and exhaustion of neurogenesis is a consequence of aging and a cause of olfactory dysfunction. Understanding the mechanisms underlying the generation of replacement neurons and non-neuronal cells is critical to any therapeutic strategy aimed at rebuilding a functional neuroepithelium. The results shown here demonstrate that canonical Notch signaling determines the balance between neurons and non-neuronal cells during restoration of the epithelium after injury. Moreover, the complexities of the multiple Notch pathways impinging on that decision are dissected in detail. Finally, RBPJ, the canonical Notch transcriptional cofactor, exhibits a heretofore unreported haploinsufficiency in setting the balance among the regenerating populations.

The Neuroregenerative Capacity of Olfactory Stem Cells Is Not Limitless: Implications for Aging.

The olfactory epithelium (OE) of vertebrates is a highly regenerative neuroepithelium that is maintained under normal conditions by a population of stem and progenitor cells, globose basal cells (GBCs), which also contribute to epithelial reconstitution after injury. However, aging of the OE often leads to neurogenic exhaustion, the disappearance of both GBCs and olfactory sensory neurons (OSNs). Aneuronal tissue may remain as olfactory, with an uninterrupted sheet of apically arrayed microvillar-capped sustentacular cell, or may undergo respiratory metaplasia. We have generated a transgenic mouse model for neurogenic exhaustion using olfactory marker protein-driven Tet-off regulation of the A subunit of Diphtheria toxin such that the death of mature OSNs is accelerated. At as early as 2 months of age, the epithelium of transgenic mice, regardless of sex, recapitulates what is seen in the aged OE of humans and rodents. Areas of the epithelium completely lack neurons and GBCs; whereas the horizontal basal cells, a reserve stem cell population, show no evidence of activation. Surprisingly, other areas that were olfactory undergo respiratory metaplasia. The impact of accelerated neuronal death and reduced innervation on the olfactory bulb (OB) was also examined. Constant neuronal turnover leaves glomeruli shrunken and affects the dopaminergic interneurons in the periglomerular layer. Moreover, the acceleration of OSN death can be reversed in those areas where some GBCs persist. However, the projection onto the OB recovers incompletely and the reinnervated glomeruli are markedly altered. Therefore, the capacity for OE regeneration is tempered when GBCs disappear.SIGNIFICANCE STATEMENT A large percentage of humans lose or suffer a significant decline in olfactory function as they age. Therefore, quality of life suffers and safety and nutritional status are put at risk. With age, the OE apparently becomes incapable of fully maintaining the neuronal population of the epithelium despite its well known capacity for recovering from most forms of injury when younger. Efforts to identify the mechanism by which olfactory neurogenesis becomes exhausted with age require a powerful model for accelerating age-related tissue pathology. The current OMP-tTA;TetO-DTA transgenic mouse model, in which olfactory neurons die when they reach maturity and accelerated death can be aborted to assess the capacity for structural recovery, satisfies that need.

Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors.

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.

Replication of JC Virus DNA in the G144 Oligodendrocyte Cell Line Is Dependent Upon Akt.

Progressive multifocal leukoencephalopathy (PML) is an often-fatal demyelinating disease of the central nervous system. PML results when oligodendrocytes within immunocompromised individuals are infected with the human JC virus (JCV). We have identified an oligodendrocyte precursor cell line, termed G144, that supports robust levels of JCV DNA replication, a central part of the JCV life cycle. In addition, we have determined that JC virus readily infects G144 cells. Furthermore, we have determined that JCV DNA replication in G144 cells is stimulated by myristoylated (i.e., constitutively active) Akt and reduced by the Akt-specific inhibitor MK2206. Thus, this oligodendrocyte-based model system will be useful for a number of purposes, such as studies of JCV infection, establishing key pathways needed for the regulation of JCV DNA replication, and identifying inhibitors of this process.IMPORTANCE The disease progressive multifocal leukoencephalopathy (PML) is caused by the infection of particular brain cells, termed oligodendrocytes, by the JC virus. Studies of PML, however, have been hampered by the lack of an immortalized human cell line derived from oligodendrocytes. Here, we report that the G144 oligodendrocyte cell line supports both infection by JC virus and robust levels of JCV DNA replication. Moreover, we have established that the Akt pathway regulates JCV DNA replication and that JCV DNA replication can be inhibited by MK2206, a compound that is specific for Akt. These and related findings suggest that we have established a powerful oligodendrocyte-based model system for studies of JCV-dependent PML.

