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

Abstract 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 Gnat3. Skn-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.

Sodium chloride (NaCl) evokes salty taste via amiloridesensitive (AS) and amiloride-insensitive (AI) mechanisms in taste cells. The AS mechanisms are specific for NaCl and are involved in the attractive responses to NaCl (Chandrashekar et al., 2010;Tordoff et al., 2014;Nomura et al., 2020). In contrast, the AI mechanisms respond to many salts and mediate aversive responses (Oka et al., 2013). The taste cells that mediate AS and AI salt-sensing mechanisms represent distinct populations (Yoshida et al., 2009;Chandrashekar et al., 2010;Roebber et al., 2019). The AS NaCl-sensing taste cells responsible for sodium preference (hereafter referred to as sodium-taste cells) reside in taste buds of fungiform papillae (FuP), but not in circumvallate papillae (CvP), and express the epithelial sodium channel ENaC as the sodium sensor and CALHM1/3 (Chandrashekar et al., 2010;Tordoff et al., 2014;Nomura et al., 2020). In the CvP all taste cells that co-express Calhm1, Calhm3, and Trpm5 depend on Skn-1a for their generation (Taruno et al., 2013;Ma et al., 2018). Furthermore, Skn-1a-deficient mice showed a partial loss of sodium taste responses of gustatory nerves (Larson et al., 2020). Nevertheless, it has been suggested that sodium-taste cells are distinct from Trpm5-expressing (Trpm5 1 ) cells and sour taste cells (Chandrashekar et al., 2010). Thus, whereas the specific cell type involved in attractive responses to NaCl share some features with sweet, umami, and bitter taste cells, it appears to be distinct from them. The identity of specific cell types responsible for the aversive AI salt-sensing mechanisms is also uncertain. It has been reported that taste cells that respond to all Cl --containing solutions, including HCl, are AI and lack ENaCa expression (Yoshida et al., 2009). Alternately, it has been suggested that AI salt-taste mechanisms reside in bitter taste cells and sour taste cells that express ENaCa (Chandrashekar et al., 2010;Oka et al., 2013). In contrast, it was recently proposed that AI NaCl taste resides in sweet and bitter taste cells but not in sour taste cells (Roebber et al., 2019).
Taste cells are epithelial sensory cells that are maintained in taste buds by continuous turnover (Beidler and Smallman, 1965). They are derived from local epithelial stem cells that express Sox2 and Krt5 commonly (Stone et al., 1995;Ohmoto et al., 2017Ohmoto et al., , 2020. At the precursor cell stage of taste cell differentiation, a POU homeodomain transcription factor Skn-1a (also known as Pou2f3) specifies the fate of a cell as a sweet, umami, and bitter cell lineage . Interestingly, Skn-1a is also required for differentiation of putative sensory cells in other tissues including microvillus cells in the main olfactory epithelium, solitary chemosensory cells in the respiratory epithelium, and tuft cells in the intestine, all of which commonly express Trpm5 similar to sweet, umami, and bitter taste cells Yamaguchi et al., 2014;Gerbe et al., 2016). Skn-1a therefore appears to be a master regulator of Trpm5-expressing sensory cells (Yamashita et al., 2017).
In the present study, we found that taste cells responsible for AS avidity to NaCl share some molecular features with sweet, umami, and bitter taste cells but are distinct from them. AS responses of the chorda tympani nerve to NaCl are abolished in both Skn-1a-deficient and Calhm3deficient mice that also lack perception of sweet, umami, and bitter tastes. The loss of AS neural responses in Skn-1a-deficient mice was correlated with the disappearance of taste cells defined by a Calhm3 1 Trpm5molecular identity. Thus, Skn-1a governs the generation of sodiumtaste cells in addition to sweet, umami, and bitter taste cells.

Animals
C57BL/6J (stock #000664) mice were purchased from The Jackson Laboratory. Heterozygous Skn-1a 1/mice in a 129/B6 mixed background  were crossed with C57BL/6J mice over 10 generations, and resultant male and female Skn-1a 1/mice with a C57BL/6J congenic background were crossed to obtain Skn-1a -/mice with a C57BL/6J congenic background, which were maintained by crossing homozygous mice. Calhm3 -/mice have a C57BL/6J background as described previously . Both sexes were used in all animal experiments, which were conducted according to a protocol approved by the Institutional Animal Care and Use Committee.

