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Taste buds are localized in fungiform (FF), foliate (FL), and circumvallate (CV) papillae on the tongue, and taste buds also occur on the soft palate (SP). Mature elongate cells within taste buds are constantly renewed from stem cells and classified into three cell types, Types I, II, and III. These cell types are generally assumed to reside in respective taste buds in a particular ratio corresponding to taste regions. A variety of cell-type markers were used to analyze taste bud cells. NCAM is the first established marker for Type III cells and is still often used. However, NCAM was examined mainly in the CV, but not sufficiently in other regions. Furthermore, our previous data suggested that NCAM may be transiently expressed in the immature stage of Type II cells. To precisely assess NCAM expression as a Type III cell marker, we first examined Type II and III cell-type markers, IP3R3 and CA4, respectively, and then compared NCAM with them using whole-mount immunohistochemistry. IP3R3 and CA4 were segregated from each other, supporting the reliability of these markers. The ratio between Type II and III cells varied widely among taste buds in the respective regions (Pearson's r = 0.442 [CV], 0.279 [SP], and - 0.011 [FF]), indicating that Type II and III cells are contained rather independently in respective taste buds. NCAM immunohistochemistry showed that a subset of taste bud cells were NCAM(+)CA4(-). While NCAM(+)CA4(-) cells were IP3R3(-) in the CV, the majority of them were IP3R3(+) in the SP and FF., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sox2 is proposed to regulate the differentiation of bipotential progenitor cells into taste bud cells. However, detailed expression of Sox2 remains unclear. In this report, Sox2 expression during taste bud development in the fungiform (FF), circumvallate (CV) and soft palate (SP) areas is examined together with Prox1. First, we immunohistochemically checked Prox1 expression in adults and found that almost all taste bud cells are Prox1-positive. During FF development, intense Sox2 expression was restricted to taste bud primordia expressing Prox1 at E12.5. However, at E14.5, Sox2 was intensely expressed outside the developing taste buds resolving to perigemmal Sox2 expression in adults. In the SP, at E14.5, taste bud primordia emerged as Prox1-expressing cell clusters. However, intense Sox2 expression was not restricted to taste bud primordia but was detected widely in the epithelium. During development, Sox2 expression outside developing taste buds was generally down-regulated but was retained in the perigemmal region similarly to that in the FF. In the CV, the initial stage of taste bud development remained unclear because of the lack of taste bud primordia comparable to that in the FF and SP. Here, we show that Prox1-expressing cells appear in the apical epithelium at E12.5, in the inner trench wall at E17.5 and in the outer trench wall at E18.5. Sox2 was again not restricted to developing taste bud cells expressing Prox1 during CV development. The expression patterns support that Sox2 does not serve as a cell fate selector between taste bud cells and surrounding keratinocytes but rather may contribute to them both.

Background: Taste buds contain ∼60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I, glial cells; II, bitter/sweet/umami receptor cells; and III, sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turn over rapidly suggesting that Shh+ cells are post-mitotic precursors of some or all taste cell types., Results: To fate map Shh-expressing cells, mice carrying ShhCreER(T2) and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III)., Conclusions: Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds., (Copyright © 2014 Wiley Periodicals, Inc.)

Tastants reach the tip of taste bud cells through taste pores which are openings in the epithelium. We found Sprr2a is selectively expressed in the upper layer of the epithelium surrounding taste buds in the circumvallate papilla (CV) where the epithelium is organized into taste pores. Sprr2a is a member of a small proline-rich protein family, which is suggested to be involved in the restitution/migration phase of epithelial wound healing. The expression of Sprr2a was restricted to the upper layer and largely segregated with Ptch1 expression that is restricted to the basal side of the epithelium around the taste buds. Denervation resulted in the gradual loss of Sprr2a-expressing cells over 10 days similarly to that of taste bud cells which is in contrast to the rapid loss of Ptch1 expression. We also found that denervation caused an increase of Keratin (Krt)13 expression around taste buds that corresponded with the disappearance of Sprr2a and Ptch1 expression. Taste buds were surrounded by Krt13-negative cells in the CV in control mice. However, at 6 days post-denervation, taste buds were tightly surrounded by Krt13-positive cells. During taste bud development, taste bud cells emerged together with Krt13-negtive cells, and Sprr2a expression was increased along with the progress of taste bud development. These results demonstrate that regional gene expression surrounding taste buds is associated with taste bud formation and controlled by the innervating taste nerve., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