Transcription factor p63 controls the reserve status but not the stemness of horizontal basal cells in the olfactory epithelium.

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.

Lysine-specific demethylase-1 (LSD1) is compartmentalized at nuclear chromocenters in early post-mitotic cells of the olfactory sensory neuronal lineage.

Mammalian olfaction depends on the development of specialized olfactory sensory neurons (OSNs) that each express one odorant receptor (OR) protein from a large family of OR genes encoded in the genome. The lysine-specific demethylase-1 (LSD1) protein removes activating H3K4 or silencing H3K9 methylation marks at gene promoters and is required for proper OR regulation. We show that LSD1 protein exhibits variable organization within nuclei of developing OSNs, and tends to consolidate into a single dominant compartment at the edges of chromocenters within nuclei of early post-mitotic cells of the mouse olfactory epithelium (MOE). Using an immortalized cell line derived from developing olfactory placode, we show that consolidation of LSD1 appears to be cell-cycle regulated, with a peak occurrence in early G1. LSD1 co-compartmentalizes with CoREST, a protein known to collaborate with LSD1 to carry out a variety of chromatin-modifying functions. We show that LSD1 compartments co-localize with 1-3 OR loci at the exclusion of most OR genes, and commonly associate with Lhx2, a transcription factor involved in OR regulation. Together, our data suggests that LSD1 is sequestered into a distinct nuclear space that might restrict a histone-modifying function to a narrow developmental time window and/or range of OR gene targets.

Primary Cilia on Horizontal Basal Cells Regulate Regeneration of the Olfactory Epithelium.

The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the mammalian lifespan. It is composed of multiple cell types including olfactory sensory neurons (OSNs) that are readily replaced by two populations of basal stem cells, frequently dividing globose basal cells and quiescent horizontal basal cells (HBCs). However, the precise mechanisms by which these cells mediate OE regeneration are unclear. Here, we show for the first time that the HBC subpopulation of basal stem cells uniquely possesses primary cilia that are aligned in an apical orientation in direct apposition to sustentacular cell end feet. The positioning of these cilia suggests that they function in the detection of growth signals and/or differentiation cues. To test this idea, we generated an inducible, cell type-specific Ift88 knock-out mouse line (K5rtTA;tetOCre;Ift88(fl/fl)) to disrupt cilia formation and maintenance specifically in HBCs. Surprisingly, the loss of HBC cilia did not affect the maintenance of the adult OE but dramatically impaired the regeneration of OSNs following lesion. Furthermore, the loss of cilia during development resulted in a region-specific decrease in neurogenesis, implicating HBCs in the establishment of the OE. Together, these results suggest a novel role for primary cilia in HBC activation, proliferation, and differentiation. SIGNIFICANCE STATEMENT: We show for the first time the presence of primary cilia on a quiescent population of basal stem cells, the horizontal basal cells (HBCs), in the olfactory epithelium (OE). Importantly, our data demonstrate that cilia on HBCs are necessary for regeneration of the OE following injury. Moreover, the disruption of HBC cilia alters neurogenesis during the development of the OE, providing evidence that HBCs participate in the establishment of this tissue. These data suggest that the mechanisms of penetrance for ciliopathies in the OE extend beyond that of defects in olfactory sensory neurons and may include alterations in OE maintenance and regeneration.

The regeneration of P2 olfactory sensory neurons is selectively impaired following methyl bromide lesion.

The capacity of the peripheral olfactory system to recover after injury has not been thoroughly explored. P2-IRES-tauLacZ mice were exposed to methyl bromide, which causes epithelial damage and kills 90% of the P2 neurons. With subsequent neuronal regeneration, P2 neurons recover within their usual territory to equal control numbers by 1 month but then decline sharply to roughly 40% of control by 3 months. At this time, the P2 projection onto the olfactory bulb is erroneous in several respects. Instead of converging onto 1 or 2 glomeruli per surface, small collections of P2 axons innervate multiple glomeruli at roughly the same position in the bulb as in controls. Within these glomeruli, the P2 axons are aggregated near the edge, whereas the remainder of the glomerulus contains olfactory marker protein (+), non-P2 axons, violating the one receptor-one glomerulus rule normally observed. The aggregates are denser than found in control P2-innervated glomeruli, suggesting that the P2 axons may not be synaptically connected. Based on published literature and other data, we hypothesize that P2 neurons lose out in an activity-based competition for synaptic territory within the glomeruli and are not maintained at control numbers due to a lack of trophic support from the bulb.