Tissue preparation
For fresh-frozen tissue samples, mice were deeply anesthetized with urethane, and the oral epithelia were dissected and embedded in O.C.T. compound (Sakura Finetech). For tissues fixed with 4% paraformaldehyde (PFA), mice were deeply anesthetized with urethane and transcardially perfused with PBS followed by 4% PFA in PBS. Dissected oral epithelia were postfixed, cryoprotected, and frozen as previously described (Ohmoto et al., 2008). Cryosections (8-mm thickness) were prepared using a Leica CM1900 cryostat (Leica Microsystems), mounted on tissue adhesive-coated glass slides (Fisher Scientific), and preserved at -80°C until use.

In situ hybridization
In situ hybridization using fresh-frozen sections was conducted as previously described (Ohmoto et al., 2008;Taruno et al., 2013). Digoxigenin-labeled and fluorescein-labeled antisense RNAs were synthesized and used as probes after fragmentation to ;150 bases under alkaline conditions. The probe regions are shown in Table 1. Sections were fixed with 4% PFA, treated with diethylpyrocarbonate, prehybridized with salmon testis DNA, and hybridized with the riboprobes for 40 h. After hybridization, the sections were washed in 0.2Â SSC. Prehybridization, hybridization, and washing were performed at 58°C except when using the riboprobes for Calhm1, Itpr3, and ENaCa, which were performed at 65°C. After washing, chromogenic and/or fluorescence signals were developed as follows: For single-label in situ hybridization, hybridized probes were detected using alkaline phosphatase-conjugated antidigoxigenin antibodies (Roche Diagnostics, 11093274910, RRID:AB_514497), and chromogenic signals were developed using 4-nitro blue tetrazolium chloride/5-bromo-4chloro-3-indolyl phosphate as a substrate for 3 h (to Plcb2 and Itpr3) or two overnights (to Calhm1). Stained images were obtained using a Nikon Eclipse 80i microscope (Nikon Instruments) equipped with a DXM1200C digital camera (Nikon).
For double-label fluorescence in situ hybridization, the fluorescence signals of the riboprobes were developed using an alkaline phosphatase-conjugated anti-digoxigenin antibody followed by the HNPP Fluorescent Detection set (Roche Diagnostics) and a biotin-conjugated anti-fluorescein antibody (Vector Laboratories, BA-0601, RRID:AB_2336069) followed by an avidinbiotin complex (Vector Laboratories), a TSA Biotin Tyramide Reagent (PerkinElmer), and an Alexa Fluor 488-conjugated streptavidin (Thermo Fisher Scientific). Fluorescence single-plane confocal images were acquired with a Leica TCS SP2 confocal microscope (Leica Microsystems). Optical confocal images were processed with Photoshop (Adobe Systems). For quantification of cells with fluorescence signals, taste buds on every 8, 12, or 16 sections of the FuP and soft palate from three mice were analyzed. For the frequencies of expression of Skn-1a and Entpd21Pkd2l1 in taste bud cells, sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI). The ratios of Skn-1a-expressing cells or Pkd2l1-expressing or Entpd2-expressing cells to the taste bud cells as judged from DAPI and DIC images were calculated using every 8, 12, or 16 sections of the FuP and soft palate of wild-type (WT; n = 3) and Skn-1a -/-(n = 3) mice.
For double-labeling of Calhm1 or ENaCa with other genes, fluorescence and chromogenic signals were developed as previously described (Taruno et al., 2013). Prehybridization, hybridization, and washing were performed at 65°C for any probes, and the fluorescence signals were first developed using a biotin-conjugated antifluorescein antibody (Vector Laboratories) followed by an avidin-biotin complex (Vector Laboratories), a TSA Biotin Tyramide Reagent (PerkinElmer), and an Alexa Fluor 488conjugated streptavidin (Thermo Fisher Scientific). After capturing the fluorescence signals with a Leica TCS SP2 confocal microscope (Leica Microsystems), the chromogenic signals of Calhm1 or ENaCa were detected using an alkaline phosphatase-conjugated anti-digoxigenin antibody and 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate. Stained images were obtained as described above. Fluorescence and stained images were processed with Photoshop (Adobe Systems). For quantification of cells with fluorescence and stained signals, taste buds on every 8, 12, or 16 sections of the FuP and soft palate from three mice were analyzed.

