Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.     

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh⁺ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.     

Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds

The epithelium of mammalian tongue hosts most of the taste buds that transduce gustatory stimuli into neural signals. In the field of taste biology, taste bud cells have been described as arising from "local epithelium", in distinction from many other receptor organs that are derived from neurogenic ectoderm including neural crest (NC). In fact, contribution of NC to both epithelium and mesenchyme in the developing tongue is not fully understood. In the present study we used two independent, well-characterized mouse lines, Wnt1-Cre and P0-Cre that express Cre recombinase in a NC-specific manner, in combination with two Cre reporter mouse lines, R26R and ZEG, and demonstrate a contribution of NC-derived cells to both tongue mesenchyme and epithelium including taste papillae and taste buds. In tongue mesenchyme, distribution of NC-derived cells is in close association with taste papillae. In tongue epithelium, labeled cells are observed in an initial scattered distribution and progress to a clustered pattern between papillae, and within papillae and early taste buds. This provides evidence for a contribution of NC to lingual epithelium. Together with previous reports for the origin of taste bud cells from local epithelium in postnatal mouse, we propose that NC cells migrate into and reside in the epithelium of the tongue primordium at an early embryonic stage, acquire epithelial cell phenotypes, and undergo cell proliferation and differentiation that is involved in the development of taste papillae and taste buds. Our findings lead to a new concept about derivation of taste bud cells that include a NC origin.     

Estimation of the junctional resistance between electrically coupled receptor cells in Necturus taste buds

Junctional resistance between coupled receptor cells in Necturus taste buds was estimated by modeling the results from single patch pipette voltage clamp studies on lingual slices. The membrane capacitance and input resistance of coupled taste receptor cells were measured to monitor electrical coupling and the results compared with those calculated by a simple model of electrically coupled taste cells. Coupled receptor cells were modeled by two identical receptor cells connected via a junctional resistance. On average, the junctional resistance was approximately 200-300 M omega. This was consistent with the electrophysiological recordings. A junctional resistance of 200-300 M omega is close to the threshold for Lucifer yellow dye-coupling detection (approximately 500 M omega). Therefore, the true extent of coupling in taste buds might be somewhat greater than that predicted from Lucifer yellow dye coupling. Due to the high input resistance of single taste receptor cells (> 1 G omega), a junctional resistance of 200-300 M omega assures a substantial electrical communication between coupled taste cells, suggesting that the electrical activity of coupled cells might be synchronized.     

Expression of T1Rs and gustducin in palatal taste buds of mice

The palatal region of the oral cavity in rodents houses 100-300 taste buds and is particularly sensitive to sweet and umami compounds; yet, few studies have examined the expression patterns of transduction-related molecules in this taste field. We investigated the interrelationships between members of the T1R family and between each T1R and gustducin in palatal taste buds. Similar to lingual taste buds, T1R1 and T1R2 are generally expressed in separate palatal taste cells. In contrast to lingual taste buds, however, T1R2 and T1R3-positive palatal taste cells almost always coexpress gustducin, suggesting that sweet taste transduction in the palate is almost entirely dependent on gustducin. T1R1-positive palate taste cells coexpress gustducin about half the time, suggesting that other G proteins may contribute to the transduction of umami stimuli in this taste field.     

Isolation of single taste cells from lingual epithelium

A method is described for obtaining large numbers of isolatedtaste cells with identified polarity from lingual epithelium.The procedure involves incubating lingual epithelium in collagenase,staining the apical surface with fluorescein-conjugated wheatgerm agglutinin (FTTC-WGA), peeling non-gustatory surface epitheliumfrom the underlying taste buds and connective tissue, and dissociatingisolated taste buds with Ca²⁺-free saline. Isolated taste cellsretain their characteristic morphology for at least 30 min afterdissociation, and the apical specialization can be identifiedas a single patch of fluorescence usually located at the tipof an elongate process. Isolated taste cells are amenable tostudy with the patch-clamp technique, and whole-cell patch-clamprecordings show that isolated taste cells have membrane propertiessimilar to taste cells of intact lingual epithelium. Evidenceis presented that FITC-WGA staining does not alter the voltage-dependentionic currents of the taste cell membrane.     

Solitary chemoreceptor cells of Ciliata mustela (Gadidae, Teleostei) are tuned to mucoid stimuli

Summed potentials were recorded from the dorsal recurrent facialnerve innervating the solitary chemoreceptor cells on the anteriordorsal fin (ADF), from the ventral recurrent facial nerve innervatingboth taste buds and solitary chemoreceptor cells on the pectoral(PEC) and pelvic (PEL) fins, and from the anterior dorsal finmuscles in the rockling, Ciliata mustela. There is little overlapbetween the sumulus spectra of solitary chemoreceptor cellsand taste buds. The ADF solitary cells are particularly sensitiveto body mucus (skin water) of non-congeners like Gadus, Solea,Cottus, Mugil, Zoarces, Gaidropsarus, and Encheliopus, but insensitiveto amino acids and a variety of body fluids of fish, invertebrates,and extracts of potential stimuli like algae and sand. Pectoraland pelvic fins are particularly sensitive to amino acids, bodyfluids of fish and invertebrates, but less sensitive to skinmucus of fish, probably due to the abundance of taste buds.Active sampling by undulation of the anterior dorsal fin isessential for proper functioning; it induces disadaptation ofthe receptor elements. Solitary chemoreceptor cells provide,apparently, cues to discriminate between conspecifics and non-conspecifics.It is unlikely that they are involved in pheromone detection.     

