A Transcription Factor Etv1/Er81 Is Involved in the Differentiation of Sweet, Umami, and Sodium Taste Cells.

Taste cells are maintained by continuous turnover throughout a lifetime, yet the mechanisms of taste cell differentiation, and how taste sensations remain constant despite this continuous turnover, remain poorly understood. Here, we report that a transcription factor Etv1 (also known as Er81) is involved in the differentiation of taste cells responsible for the preference for sweet, umami, and salty tastes. Molecular analyses revealed that Etv1 is expressed by a subset of taste cells that depend on Skn-1a (also known as Pou2f3) for their generation and express T1R genes (responsible for sweet and umami tastes) or Scnn1a (responsible for amiloride-sensitive salty taste). Etv1CreERT2/CreERT2 mice express Etv1 isoform(s) but not Etv1 in putative proprioceptive neurons as comparable to wild-type mice, yet lack expression of Etv1 or an isoform in taste cells. These Etv1CreERT2/CreERT2 mice have the same population of Skn-1a-dependent cells in taste buds as wild-type mice but have altered gene expression in taste cells, with regional differences. They have markedly decreased electrophysiological responses of chorda tympani nerves to sweet and umami tastes and to amiloride-sensitive salty taste evoked by sodium cation, but they have unchanged responses to bitter or sour tastes. Our data thus show that Etv1 is involved in the differentiation of the taste cells responsible for sweet, umami, and salty taste preferences.

Maintenance and turnover of Sox2+ adult stem cells in the gustatory epithelium.

Continuous turnover of taste bud cells in the oral cavity underlies the homeostasis of taste tissues. Previous studies have demonstrated that Sox2+ stem cells give rise to all types of epithelial cells including taste bud cells and non-gustatory epithelial cells in the oral epithelium, and Sox2 is required for generating taste bud cells. Here, we show the dynamism of single stem cells through multicolor lineage tracing analyses in Sox2-CreERT2; Rosa26-Confetti mice. In the non-gustatory epithelium, unicolored areas populated by a cluster of cells expressing the same fluorescent protein grew over time, while epithelial cells were randomly labeled with multiple fluorescent proteins by short-term tracing. Similar phenomena were observed in gustatory epithelia. These results suggest that the Sox2+ stem cell population is maintained by balancing the increase of certain stem cells with the reduction of the others. In the gustatory epithelia, many single taste buds contained cells labeled with different fluorescent proteins, indicating that a single taste bud is composed of cells derived from multiple Sox2+ stem cells. Our results reveal the characteristics of Sox2+ stem cells underlying the turnover of taste bud cells and the homeostasis of taste tissues.

R-spondin substitutes for neuronal input for taste cell regeneration in adult mice.

Taste bud cells regenerate throughout life. Taste bud maintenance depends on continuous replacement of senescent taste cells with new ones generated by adult taste stem cells. More than a century ago it was shown that taste buds degenerate after their innervating nerves are transected and that they are not restored until after reinnervation by distant gustatory ganglion neurons. Thus, neuronal input, likely via neuron-supplied factors, is required for generation of differentiated taste cells and taste bud maintenance. However, the identity of such a neuron-supplied niche factor(s) remains unclear. Here, by mining a published RNA-sequencing dataset of geniculate ganglion neurons and by in situ hybridization, we demonstrate that R-spondin-2, the ligand of Lgr5 and its homologs Lgr4/6 and stem-cell-expressed E3 ligases Rnf43/Znrf3, is expressed in nodose-petrosal and geniculate ganglion neurons. Using the glossopharyngeal nerve transection model, we show that systemic delivery of R-spondin via adenovirus can promote generation of differentiated taste cells despite denervation. Thus, exogenous R-spondin can substitute for neuronal input for taste bud cell replenishment and taste bud maintenance. Using taste organoid cultures, we show that R-spondin is required for generation of differentiated taste cells and that, in the absence of R-spondin in culture medium, taste bud cells are not generated ex vivo. Thus, we propose that R-spondin-2 may be the long-sought neuronal factor that acts on taste stem cells for maintaining taste tissue homeostasis.

