Type II/III cell composition and NCAM expression in taste buds.

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.

During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages.

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.

Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells.

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.

Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans.

Male embryonic mice with mutations in the X-linked aristaless-related homeobox gene (Arx) developed with small brains due to suppressed proliferation and regional deficiencies in the forebrain. These mice also showed aberrant migration and differentiation of interneurons containing gamma-aminobutyric acid (GABAergic interneurons) in the ganglionic eminence and neocortex as well as abnormal testicular differentiation. These characteristics recapitulate some of the clinical features of X-linked lissencephaly with abnormal genitalia (XLAG) in humans. We found multiple loss-of-function mutations in ARX in individuals affected with XLAG and in some female relatives, and conclude that mutation of ARX causes XLAG. The present report is, to our knowledge, the first to use phenotypic analysis of a knockout mouse to identify a gene associated with an X-linked human brain malformation.

Gα-gustducin is extensively coexpressed with sweet and bitter taste receptors in both the soft palate and fungiform papillae but has a different functional significance.

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.

FXYD6, a Na,K-ATPase regulator, is expressed in type II taste cells.

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 ß1. These results indicate the presence of an inherent mechanism that regulated transmembrane Na(+) dynamics in type II taste cells.

Lrmp/Jaw1 is expressed in sweet, bitter, and umami receptor-expressing cells.

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.

G alpha14 is a candidate mediator of sweet/umami signal transduction in the posterior region of the mouse tongue.

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.

Leptin modulates behavioral responses to sweet substances by influencing peripheral taste structures.

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.

Co-expression pattern of Shh with Prox1 and that of Nkx2.2 with Mash1 in mouse taste bud.

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.

Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays).

Polymerase Chain Reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. In this paper, recombinant DNAs introduced into the seven lines of GM maize, such as Event 176, Bt11, T25, MON810, GA21, DLL25, and MON802, are sequenced. On the basis of the obtained sequence, 14 primer pairs for the detection of the segments, such as promoter, terminator regions, and construct genes, were designed. To confirm the specificities of the designed primer pairs, PCR was performed on genomic DNAs extracted from GM and non-GM maize, GM and non-GM soy, and other cereal crops. Because the presence of the corresponding DNA segments was specifically detected in GM crops by the designed primer pairs, it was concluded that this method is useful for fast and easy screening of GM crops including unauthorized ones.

Decline of umami preference in aged rats.

The effects of aging on the umami sensation were compared between the preference and neural responses from the greater superficial petrosal nerve (GSP innervating the soft palate) and the chorda tympani nerve (CT innervating the fungiform papillae) in the Sprague Dawley rat. A two-bottle preference test revealed that younger rats (5-12 weeks) preferred significantly 0.001 M 5'-inosine monophosphate (IMP), 0.01 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP+0.01 M MSG than deionized water. However, aged rats (21-22 months) showed no significant preference to these umami solutions compared to deionized water. Among the other four basic taste stimuli, there were no significant differences in preference between young and aged rats. Regardless of the age of the rat, neural responses from the GSP and CT produced robust integrated responses to all three umami solutions used in the two-bottle tests. These results indicate that the lack of preference to umami in aged rats is a central nervous system phenomenon and suggests that the loss of preference to umami taste in aged rats is caused by homeostatic changes in the brain incurred by aging.

Diverse contributions of Tas1r2/Tas2rs within the rat and mouse soft palate to sweet and bitter neural responses.

Neural responses to sweet and bitter stimuli in the rat and mouse are compared to the expression of the molecular taste receptors, Tas1r2/Tas2rs. Integrated taste responses from the greater superficial petrosal nerve (GSP) innervating the soft palate (SP) and the chorda tympani (CT) nerve innervating the fungiform papillae (FF) were recorded in C57BL mice and SD rats. The sum of the phasic and tonic response magnitudes (SRM) was calculated by summating all relative mean responses to a concentration series of QHCl (10(-6)-10(-2)M) or Suc (10(-4)-1.0M). Molecular expression was analyzed by double-colored in situ hybridization for Gα-gustducin with Tas1r2 or Tas2rs in the SP and FF. The vast majority of cells expressing Tas1r2 or Tas2rs were included in Gα-gustducin-expressing cells in the SP of both species. Unexpectedly, a comparison between species revealed that the SRM from the GSP is not positively correlated with receptor expression in the SP. In the rat SP, the percentage of Tas2rs with Gα-gustducin (Tas2rs/gust, 65%) was twice larger than that for Tas1r2/gust (33%), while the SRM to Suc in the rat GSP was 1.5 times (tonic and phasic) larger than that to QHCl. In the mouse SP, the percentage of Tas2rs/gust (46%) was less than that in the rat and similar to that of Tas1r2/gust (40%). However, the SRM to QHCl in the mouse GSP was 2.4 (phasic) and 4.7 (tonic) times larger than to Suc. On the other hand, threshold to Suc in the rat GSP was 10(-3)M, one log unit lower than in mouse, and the threshold to QHCl in the mouse GSP was 10(-6)M, one log unit lower than in rat. These results suggest that the robust GSP response to Suc in rat and to QHCl in mouse likely do not depend upon a large number of taste cells expressing the taste receptors Tas1r2 for Suc or Tas2rs for QHCl, but upon a higher density of Tas1r2/Tas2rs within the respective taste cells of the two species.

The glossopharyngeal nerve controls epithelial expression of Sprr2a and Krt13 around taste buds in the circumvallate papilla.

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.

Expression of the basal cell markers of taste buds in the anterior tongue and soft palate of the mouse embryo.

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.

Expression of gustducin overlaps with that of type III IP3 receptor in taste buds of the rat soft palate.

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.