Whole chorda tympani nerve recordings
We investigated the electrophysiological response of the chorda tympani nerve in mice of the Skn-1a knock-out (Skn-1a -/-) and Calhm3 knock-out (Calhm3 -/-) strains, using C57BL/6J mice as WT controls (see above). The experimenters were blinded to the genotype of the mice during testing. The mice were anesthetized with an intraperitoneal injection of a mixture of 4.28 mg/ml ketamine, 0.86 mg/ml xylazine, and 0.14 mg/ml acepromazine in saline (5 ml/g body weight). Anesthesia was maintained with additional injections. Each mouse was fixed with a head holder after its trachea was cannulated, and the chorda tympani nerve was dissected free from its junction with the lingual nerve near the tympanic bulla; then the nerve was cut and the central part was placed on a platinum wire recording electrode. An indifferent electrode touched the walls of the wound. Taste stimuli were delivered to the tongue with a computer-controlled open flow system under constant flow and temperature (25°C) conditions. Each stimulation lasted for 30 s with a 60-s rinse between stimulations. Care was taken to ensure that the flow was directed over the FuP. The nerve impulses were fed into a custom-made amplifier, monitored over a loudspeaker and with an oscilloscope, and recorded (PowerLab/sp4; AD Instruments). The integrated response during stimulation was calculated by subtracting the area of nerve activity preceding the stimulation from that during stimulation. Thus, the data reflect the level of activity during the stimulation period. The responses to all compounds were expressed relative to the response to 0.1 M NH 4 Cl, which is derived from solely sour taste cells (Oka et al., 2013), for each mouse as previously described Ma et al., 2018). The averages for each animal and group were calculated for the statistical analyses.

Statistical analyses
Data are shown as the mean 6 SEM. A Welch's t test (for histochemical analyses) or repeated-measures twoway ANOVA (for gustatory nerve recordings) was used to determine the effects of genotype using Prism 6 software (GraphPad Software). When a significant interaction was detected between a genotype and a taste solution concentration, Tukey-Kramer multiple comparison tests were conducted to identify significant differences between pairs of mean values.

Results
Previous studies demonstrated that Skn-1a is necessary for the generation of sweet, umami, and bitter taste cells, and that Calhm1, Calhm3, and Trpm5 mRNAs are co-expressed only in Skn-1a-dependent taste cells in the CvP Ma et al., 2018). Intriguingly, Calhm1 has been implicated in salty taste (Tordoff et al., 2014), whereas it has been suggested that AS NaCl responses arise from cells that lack Trpm5 expression (Chandrashekar et al., 2010). Recent efforts to identify AS sodium taste cells in the FuP have produced conflicting results. For example, it was suggested that Calhm3 expressed in Skn-1a-dependent taste cells  is required for AS NaCl responses (Nomura et al., 2020), whereas it has also been suggested that Skn-1a-deficient mice that do not express either Calhm1 or Calhm3 (Taruno et al., 2013;Ma et al., 2018) still exhibited AS NaCl responses as much as a half of those of WT mice (Larson et al., 2020). To better understand the identity and molecular features of AS NaCl-sensing taste (i.e., as sodium-taste) cells, we conducted in situ hybridization analyses in FuP taste buds where sodium-taste cells reside and soft palate and gustatory nerve recordings of the chorda tympani nerve innervating FuP taste buds.