Expression of Eya1 in mouse taste buds.

Taste substances are detected by taste receptor cells in the taste buds in the oral epithelium. Individual taste receptor cells contribute to evoking one of the five taste qualities: sweet, umami, bitter, sour, and salty (sodium). They are continuously replaced every few weeks by new ones generated from local epithelial stem cells. A POU transcription factor, Pou2f3 (also known as Skn-1a), regulates the generation and differentiation of sweet, umami, and bitter cells. However, the molecular mechanisms underlying terminal differentiation into these Pou2f3-dependent taste receptor cells remain unknown. To identify the candidate molecules that regulate the differentiation of these taste receptor cells, we searched for taste receptor type-specific transcription factors using RNA-sequence data of sweet and bitter cells. No transcription factor gene showing higher expression in sweet cells than in bitter cells was found. Eyes absent 1 (Eya1) was identified as the only transcription factor gene showing higher expression in bitter cells than in sweet cells. In situ hybridization revealed that Eya1 was predominantly expressed in bitter cells and also in the putative immature/differentiating taste bud cells in circumvallate and fungiform papillae and soft palate. Eya1 is a candidate molecule that regulates the generation and differentiation of bitter cells.

Sodium-Taste Cells Require Skn-1a for Generation and Share Molecular Features with Sweet, Umami, and Bitter Taste Cells.

Taste buds are maintained via continuous turnover of taste bud cells derived from local epithelial stem cells. A transcription factor Skn-1a (also known as Pou2f3) is required for the generation of sweet, umami (savory), and bitter taste cells that commonly express TRPM5 and CALHM ion channels. Here, we demonstrate that sodium-taste cells distributed only in the anterior oral epithelia and involved in evoking salty taste also require Skn-1a for their generation. We discovered taste cells in fungiform papillae and soft palate that show similar but not identical molecular feature with sweet, umami, and bitter taste-mediated Type II cells. This novel cell population expresses Plcb2, Itpr3, Calhm3, Skn-1a, and ENaCα (also known as Scnn1a) encoding the putative amiloride-sensitive (AS) salty taste receptor but lacks Trpm5 and Gnat3Skn-1a-deficient taste buds are predominantly composed of putative non-sensory Type I cells and sour-sensing Type III cells, whereas wild-type taste buds include Type II (i.e., sweet, umami, and bitter taste) cells and sodium-taste cells. Both Skn-1a and Calhm3-deficient mice have markedly decreased chorda tympani nerve responses to sodium chloride, and those decreased responses are attributed to the loss of the AS salty taste response. Thus, AS salty taste is mediated by Skn-1a-dependent taste cells, whereas amiloride-insensitive salty taste is mediated largely by Type III sour taste cells and partly by bitter taste cells. Our results demonstrate that Skn-1a regulates differentiation toward all types of taste cells except sour taste cells.

SOX2 regulates homeostasis of taste bud cells and lingual epithelial cells in posterior tongue.

Taste bud cells arise from local epithelial stem cells in the oral cavity and are continuously replaced by newborn cells throughout an animal's life. However, little is known about the molecular and cellular mechanisms of taste cell turnover. Recently, it has been demonstrated that SOX2, a transcription factor expressed in epithelial stem/progenitor cells of the oral cavity, regulates turnover of anterior tongue epithelium including gustatory and non-gustatory papillae. Yet, the role of SOX2 in regulating taste cell turnover in the posterior tongue is unclear. Prompted by the fact that there are regional differences in the cellular and molecular composition of taste buds and stem/progenitor cells in the anterior and posterior portions of tongue, which are derived from distinct embryonic origins, we set out to determine the role of SOX2 in epithelial tissue homeostasis in the posterior tongue. Here we report the differential requirement of SOX2 in the stem/progenitor cells for the normal turnover of lingual epithelial cells in the posterior tongue. Sox2 deletion in the stem/progenitor cells neither induced active caspase 3-mediated apoptotic cell death nor altered stem/progenitor cell population in the posterior tongue. Nevertheless, morphology and molecular feature of non-gustatory epithelial cells were impaired in the circumvallate papilla but not in the filiform papillae. Remarkably, taste buds became thinner, collapsed, and undetectable over time. Lineage tracing of Sox2-deleted stem/progenitor cells demonstrated an almost complete lack of newly generated basal precursor cells in the taste buds, suggesting mechanistically that Sox2 is involved in determining stem/progenitor cells to differentiate to gustatory lineage cells. Together, these results demonstrate that SOX2 plays key roles in regulating epithelial tissue homeostasis in the posterior tongue, similar but not identical to its function in the anterior tongue.

Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D2.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. METHODS: We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. RESULTS: Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. CONCLUSIONS: In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

Erratum: Author Correction: Bcl11b controls odorant receptor class choice in mice.

[This corrects the article DOI: 10.1038/s42003-019-0536-x.].

Bcl11b controls odorant receptor class choice in mice.

Each olfactory sensory neuron (OSN) expresses a single odorant receptor (OR) gene from the class I or class II repertoire in mice. The mechanisms that regulate OR class choice in OSNs remain unknown. Here, we show that the transcription factor Bcl11b determines the OR class to be expressed in OSNs. Both loss- and gain-of-function analyses demonstrate that class I is a default fate of OSNs and that Bcl11b dictates a class II OR choice by suppressing the effect of the J-element, a class I-OR enhancer. We further demonstrate that OSN-specific genetic manipulations of Bcl11b bias the OR class choice, generating mice with "class I-dominant" and "class II-dominant" noses, which display contrasting innate olfactory behaviors to two distinct aversive odorants. Overall, these findings reveal a unique transcriptional mechanism mediating a binary switch for OR class choice that is crucial to both the anatomical and functional organization of the olfactory system.

Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine.

The hallmark features of type 2 mucosal immunity include intestinal tuft and goblet cell expansion initiated by tuft cell activation. How infectious agents that induce type 2 mucosal immunity are detected by tuft cells is unknown. Published microarray analysis suggested that succinate receptor 1 (Sucnr1) is specifically expressed in tuft cells. Thus, we hypothesized that the succinate-Sucnr1 axis may be utilized by tuft cells to detect certain infectious agents. Here we confirmed that Sucnr1 is specifically expressed in intestinal tuft cells but not in other types of intestinal epithelial cells, and demonstrated that dietary succinate induces tuft and goblet cell hyperplasia via Sucnr1 and the tuft cell-expressed chemosensory signaling elements gustducin and Trpm5. Conventional mice with a genetic Sucnr1 deficiency (Sucnr1-/-) showed diminished immune responses to treatment with polyethylene glycol and streptomycin, which are known to enhance microbiota-derived succinate, but responded normally to inoculation with the parasitic worm Nippostrongylus brasiliensis that also produces succinate. Thus, Sucnr1 is required for microbiota-induced but not for a generalized worm-induced type 2 immunity.

CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes.

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.

Skn-1a/Pou2f3 functions as a master regulator to generate Trpm5-expressing chemosensory cells in mice.

Transient receptor potential channel M5 (Trpm5)-expressing cells, such as sweet, umami, and bitter taste cells in the oropharyngeal epithelium, solitary chemosensory cells in the nasal respiratory epithelium, and tuft cells in the small intestine, that express taste-related genes function as chemosensory cells. Previous studies demonstrated that Skn-1a/Pou2f3, a POU homeodomain transcription factor is expressed in these Trpm5-expressing chemosensory cells, and is necessary for their generation. Trpm5-expressing cells have recently been found in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. They are considered to be involved in protective responses to potential hazardous compounds as Skn-1a-dependent bitter taste cells, respiratory solitary chemosensory cells, and intestinal tuft cells are. In this study, we examined the expression and function of Skn-1a/Pou2f3 in Trpm5-expressing cells in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. Skn-1a/Pou2f3 is expressed in a majority of Trpm5-expressing cells in all tissues examined. In Skn-1a/Pou2f3-deficient mice, the expression of Trpm5 as well as marker genes for Trpm5-expressing cells were absent in all tested tissues. Immunohistochemical analyses demonstrated that two types of microvillous cells exist in trachea, urethra, and thymus, Trpm5-positive and Trpm5-negative cells. In Skn-1a/Pou2f3-deficient mice, a considerable proportion of Trpm5-negative and villin-positive microvillous cells remained present in these tissues. Thus, we propose that Skn-1a/Pou2f3 is the master regulator for the generation of the Trpm5-expressing microvillous cells in multiple tissues.

Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain.

Taste cells in taste buds are epithelial sensory cells. Old taste bud cells die and are replaced by new ones generated from taste stem cells. Identifying and characterizing adult taste stem cells is therefore important to understand how peripheral taste tissues are maintained. SOX2 is expressed in oral epithelium including gustatory papillae and has been proposed to be a marker of adult taste stem/progenitor cells. Nevertheless, this hypothesis has never been directly tested. Here, by single-color genetic lineage tracing using Sox2-CreERT2 strain, we reveal that all types of taste bud cells distributed throughout the oral epithelium are derived from stem cells that express SOX2. Short-term tracing shows that SOX2-positive taste stem cells actively supply taste bud cells. At the base of epithelium outside taste buds are distributed proliferation marker- and SOX2-positive cells. Consistently, taste stem cells identified by Lgr5 expression in the circumvallate papillae also express SOX2. Together, taste stem cells distributed in oral epithelia express SOX2.

Bcl11b/Ctip2 is required for development of lingual papillae in mice.

Molecular mechanisms underlying the development and morphogenesis of oral epithelia, comprising the gustatory and nongustatory epithelium, remain unclear. Here, we show that Bcl11b, a zinc finger transcription factor, plays an important role in the development of lingual papillae, especially filiform papillae. In both gustatory and nongustatory epithelium, Bcl11b was expressed in keratin 14-positive epithelial basal cells, which differentiate into keratinocytes and/or taste cells. Loss of Bcl11b function resulted in abnormal morphology of the gustatory papillae: flattened fungiform papillae, shorter trench wall in the foliate and circumvallate papillae, and ectopic invagination in more than half of circumvallate papillae. However, Bcl11b loss caused no effect on differentiation of taste receptor cells. In nongustatory epithelium, the impact of Bcl11b deficiency was much more striking, resulting in a smooth surface on the tongue tip and hypoplastic filiform papillae in the dorsal lingual epithelium. Immunohistochemical analyses revealed that a keratinocyte differentiation marker, Tchh expression was severely decreased in the Bcl11b(-/-) filiform papillae. In addition, expression of Pax9, required for morphogenesis of filiform papillae and its downstream target genes, hard keratins, almost disappeared in the tongue tip and was decreased in the dorsal tongue of Bcl11b(-/-) mice. Gene expression analyses demonstrated a delayed onset of expression of epithelial differentiation complex genes, which disturbed barrier formation in the mutant tongue. These results indicate that Bcl11b regulates the differentiation of keratinocytes in the tongue and identify Bcl11b as an essential factor for the lingual papilla morphogenesis.

CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice.

CALHM1 is a cell surface calcium channel expressed in cerebral neurons. CALHM1 function in the brain remains unknown, but recent results showed that neuronal CALHM1 controls intracellular calcium signaling and cell excitability, two mechanisms required for synaptic function. Here, we describe the generation of Calhm1 knockout (Calhm1(-/-)) mice and investigate CALHM1 role in neuronal and cognitive functions. Structural analysis revealed that Calhm1(-/-) brains had normal regional and cellular architecture, and showed no evidence of neuronal or synaptic loss, indicating that CALHM1 deficiency does not affect brain development or brain integrity in adulthood. However, Calhm1(-/-) mice showed a severe impairment in memory flexibility, assessed in the Morris water maze, and a significant disruption of long-term potentiation without alteration of long-term depression, measured in ex vivo hippocampal slices. Importantly, in primary neurons and hippocampal slices, CALHM1 activation facilitated the phosphorylation of NMDA and AMPA receptors by protein kinase A. Furthermore, neuronal CALHM1 activation potentiated the effect of glutamate on the expression of c-Fos and C/EBPß, two immediate-early gene markers of neuronal activity. Thus, CALHM1 controls synaptic activity in cerebral neurons and is required for the flexible processing of memory in mice. These results shed light on CALHM1 physiology in the mammalian brain.