To clarify the regional differences in the expression and functional significance of Gα-gustducin in soft palate (SP) and fungiform (FF) taste buds, we examined the coexpression of Gα-gustducin with taste receptors and the impact of Gα-gustducin knockout (gKO) on neural responses to several sweet and bitter compounds. Sweet responses from both the greater superficial petrosal (GSP) and chorda tympani (CT) nerves in gKO mice were markedly depleted, reflecting overlapping expression of Gα-gustducin and Tas1r2. However, although Gα-gustducin was expressed in 87% and 88% of Tas2rs cells in the SP and FF, respectively, there were no statistically significant differences in the CT responses to quinine-HCl (QHCl) and denatonium (Den) between gKO and wild-type (WT) mice. In contrast, GSP responses to these compounds were markedly reduced in gKO mice with an apparent elevation of thresholds (>10-fold). These results suggest that 1) Gα-gustducin plays a critical role in sweet transduction in both the SP and the FF, 2) other Gα subunits coexpressed with Gα-gustducin in the FF are sufficient for responses to QHCl and Den, and 3) robust GSP responses to QHCl and Den occur in the SP by a Gα-gustducin-dependent mechanism, which is absent in the FF.

Taste buds contain three types of taste cells. Each type can respond to taste stimulation, and type II and III taste cells are electrically excitable. However, there are differences between the properties of type II and III taste cells. In this study, we found that Fxyd6, an Na,K-ATPase regulator gene, is expressed in type II taste cells in the taste buds of mice. Double-labeled in situ hybridization analysis showed that Fxyd6 was coexpressed with transient receptor potential cation channel, subfamily M, member 5 (Trpm5), a critical component of the sweet, bitter, and umami taste signal transduction pathways and that it was specifically expressed in type II taste cells. We also found that taste cells frequently coexpressed Fxyd6 and Na,K-ATPase β1. These results indicate the presence of an inherent mechanism that regulated transmembrane Na(+) dynamics in type II taste cells.

While the taste periphery has been studied for over a century, we are only beginning to understand how this important sensory system is maintained throughout adult life. With the advent of molecular genetics in rodent models, and the upswing in translational approaches that impact human patients, we expect the field will make significant advances in the near future.

Inositol 1,4,5-triphosphate-mediated calcium (IP3-Ca2+) signal cascade is an essential process in sweet, bitter, and umami taste signal transduction. Although the main components of this cascade have been identified, the candidate regulators of them in taste tissues are still unclear. In an effort to identify genes involved in taste signal transduction, we found that a gene encoding lymphoid-restricted membrane protein (Lrmp/Jaw1) was expressed in mouse taste tissues. Here we report that Lrmp/Jaw1 is specifically expressed in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. In addition to this specific expression patterns, we found that Lrmp/Jaw1 is associated with type III IP3 receptor (IP3R3) via its coiled-coil domain in the COS7 heterologous expression system. These results raise the possibility that Lrmp/Jaw1 interacts with IP3R3 in taste cells and suggest an important role for Lrmp/Jaw1 in the IP3-Ca2+ signal cascade in sweet, bitter, and umami taste signal transduction.

Gustducin, a G alpha subunit expressed in taste cells, is known as a key molecule for sweet, umami and bitter taste signal transduction. However, previous studies demonstrated that the contribution of gustducin to the sweet/umami responses in the posterior region of the tongue is less than that in the anterior region, implying the existence of another G alpha subunit mediating sweet/umami taste signal transduction. Here, we propose G alpha14, a member of G alpha q family, as the candidate mediator. G alpha14 was found in our subtracted full-length cDNA library derived from mouse circumvallate papillae (CV) and expressed in a subset of taste cells in CV and foliate papillae, but not in fungiform papillae and soft palate. G alpha14 was co-expressed with T1r3, a sweet/umami taste receptor, but not with gustducin in CV. These results suggest the important roles of G alpha14 in sweet/umami taste signal transduction in the posterior region of the tongue.