Taste cell gene expression in taste buds of FuP
First, we asked whether CALHM and Trpm5 channel genes are always co-expressed in the same cells of FuP by double-fluorescence in situ hybridization using Calhm3 and Trpm5 as probes. In taste buds of FuP where the chorda tympani nerve innervates, Trpm5 signals were observed only in cells showing Calhm3 signals. However, ;20% of Calhm3 1 cells did not generate Trpm5 signals (Fig. 1A). This result is in contrast to their complete co-expression in taste bud cells of the CvP . Accordingly, these data are consistent with previous findings that sodium taste is mediated by a taste cell subset distinct from sweet, umami, and bitter taste cells but nevertheless requires CALHM channel genes for neurotransmission (Chandrashekar et al., 2010;Tordoff et al., 2014;Nomura et al., 2020).
Then, we examined whether other genes encoding sweet, umami, and bitter taste signaling molecules are expressed in both the Trpm5 1 and Trpm5taste cells. Of note, it has been suggested that sodium-taste cells do not require Ca 21 signaling evoked by phosphatidylinositol (PI) turnover, unlike Type II cells (Nomura et al., 2020). Surprisingly, in taste buds of the FuP, Plcb2 and Itpr3 were expressed in Trpm5cells and always co-expressed with Calhm3, whereas Gnat3 was expressed only in Trpm5 1 taste cells (Fig. 1). These results strongly suggest that Calhm3-expressing taste cells can be classified into two subsets: those that are all positive (i.e., for the expression of Plcb2, Itpr3, Calhm3, Gnat3, and Trpm5) and those that are Plcb2 1 Itpr3 1 Calhm3 1 Gnat3 -Trpm5 -(hereafter referred to as Calhm3 1 Trpm5cells in this study). In agreement, anti-Itpr3 and anti-Gnat3 antibodies identified Itpr3 1 Gnat3 1 and Itpr3 1 Gnat3cells in taste buds of the FuP, whereas in taste buds of the CvP, where cells expressing Gnat3 and/or Tas1r3 are identical to Trpm5 1 cells , Itpr3 1 cells are always positive for Gnat3 and/or T1R3 (Extended Data Fig. 1-1A). The ratios of Itpr3 1 Gnat3 1 and Itpr3 1 Gnat3cells to total Itpr3 1 cells (Itpr3 1 Gnat3 1 , 77.8%; Itpr3 1 Gnat3cells, 22.2%) are comparable to those of Calhm3 1 Trpm5 1 (78.3%) and Calhm3 1 Trpm5 -(21.7%) cells to Calhm3 1 cells ( Fig. 1; Extended Data Fig. 1-1A). Consistent with mRNA expression profiles, Itpr3-immunoreactive signals were observed in Skn-1a 1 cells but not in cells positive for sour taste cell marker Ddc (i.e., DDC 1 cells; Extended Data Fig. 1-1B). Interestingly, cells exhibiting similar molecular features were also detected in taste buds of soft palate that are innervated by the greater superficial petrosal nerves (Extended Data Figs. 1-1, 1-2). Accordingly, the Calhm3 1 Trpm5cells found in taste buds in the FuP and soft palate but not in the CvP are predicted to be involved in a taste that is specifically transmitted by the chorda tympani and greater superficial petrosal nerves. Because neurophysiological studies in rats suggest the existence of AS NaCl-sensing taste cells in the greater superficial petrosal nerve-innervated taste buds in soft palate (Harada et al., 1997;Sollars and Hill, 1998), the Calhm3 1 Trpm5cells are likely to be sodium-taste cells in the FuP and soft palate taste buds.
In taste buds of Skn-1a-deficient mice, Calhm3 expression was not detected in the FuP, palate, or CvP . Similarly, the expression of Plcb2, Itpr3, and Calhm1 was not detected in taste buds in any gustatory areas ( Fig. 2; Extended Data Fig. 2-1; Matsumoto et al., 2011;Taruno et al., 2013). Notably, Plcb2, Itpr3, and Calhm3 were always co-expressed with Skn-1a, and the frequencies of Skn-1a signal are comparable to those of Plcb2, Itpr3, and Calhm3 signals ( Fig. 3; Extended Data Fig. 3-1). Consistent with the relationship of expression of Trpm5 and Plcb2, Itpr3, and Calhm3, Trpm5 signals were detected in 77.0 6 3.2% and 87.2 6 3.2% of Skn-1a 1 cells in the FuP and soft palate, respectively ( Fig. 3; Research Article: New Research Extended Data Fig. 3-1). These results indicate that Skn-1a 1 cells in the FuP and soft palate can be classified into the same two differentiated cell subsets as Calhm3-expressing taste cells: all positive and Calhm3 1 Trpm5subsets. The somewhat greater number of cells expressing Skn-1a than Plcb2, Itpr3, and Calhm3 can be explained by Skn-1a expression in putative precursor cells in taste buds in addition to the differentiated taste cells such as sweet, umami, and bitter taste cells.