Expression of serotonin receptor genes in cranial ganglia.

Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

Normal Taste Acceptance and Preference of PANX1 Knockout Mice.

Taste compounds detected by G protein-coupled receptors on the apical surface of Type 2 taste cells initiate an intracellular molecular cascade culminating in the release of ATP. It has been suggested that this ATP release is accomplished by pannexin 1 (PANX1). However, we report here that PANX1 knockout mice do not differ from wild-type controls in response to representative taste solutions, measured using 5-s brief-access tests or 48-h two-bottle choice tests. This implies that PANX1 is unnecessary for taste detection and consequently that ATP release from Type 2 taste cells does not require PANX1.

Expression of the synaptic exocytosis-regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction.

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2-knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2-knockout mice were significantly lower than those in wild-type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons. A part of taste information is thought to be transmitted via synapses. However, the molecular mechanisms have not been fully elucidated. To identify molecules that participate in synaptic taste transduction, we investigated complexins (Cplxs) expression in taste bud cells. Strong expression of Cplx2 mRNA was detected in taste bud cells. Furthermore, taste responses to some sour stimuli in CPLX2- knockout mice were significantly lower than those in wild-type mice. These suggested that CPLX2 participated in synaptic taste transduction.

Skn-1a/Pou2f3 is required for the generation of Trpm5-expressing microvillous cells in the mouse main olfactory epithelium.

BACKGROUND: The main olfactory epithelium (MOE) in mammals is a specialized organ to detect odorous molecules in the external environment. The MOE consists of four types of cells: olfactory sensory neurons, supporting cells, basal cells, and microvillous cells. Among these, development and function of microvillous cells remain largely unknown. Recent studies have shown that a population of microvillous cells expresses the monovalent cation channel Trpm5 (transient receptor potential channel M5). To examine functional differentiation of Trpm5-expressing microvillous cells in the MOE, we investigated the expression and function of Skn-1a, a POU (Pit-Oct-Unc) transcription factor required for functional differentiation of Trpm5-expressing sweet, umami, and bitter taste bud cells in oropharyngeal epithelium and solitary chemosensory cells in nasal respiratory epithelium. RESULTS: Skn-1a is expressed in a subset of basal cells and apical non-neuronal cells in the MOE of embryonic and adult mice. Two-color in situ hybridization revealed that a small population of Skn-1a-expressing cells was co-labeled with Mash1/Ascl1 and that most Skn-1a-expressing cells coexpress Trpm5. To investigate whether Skn-1a has an irreplaceable role in the MOE, we analyzed Skn-1a-deficient mice. In the absence of Skn-1a, olfactory sensory neurons differentiate normally except for a limited defect in terminal differentiation in ectoturbinate 2 of some of MOEs examined. In contrast, the impact of Skn-1a deficiency on Trpm5-expressing microvillous cells is much more striking: Trpm5, villin, and choline acetyltransferase, cell markers previously shown to identify Trpm5-expressing microvillous cells, were no longer detectable in Skn-1a-deficient mice. In addition, quantitative analysis demonstrated that the density of superficial microvillous cells was significantly decreased in Skn-1a-deficient mice. CONCLUSION: Skn-1a is expressed in a minority of Mash1-positive olfactory progenitor cells and a majority of Trpm5-expressing microvillous cells in the main olfactory epithelium. Loss-of-function mutation of Skn-1a resulted in complete loss of Trpm5-expressing microvillous cells, whereas most of olfactory sensory neurons differentiated normally. Thus, Skn-1a is a critical regulator for the generation of Trpm5-expressing microvillous cells in the main olfactory epithelium in mice.