Although embryonic expression of Shh in the fungiform papilla placodes has a critical role in fungiform papilla patterning, it remains unclear whether its appearance indicates the differentiation of the basal cells of taste buds. To examine the embryonic development of the basal cells, the expression of Shh, Prox1, and Mash1 was determined in the anterior tongue and soft palate in mouse embryos by in situ hybridization. In the anterior tongue, Prox1 was coexpressed with Shh from the beginning of Shh expression in the fungiform papilla placodes at E12.5. Shh was expressed in the soft palate in a band-like pattern in the anteriormost region and in a punctate pattern in the posterior region at E14.5. The number (21.4 +/- 4.3, at E14.5) of locations where Shh was observed (i.e., spots) rapidly increased and reached a peak level (54.8 +/- 4.0 at E15.5). Also in the soft palate, Prox1 was coexpressed with Shh from the beginning of Shh expression. These results suggest that basal cell differentiation occurs synchronously with the patterning of Shh spots both in the anterior tongue and in the soft palate. In contrast, Mash1 expression lagged behind the expression of Shh and Prox1 and began after the number of Shh spots had reached its peak level in the soft palate. Furthermore, immunohistochemistry of PGP9.5 and Shh revealed that epithelial innervation slightly preceded Mash1 expression both in the tongue and in the soft palate. This is the first report describing the time courses of the embryonic expression of basal cell markers of taste buds., ((c) 2008 Wiley-Liss, Inc.)

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.

Mammalian taste buds are maintained through continuous cell renewal so that taste bud cells are constantly generated from progenitor cells throughout life. Taste bud cells are composed of basal cells and elongated cells. Elongated cells are derived from basal cells and contain taste receptor cells (TRC). Morphologically, elongated cells consist of three distinct types of cells: Types I, II and III. In contrast to the remarkable progress in understanding of the molecular basis for taste reception, the mechanisms of taste bud maintenance have remained a major area of inquiry. In this article, we review the expression of regulatory genes in taste buds and their involvement in taste bud cell differentiation. Three major topics include: 1) the Sonic hedgehog (Shh)-expressing cell in the basal cell in taste buds as a transient precursor of elongated cells and as a signal center for the proliferation of progenitor cells; 2) the Mash1-expressing cell as an immature cell state of both Type II and Type III cells and as a mature cell state of Type III cell; and 3) the nerve dependency of gene expression in taste buds. Problems in the application of NCAM for the type III cell marker are also discussed.

Neural cell adhesion molecule (NCAM) is a type III cell marker in the taste buds. In order to clarify the cell type of Mash1-expressing cells in taste buds, expression of NCAM was examined in Mash1-expressing taste cells of adult mice in comparison with gustducin- and T1r3-expressing cells, using a combination of NCAM immunohistochemistry and in situ hybridization. About 98% of Mash1-expressing cells were NCAM immunopositive (IP), suggesting that Mash1-expressing cells should be categorized as type III cells. Unexpectedly, small subsets of gustducin- and T1r3-expressing cells were also found to be NCAM-IP, contradicting previous immunohistochemical studies in rats, in which gustducin-IP cells were observed specifically in type II cells, which do not have NCAM immunoreactivity. Examinations of developing taste buds showed temporal changes in the ratio of NCAM-IP cells in gustducin- and T1r3-expressing cells; the ratio of NCAM-IP cells in these gene-expressing cells were approximately 90% at 0.5 days after birth and decreased markedly during development. In contrast, the majority of Mash1-expressing cells showed constant NCAM immunoreactivity throughout development. In addition, BrdU-labeling experiments showed that the differentiation of Mash1-expressing cells precedes those of gustducin- and T1r3-expressing cells in taste buds of adult mice. These results suggest that T1r3- and gustducin-expressing cells are NCAM-IP at the beginning of cell differentiation, and that NCAM immunoreactivity in gustducin- and T1r3-expressing cells might remain from the previous developmental stage expressing Mash1.