Spatiotemporal dynamics exhibited by horizontal basal cells reveal a pro-neurogenic pathway during injury-induced olfactory epithelium regeneration.

Horizontal basal cells (HBCs) mediate olfactory epithelium (OE) regeneration following severe tissue injury. The dynamism of the post-injury environment is well illustrated by in silico modeling of RNA sequencing data that demonstrate an evolving HBC transcriptome. Unfortunately, spatiotemporally dynamic processes occurring within HBCs in situ remain poorly understood. Here, we show that HBCs at 24 h post-OE injury spatially redistribute a constellation of proteins, which, in turn, helped to nominate Rac1 as a regulator of HBC differentiation during OE regeneration. Using our primary culture model to activate HBCs pharmacologically, we demonstrate that concurrent Rac1 inhibition attenuates HBC differentiation potential. This in vitro functional impairment manifested in vivo as decreased HBC differentiation into olfactory sensory neurons following HBC-specific Rac1 conditional knockout. Taken together, our data potentiate the design of hyposmia-alleviating therapies and highlight aspects of in situ HBC spatiotemporal dynamics that deserve further investigation.

Canonical Wnt signaling promotes the proliferation and neurogenesis of peripheral olfactory stem cells during postnatal development and adult regeneration.

The mammalian olfactory epithelium (OE) has a unique stem cell or progenitor niche, which is responsible for the constant peripheral neurogenesis throughout the lifespan of the animal. However, neither the signals that regulate the behavior of these cells nor the lineage properties of the OE stem cells are well understood. Multiple Wnt signaling components exhibit dynamic expression patterns in the developing OE. We generated Wnt signaling reporter TOPeGFP transgenic mice and found TOPeGFP activation predominantly in proliferating Sox2(+) OE basal cells during early postnatal development. FACS-isolated TOPeGFP(+) OE basal cells are required, but are not sufficient, for formation of spheres. Wnt3a significantly promotes the proliferation of the Sox2(+) OE sphere cells. Wnt-stimulated OE sphere cells maintain their multipotency and can differentiate into most types of neuronal and non-neuronal epithelial cells. Also, Wnt activators shift the production of differentiated cells toward olfactory sensory neurons. Moreover, TOPeGFP(+) cells are robustly increased in the adult OE after injury. In vivo administration of Wnt modulators significantly alters the regeneration potential. This study demonstrates the role of the canonical Wnt signaling pathway in the regulation of OE stem cells or progenitors during development and regeneration.

Mechanisms of permanent loss of olfactory receptor neurons induced by the herbicide 2,6-dichlorobenzonitrile: effects on stem cells and noninvolvement of acute induction of the inflammatory cytokine IL-6.

We explored the mechanisms underlying the differential effects of two olfactory toxicants, the herbicide 2,6-dichlorobenzonitrile (DCBN) and the anti-thyroid drug methimazole (MMZ), on olfactory receptor neuron (ORN) regeneration in mouse olfactory epithelium (OE). DCBN, but not MMZ, induced inflammation-like pathological changes in OE, and DCBN increased interleukin IL-6 levels in nasal-wash fluid to much greater magnitude and duration than did MMZ. At 24h after DCBN injection, the population of horizontal basal cells (HBCs; reserve, normally quiescent OE stem cells) lining the DMM became severely depleted as some of them detached from the basal lamina, and sloughed into the nasal cavity along with the globose basal cells (GBCs; heterogeneous population of stem and progenitor cells), neurons, and sustentacular cells of the neuroepithelium. In contrast, the layer of HBCs remained intact in MMZ-treated mice, as only the mature elements of the neuroepithelium were shed. Despite the respiratory metaplasia accompanying the greater severity of the DCBN lesion, residual HBCs that survived intoxication were activated by the injury and contributed to the metaplastic respiratory epithelium, as shown by tracing their descendants in a K5CreEr(T2)::fl(stop)TdTomato strain of mice in which recombination causes HBCs to express TdTomato in advance of the lesion. But, contrary to published observations with MMZ, the HBCs failed to form ORNs. A role for IL-6 in suppressing ORN regeneration in DCBN-treated mice was rejected by the failure of the anti-inflammatory drug dexamethasone to prevent the subsequent respiratory metaplasia in the DMM, suggesting that other factors lead to HBC neuro-incompetence.

An in vitro model of acute horizontal basal cell activation reveals dynamic gene regulatory networks underlying the acute activation phase.