Skn-1a is required for gustatory nerve responses to NaCl
Because the existence of Calhm3 1 cells depends on Skn-1a, the phenotype by Calhm3 deficiency in gustatory nerve responses is expected to be recapitulated in the Skn-1a-deficient mice. However, recent findings by two groups presented conflicting results, although they both showed the requirement, at least in part, of Calhm3 and Skn-1a in the AS NaCl responses of chorda tympani nerves (Larson et al., 2020;Nomura et al., 2020). We interrogated whether Skn-1a and Calhm3 are equally required for NaCl taste by recording chorda tympani nerve responses. Genetic deletion of Skn-1a reduced responses to 300 and 1000 mM NaCl (p , 0.0001). AS responses were eliminated at all concentrations over 100 mM (p , 0.0001 for 100, 300, and 1000 mM), whereas AI responses were diminished only at 1000 mM (p , 0.0001; Fig. 5A,B; Table 2). The decrease of AI responses in the Skn-1a-deficient mice can most likely be accounted for by the absence of bitter taste cells in these mice that contribute to the taste of high salt concentrations Oka et al., 2013). These results demonstrate that both AS and AI NaCl tastes are largely mediated by Skn-1a-dependent taste bud cells. Consistent with this, AS responses of the chorda tympani nerve to NaCl were eliminated in Calhm3-deficient mice (Fig. 5C), and Calhm3 is involved in AI chorda tympani nerve responses in bitter taste cells . Together, our results indicate that the Calhm3 1 Trpm5cells mediate sodium taste, whereas sour taste cells together with bitter taste cells mediate AI salt taste, as previously demonstrated (Oka et al., 2013). These results support the finding by Nomura et al. (2020) and are partially consistent with the results by Larson et al. (2020), with regard to AS NaCl responses. Furthermore, they question the claim that AI NaCl taste is mediated by Type II cells including sweet taste cells (Roebber et al., 2019).

Calhm3 1 Trpm5cells express ENaCa
Sodium taste deficiency by the conditional deletion of ENaCa in Calhm3 1 taste cells (Nomura et al., 2020) and the lack of Trpm5 expression in the putative ENaCa 1 cells identified by a reporter expression in transgenic mice (Chandrashekar et al., 2010) suggest that ENaCa is expressed in Calhm3 1 Trpm5cells. However, it was not confirmed that the reporter recapitulates the ENaCa expression in the FuP and/or soft palate, since reginal expression may be regulated by a distinct enhancer that may not be included in the transgene, as shown for Shh expression (Sagai et al., 2009). Thus, we tested the possibility that ENaCa is expressed in Calhm3 1 Trpm5cells.
Because ENaCa signals in taste buds in the FuP were too weak to detect by double-fluorescence in situ hybridization, we employed long-term signal development using chromogenic substrate to detect ENaCa expression in combination with fluorescence signal detection for other taste cell genes. This method was previously shown to be as efficacious as double-fluorescence in situ hybridization in an analysis of the relationship of weak Calhm1 expression with taste cell marker gene expression (Taruno et al., 2013). Employing this method, we found that Calhm1 was always co-expressed with Calhm3 (Extended Data Fig.   Figure 4. Disappearance of Skn-1a-dependent taste bud cells by Skn-1a deficiency. Populations of Skn-1a 1 and Skn-1acells (i.e., positive to a mixed probe to Entpd2 and Pkd2l1) in taste buds in FuP were quantified by double-fluorescence in situ hybridization analyses. Taste bud profiles are outlined by broken white line. Asterisk indicates the ratio expressing mutant Skn-1a mRNA. The decrease of the Skn-1a 1 cell population was statistically evaluated by Welch's t test: p = 0.0056. Scale bar: 25 mm.
6-1), consistent with the same phenotypes of the knockouts in gustatory nerve recordings ( Fig. 5; Taruno et al., 2013;Tordoff et al., 2014;Ma et al., 2018;Nomura et al., 2020). Similarly, we observed partial overlap of ENaCa with Skn-1a and Calhm3 localizations (Fig. 6A,B; Extended Data Fig. 6-2A,B; Table 3). Although fluorescence signals of a cell may possibly overlap with chromogenic signals of another cell located above or below of fluorescence signal 1 cell, we never observed any overlap of ENaCa with Trpm5 in the FuP or soft palate ( Fig. 6C; Extended Data Figure 5. Skn-1a deficiency extinguishes AS chorda tympani nerve responses to NaCl. A, Representative charts of chorda tympani nerve responses of WT and Skn-1a -/mice to NaCl in the presence (green traces) or absence of 100 mM amiloride. Shaded rectangles depict the AS (blue) and AI (green) components in response to NaCl. The bars under the traces show the duration (30 s) of the taste stimulus. B, C, Whole chorda tympani nerve responses of Skn-1a -/-(n = 3) and WT (n = 4) mice (B) and Calhm3 -/-(n = 6) and WT (n = 5) mice (C) to NaCl. AS salt responses (AS component; B, middle) were measured by subtracting the AI response (AI component; B, right) from the whole salt response (B, left). Significance was assessed by a repeated-measures two-way ANOVA and the Tukey-Kramer test: *p , 0.05. Data are expressed as the mean 6 SEM; where error bars are not visible, they are smaller than the symbol depicting the mean. For details, see Table 2.    Table 3), strongly suggesting that ENaCa and Trpm5 are not co-expressed in any taste cells. In addition, ENaCa expression was detected in both sour and nonsour taste cells (Fig. 6D), consistent with previous findings in transgenic mice (Chandrashekar et al., 2010) but incompatible with a previous single cell-PCR analysis (Yoshida et al., 2009). The ENaCa expression in nonsour taste cells was absent in Skn-1a-deficient mice (in FuP, p = 0.0422; in soft palate, p = 0.0009; Fig. 6; Extended Data Fig. 6-2; Table 3). These results indicate that Skn-1a-dependent Calhm3 1 Trpm5cells express ENaCa and serve as sodium-taste cells.