The nerve-dependency of gene expression in mouse taste bud was examined through an analysis of changes in gene expression in and around the taste buds in circumvallate papillae after surgery of cranial nerve IXth (glossopharyngeal nerve). The number of cells expressing T1r3, gustducin, Mash1 and Nkx2.2 gradually decreased after denervation. However, the expression intensity of these genes was barely influenced by denervation, and strong expression was observed at 6 days after denervation. In contrast, the basal cell-specific Sonic hedgehog (Shh) expression in the taste buds was decreased markedly at 6 h after denervation. In the regeneration process of taste buds, Shh expression was observed during a very early phase before taste bud formation. These results indicate the autonomous transcriptional control of genes in differentiated taste cells and the strong nerve-dependency of Shh expression in basal cells. Furthermore, in order to reveal the mitotic activity of Shh-expressing cells in taste buds, the BrdU-labeling experiments were performed using a combination of BrdU-immunohistochemistry and in situ hybridization. BrdU-signal was very rarely observed in Shh-expressing cells immediately after BrdU injection, and the signals were noted mainly in Ptc-expressing cells. BrdU signals rapidly increased in Shh-expressing cells in following 12 h and began to decrease after 2 days post-injection. These results suggest that most Shh-expressing cells are not mitotically active, but that Shh-expressing cells may be in the early transient developmental state of taste cells in taste buds.

Leptin is a hormone that regulates body weight homeostasis mainly via the hypothalamic functional leptin receptor Ob-Rb. Recently, we proposed that the taste organ is a new peripheral target for leptin. Leptin selectively inhibits mouse taste cell responses to sweet substances and thereby may act as a sweet taste modulator. The present study further investigated leptin action on the taste system by examining expression of Ob-Rb in taste cells and behavioral responses to sweet substances in leptin-deficient ob/ob, and Ob-Rb-deficient db/db mice and their normal litter mates. RT-PCR analysis showed that Ob-Rb was expressed in taste cells in all strains tested. The db/db mice, however, had a RT-PCR product containing an abnormal db insertion that leads to an impaired shorter intracellular domain. In situ hybridization analysis showed that the hybridization signals for normal Ob-Rb mRNA were detected in taste cells in lean and ob/ob mice but not in db/db mice. Two different behavioral tests, one using sweet-bitter mixtures as taste stimuli and the other a conditioned taste aversion paradigm, demonstrated that responses to sucrose and saccharin were significantly decreased after ip injection of leptin in ob/ob and normal littermates, but not in db/db mice. These results suggest that leptin suppresses behavioral responses to sweet substances through its action on Ob-Rb in taste cells. Such taste modulation by leptin may be involved in regulation for food intake.

In mammals, taste buds are maintained by continuous turnover of cells, even in adulthood. Cell proliferation and differentiation continue to produce taste cells, which express various genes related to taste reception. We found the co-expression of Sonic hedgehog (Shh) with Prox1 and that of Nkx2.2 with Mash1 in adult mouse taste buds. Whereas Prox1was expressed strongly in cells in the basal region of mouse taste buds where Shh was co-expressed, it was expressed weakly in almost all taste bud cells lacking Shh expression. At 0.5 day after birth, when taste cells have not yet differentiated, the expressions of Shh and Prox1 completely overlapped in the epithelium of circumvallate papillae. Nkx2.2 was observed in cells expressing Mash1, but not in cells expressing genes related to taste reception, such as gustducin and T1R3. Almost all fusiform cells expressing Mash1 co-expressed Nkx2.2, while the majority of round cells expressing Mash1 in the basal region of taste buds lacked Nkx2.2 expression.

Leptin is a hormone that regulates food intake, energy expenditure and body weight. Our previous studies have demonstrated that the taste organ is a new peripheral target for leptin in mice. Leptin selectively inhibits the responses of taste nerves and receptor cells to sweet substances without affecting responses to sour, salty, and bitter substances. Still, there is no convincing evidence for the existence of leptin receptors (Ob-Rs) in taste receptor cells, especially the functional isoform Ob-Rb. We investigated the expression of 5 different Ob-R isoforms (a-e) and 6 STAT (signal transducers and activators of transcription) members in mouse taste cells. STATs are considered to be involved in the leptin signaling via Ob-Rb. Semiquantitative RT-PCR analysis showed that Ob-Rb was expressed in the taste buds of the fungiform and circumvallate papillae, but not so clearly in the surrounding epithelial tissue. The expression pattern among the three different tissues was similar to that of the taste cell specific G-protein, alpha-gustducin. The other Ob-R isoforms were widely detected in either the taste papillae or the epithelial tissue. Among 6 STAT members, STAT3 showed the highest relative abundance of mRNA in the taste buds. Consistently, in situ hybridization analysis showed that while Ob-Rb and STAT3 signals were detected in some taste bud cells, the signals were not clearly observed in the epithelial tissue cells. In conclusion, the present study provides evidence of the existence of the leptin receptor, Ob-Rb, and STAT3 in the mouse taste bud cells. This finding further confirms the involvement of leptin in the control of taste sensitivities to sweet substances in mice.