Horizontal basal cells (HBCs) activate only in response to severe olfactory epithelium (OE) injury. This activation is mediated by the loss of the transcription factor TP63. Using the compound phorbol 12-myristate 13-acetate (PMA), we find that we can model the process of acute HBC activation. First, we find that PMA treatment induces a rapid loss in TP63 protein and induces the expression of HOPX and the nuclear translocation of RELA, previously identified to mediate HBC activation. Using bulk RNA sequencing, we find that PMA-treated HBCs pass through various stages of acute activation identifiable by transcriptional regulatory signatures that mimic stages identified in vivo . These temporal stages are associated with varying degrees of engraftment and differentiation potential in transplantation assays. Together, this data shows that our model can model physiologically relevant features of HBC activation and identifies new candidates for mechanistic testing.

DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium.

The ability of the olfactory epithelium (OE) to regenerate after injury is mediated by at least two populations of presumed stem cells-globose basal cells (GBCs) and horizontal basal cells (HBCs). Of the two, GBCs are molecularly and phenotypically analogous to the olfactory progenitors of the embryonic placode (OPPs). In contrast, HBCs are a reserve stem cell population that appears later in development and requires activation by severe epithelial damage before contributing to epithelial reconstitution. Neither HBC emergence nor the mechanism of activation after injury is understood. Here we show that the transcription factor p63 (Trp63), which is expressed selectively by adult HBCs, is required for HBC differentiation. The first evidence of HBC differentiation is the expression of p63 by cells that closely resemble embryonic OPPs and adult GBCs by morphology and expression of the transcription factors Sox2, Ascl1, and Hes1. HBC formation is delayed in Ascl1 knock-out OE and is completely abrogated in p63-null mice. Strikingly, other cell types of the OE form normally in the p63 knock-out OE. The role of p63 in HBC differentiation appears to be conserved in the regenerating rat OE, where HBCs disappear and then reappear after tissue lesion. Finally, p63 protein is downregulated in HBCs activated by lesion to become multipotent progenitor cells. Together, our data identify a novel mechanism for the generation of a reserve stem cell population and suggest that a p63-dependent molecular switch is responsible for activating reserve stem cells when they are needed.

Lifespan of mature olfactory sensory neurons varies with location in the mouse olfactory epithelium and age of the animal.

The olfactory sensory neurons (OSNs) of the olfactory epithelium (OE) exhibit a remarkable regenerative capability, which protects the population against environmental insult and enables adjustment to new odors. The lifespan of OSNs is still open to question, with estimates ranging from 1 month to at least 1 year. However, the estimates come with some caveats, including low labeling efficiency and a focus solely on newborn neurons. We revisited the issue via the use of OMP-tTA; TetO-Cre; Rosa26-fl(stop)-Tdtomato (OMP-tTA;TdT) mice, which allowed us to selectively label ∼95% of the OMP(+) OSN population that reach maturity by a given time and, by switching to doxycycline chow, to "chase" this preexisting OSN population. Two loading protocols were used: conception to 2 months old and conception to 4.5 months old. Surviving OSNs were common up to 6 months chase time in both groups, but more neurons survived when loading for 4.5 months as compared with 2 months. A spatial difference was evident: higher percentages of OSNs survived in the dorsomedial OE as compared with ventrolateral and in posterior versus anterior OE regions. Finally, proliferation rates anticorrelated with the spatial differences in OSN survival; higher proliferation rates were observed ventrally. Together, these results demonstrate spatial and temporal differences in OSN survival, highlighting it as a dynamic system that can be studied for factors affecting neuronal survival.

Rapid fluorescent vital imaging of olfactory epithelium.

Olfactory epithelium (OE) undergoes degeneration in disorders such as age-related and post-viral olfactory dysfunction. However, methods for real-time in vivo detection of OE and assessment of total extent within the nasal cavity are currently unavailable. We identified two fluorescence probes for rapidly detecting and evaluating the entire extent of mice OE with topical application. Taking advantage of the differential expression of the enzymes cytochrome p450 (CYP) and γ-glutamyltranspeptidase (GGT) in OE relative to respiratory epithelium, we utilized the conversion of coumarin (a substrate of various CYP subtypes) and gGlu-HRMG (a substrate of GGT) by these enzymes to form metabolites with fluorescent emissions in the duct cells and sustentacular cells of neuron-containing OE. In depleted and regenerated OE model, the emission of these probes remained absent in respiratory metaplasia but appeared in regenerated OE. These substrates could be used to monitor OE degeneration and follow regenerative response to therapeutic interventions.