Discussion
The results of the present study demonstrate that sodium-taste cells and Type II sweet, umami, and bitter taste cells have shared molecular expression features, and a similar reliance on Skn-1a for their generation. These findings advance our understanding of the molecular mechanisms of taste cell differentiation, provide new insights into classification of taste cell lineage, and reveal a cellular mechanism that elicits salt taste.

Cellular mechanism of taste by NaCl
Salts are dissolved in saliva, and either cations or anions could activate different taste cells independently. In case of NaCl, Na 1 activates ENaCa-mediated AS mechanisms in a specific population of taste cells characterized by their ENaCa 1 Pkd2l1 -Trpm5expression profile (Chandrashekar et al., 2010). Although it has been suggested that these AS salt taste cells do not possess voltage-gated Na 1 currents (Vandenbeuch et al., 2008), several studies have demonstrated that sodium-taste cells fire Na 1 action potentials (Bigiani and Cuoghi, 2007;Yoshida et al., 2009;Nomura et al., 2020) and that they are responsible for the avidity to NaCl (Chandrashekar et al., 2010;Nomura et al., 2020). They are most likely sensory cells.
Various chloride salts are sensed by yet-to-be identified AI mechanisms that may reside in sour and bitter taste cells (Oka et al., 2013) or sweet and bitter taste cells (Roebber et al., 2019). If AI salt taste resides in only sweet and bitter taste cells, chorda tympani nerve responses to NaCl in mice deficient in Calhm1, Calhm3, and Skn-1a should be absent over all concentrations. However, chorda tympani nerve responses to NaCl in these mice are comparable to those of WT mice except at very high concentrations (Fig. 5;Tordoff et al., 2014;Ma et al., 2018). Of note, Skn-1a-deficient mice have only sour taste cells as sensory cells, and retain AI salt taste, demonstrating that the AI mechanism resides at least in part in sour taste cells. Consistent with this, Car4, which is expressed specifically in sour taste cells in taste buds, is involved in sensing a variety of chloride salts, although the mechanisms are unclear (Oka et al., 2013). Bitter taste cells have also been implicated in aversive salt taste (Oka et al., 2013). Mice deficient in any intracellular bitter signal transduction pathway molecule including Gnat3, PLCb 2, Trpm5, CALHM1, and CALHM3 exhibit deficits in neural or behavioral responses to high salt concentrations (Dotson et al., 2005;Glendinning et al., 2005;Oka et al., 2013;Taruno et al., 2013;Ma et al., 2018). It is possible that T2R bitter receptors associated with Gnat3 respond to chloride salts and trigger the bitter receptor downstream intracellular signal transduction pathway. Of note, human TAS2R7 serves as metallic cation receptor (Behrens et al., 2019;Wang et al., 2019). It is interesting to speculate that specific receptors for Clexist in sour and bitter taste cells that respond to various chloride salts.