Taste bud cells have a limited lifespan and are continuously replaced just like other epithelial cells. Although there is some evidence that taste buds may arise from the local epithelium, taste receptor cells have neuronal properties. This implies that there must be a critical stage at which the epithelial precursor cells for taste receptor cells start to exhibit neural properties during the differentiation of the taste receptor cells. The expression of the neural-specific transcription factors Mash-1 and Prox-1 in the nervous system is transient and precedes neuronal differentiation. Therefore, we examined the expression of Mash-1 and Prox-1 in the epithelium of circumvallate papillae of the tongue in order to clarify the localization of the precursor cells with neural properties and observed that both expressions are restricted to the taste buds. Two-colour in situ hybridization showed that the signals for Mash-1 did not overlap those for taste receptor cell-specific genes such as gustducin and T1R2. In the process of development and regeneration of the taste buds, the expression of Mash-1 preceded that of gustducin and T1R2. These observations suggest that Mash-1 could be a candidate for a marker of immature taste receptor cells, including the cells that express gustducin and/or T1R2 at a later stage.

In mammals, taste receptor cells are organized into taste buds on tongue. Taste buds are trophically maintained by taste neurons and under continuous renewal, even in adults. We found that the receptor for Sonic hedgehog (Shh), Patched1 (Ptc), was expressed around taste buds where cells were proliferating, and that Shh was expressed within basal cells of taste buds. Denervation caused the loss of Shh and Ptc expression before the degeneration of taste buds.

A cDNA clone encoding a novel member of the putative taste receptor T1R family, designated T1R3, was isolated from circumvallate papillae of the mouse tongue using degenerate primers. Reverse transcription-polymerase chain reaction analysis showed predominant expression of the receptor in circumvallate papillae. In situ hybridization analysis revealed that T1R3 was expressed in a subset of taste receptor cells in taste buds and that the topographic distribution of T1R3 in various taste papillae was different from those of the other T1R members. Genetic mapping of T1R3 with a mouse/hamster radiation hybrid panel located the gene on the distal end of mouse chromosome 4 correlated with the Sac locus affecting sweet sensitivity of mice. Our results indicate that T1R3 may serve as the receptor for sweet perception in mice., (Copyright 2001 Academic Press.)

Leptin acts as a potent inhibitory factor against obesity by regulating energy expenditure, food intake, and adiposity. The obese diabetic db/db mouse, which has defects in leptin receptor, displays enhanced neural responses and elevated behavioral preference to sweet stimuli. Here, we show the effects of leptin on the peripheral taste system. An administration of leptin into lean mice suppressed responses of peripheral taste nerves (chorda tympani and glossopharyngeal) to sweet substances (sucrose and saccharin) without affecting responses to sour, salty, and bitter substances. Whole-cell patch-clamp recordings of activities of taste receptor cells isolated from circumvallate papillae (innervated by the glossopharyngeal nerve) demonstrated that leptin activated outward K(+) currents, which resulted in hyperpolarization of taste cells. The db/db mouse with impaired leptin receptors showed no such leptin suppression. Taste tissue (circumvallate papilla) of lean mice expressed leptin-receptor mRNA and some of the taste cells exhibited immunoreactivities to antibodies of the leptin receptor. Taken together, these observations suggest that the taste organ is a peripheral target for leptin, and that leptin may be a sweet-sensing modulator (suppressor) that may take part in regulation of food intake. Defects in this leptin suppression system in db/db mice may lead to their enhanced peripheral neural responses and enhanced behavioral preferences for sweet substances.