Intracellular signal transduction of sodium taste
Our results suggest that taste bud cells in the FuP with the ENaCa 1 Pkd2l1 -Trpm5expression profile function as taste receptor cells responsible for sensing Na 1 (Chandrashekar et al., 2010). Similar to other tastes, NaCl taste involves ATP release in neurotransmission (Finger et al., 2005), and deficiencies of Calhm1 and Calhm3 encoding functional components of the ATPrelease channel eliminate AS salt responses (Fig. 5;Tordoff et al., 2014;Nomura et al., 2020). Sodium-taste cells also express Plcb2 and Itpr3 (Fig. 1), both of which are involved in the increases of intracellular [Ca 21 ] in sweet, umami, and bitter Type II taste cells (Zhang et al., 2003). However, unlike their involvement in Type II cells, neither PLCb 2 nor IP 3 R3 is involved in the perception of NaCl (Zhang et al., 2003;Hisatsune et al., 2007;Tordoff and Ellis, 2013), consistent with recent findings that sodium-taste cells fire action potentials without increases of intracellular [Ca 21 ] (Nomura et al., 2020). The roles of PLCb 2 and IP 3 R3 in sodium-taste cells remain to be determined. Although sodium-taste cells lack expression of Gnat3 and Trpm5, their expression of Plcb2 and Itpr3 in sodium-taste cells may suggest that they express yet-to-be-identified G protein-coupled receptor (GPCR), G proteins, and cation channels (possibly Ca 21 -dependent monovalent cation channels like Trpm5). It is therefore of some interest to understand the transcriptome of sodium-taste cells.

Sodium-taste cells and the morphologic classification of taste bud cells
Skn-1a regulates the differentiation of sweet, umami, and bitter taste cells and extra-oral taste cell-like chemosensory cells such as brush cells in the airways, urethra, and auditory tube. Like taste cells, those chemosensory cells express taste GPCRs (i.e., T1Rs and/or T2Rs), Gnat3, PLCb 2, and Trpm5 (Finger et al., 2003;Ohmoto et al., 2008;Krasteva et al., 2011Krasteva et al., , 2012Matsumoto et al., 2011;Deckmann et al., 2014;Panneck et al., 2014;Yamashita et al., 2017). On the other hand, microvillous cells in the main olfactory epithelium have little similarity to taste and taste cell-like chemosensory cells with regard to molecular feature: they express only Trpm5 but not mRNA of taste GPCRs, Gnat3, or PLCb 2 (Yamaguchi et al., 2014), although immunoreactivities to Gnat3 and PLCb 2 were somehow detected (Genovese and Tizzano, 2018). Although neither intestinal tuft cells nor olfactory microvillous cells express taste GPCRs, the former express another GPCR, Sucnr1, and are involved in sensing chemical succinate, and the latter likely detect odor chemicals and modulate olfactory sensory neuron activity (Lemons et al., 2017;Lei et al., 2018;Nadjsombati et al., 2018;Schneider et al., 2018). The only commonality among these Skn-1a-dependent chemosensory cells, including taste cells, is their expression of Trpm5 (Yamashita et al., 2017). Therefore, sodium-taste cells are the first, very unique population of Skn-1a-dependent chemosensory cells that lack of Trpm5 expression. Other unidentified Skn-1a-dependent chemosensory cells devoid of Trpm5 expression may exist. Genetic tools to mark Skn-1a 1 cells will help identify such novel chemosensory cells. Taste bud cells have been classified into four types based on their ultra-microscopic morphologic features. This morphologic classification correlates with molecular features: Type I cells appear to be non-sensory supporting cells that lack voltage-gated Na 1 currents (Medler et al., 2003) and express Entpd2 that hydrolyzes extracellular ATP released from other taste bud cells as a neurotransmitter (Finger et al., 2005;Bartel et al., 2006;Vandenbeuch et al., 2013); Type II cells are taste cells expressing GPCRs and signaling molecules including PLCb 2, IP 3 R3, and Trpm5; Type III cells are Pkd2l1 1 cells; and Type IV cells are undifferentiated basal cells (Yang et al., 2000;Clapp et al., 2004;Chaudhari and Roper, 2010). Sodium-taste cells are sensory cells, but they are distinct from these cell types. Their molecular features, including the expression of Plcb2, Itpr3, Calhm1, Calhm3, and Skn-1a, requirement of Skn-1a for their generation, and apparent Calhm1/3 requirement for neurotransmitter release are reminiscent of Type II cells. They are however distinguished from Type II cells by their lack of Trpm5 and presence of ENaCa expression. Accordingly, sodium-taste cells can be regarded as a Type II cell subset, similar to the distinctions among Type II cells by their GPCR expression profiles. Ultramicroscopic morphologic studies in combination with immunohistochemistry in FuP taste buds where sodium-taste cells reside will be necessary to determine whether sodium-taste cells constitute a morphologically-distinct cell type in taste buds.