Morphometry and cellular dynamics of denervated fungiform taste buds in the hamster

For most species and gustatory papillae denervation resultsin a virtual disappearance of taste buds. This is not the casefor hamster fungiform papillae, which contain taste buds thatsurvive denervation. To characterize these taste buds, in thisstudy, counts and measurements were made of all buds on theanterior 3 mm of the hamster tongue at 36 or 91 days after resectingthe chorda/lingual nerve. Taste bud numbers were, at both timeperiods, unaffected by denervation. However, bud dimensionswere affected with denervated buds 25–30% smaller thancontrol ones. Counts of taste bud cells indicated that decreasesin bud size may result from shrinkage, but not a loss of cells.Tritiated thymidine autoradiography was used to evaluate whetherdenervation influences the mitotic activity or the migratorypattern of bud cells. For every animal, the average number oflabelled cells per bud was slightly lower on the denervatedthan the control side of the tongue. However, when labelledcell positions were evaluated at 0.25, 3 and 6 days after thymidine,the distances from the sides of the bud increased at increasingtimes after injection for both the innervated and the denervatedbuds. Stem cells were located laterally or basally in the bud.Labelled cells that migrated into the centers of the buds werefew and seen only at 6 days post-injection time in both controland experimental buds. The moderate effects of denervation ontaste bud sizes and mitotic activities may indicate a generalizedatrophy. Remarkably intact were taste bud numbers and the migratorypatterns of cells, features of anterior tongue taste buds inthe hamster that are relatively invulnerable to resection ofthe chorda /lingual nerve.     

Chronic hypoxia alters calbindin D-28k immunoreactivity in lingual and laryngeal taste buds in the rat

The distribution and abundance of the calcium binding protein, calbindin D-28k (CB) immunoreactivity in the taste buds of the circumvallate papillae and larynx were compared between normoxic and chronically hypoxic rats (10% O2 for 8 weeks). In the normoxic rats, CB immunoreactivity was observed in some cells and fibers of the intragemmal region of the taste buds in the circumvallate papillae. In contrast, in the subgemmal region of the laryngeal taste buds, fibers but not cells were immunoreactive for CB. In chronically hypoxic rats, CB immunoreactive cells and fibers in the taste buds were decreased in the circumvallate papillae. In the laryngeal taste buds, the density of the subgemmal CB immunoreactive fibers in chronically hypoxic rats was greater than in normoxic rats. It is considered that function of the laryngeal taste buds is different from that of the lingual taste buds, so that laryngeal taste buds may be involved in chemosensation other than taste. The altered density of CB immunoreactive cells and fibers in the lingual and laryngeal taste buds is a predominant feature of hypoxic adaptation, and chronic hypoxic exposure might change the chemical sensitivity of the circumvallate papillae and larynx through the regulation of intracellular Ca2+.     

Laryngeal chemosensory clusters

The expression of molecules involved in the transductory cascade of the sense of taste (TRs, alpha-gustducin, PLCbeta2, IP3R3) has been described in lingual taste buds or in solitary chemoreceptor cells located in different organs. At the laryngeal inlet, immunocytochemical staining at the light and electron microscope levels revealed that alpha-gustducin and PLCbeta2 are mainly localized in chemosensory clusters (CCs), which are multicellular organizations differing from taste buds, being generally composed of two or three chemoreceptor cells. Compared with lingual taste buds, CCs are lower in height and smaller in diameter. In laryngeal CCs, immunocytochemistry using the two antibodies identified a similar cell type which appears rather unlike the alpha-gustducin-immunoreactive (IR) and PLCbeta2-IR cells visible in lingual taste buds. The laryngeal IR cells are shorter than the lingual ones, with poorly developed basal processes and their apical process is shorter and thicker. Some cells show a flask-like shape due to the presence of a large body and the absence of basal processes. CCs lack pores and their delimitation from the surrounding epithelium is poorly evident. The demonstration of the existence of CCs strengthens the hypothesis of a phylogenetic link between gustatory and solitary chemosensory cells.     

Occurrence of nitric oxide synthase in taste buds of the rat vallate papilla

Nitric oxide (NO) is generated by some types of cells as a membrane-permeant, short-acting paracrine signal. Its effects include activation of ion channels as well as formation of cGMP in the NO-generating and/or neighbouring cells. We have explored the possible involvement of NO in taste transduction by searching for NO synthase with histochemical and immunohistochemical methods. In taste buds of the rat vallate and foliate papilla, we found NADPH-diaphorase activity under stringent conditions that suppress the reactions of non-NO synthase enzymes. Furthermore, an antibody against neuronal NO synthase (NOS-I) labelled the basal and apical parts of taste cells, while an antibody against endothelial NO synthase (NOS-III) labelled taste buds and lingual epithelium more uniformly. The inducible macrophage enzyme NOS-II did not show immunoreactivity in taste buds. The results provide a first suggestion that NO may play a role in taste transduction. © 1998 Chapman & Hall     

Expression of adenosine A2b receptor in rat type II and III taste cells

We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.     

Ecto-calcium-dependent ATPase activity of mammalian taste bud cells.

Histochemistry was utilized to characterize Ca-ATPases associated with lingual taste buds in the golden hamster. Taste buds showed elevated staining for magnesium- or calcium-dependent ATPase (Ca-ATPase) relative to the surrounding epithelium. At low calcium concentrations (0.1-0.5 mM), intracellular staining predominated. Most of the studies were conducted at calcium concentrations of > or = 10 mM, in which most of the staining was localized to the external face of plasma membranes of taste bud cells (including receptor and basal cells) located in the core of fungiform taste buds, or the entire vallate or foliate taste buds. The peripheral fungiform taste bud cells stained much less intensely, but the peripheral cells adjacent to the core showed intermediate levels. GTP and ITP were just as effective substrates as ATP. Millimolar concentrations of magnesium were as effective as calcium. Inhibitors of intracellular ATPases, including quercetin, sodium azide, and 2,4-dinitrophenol, had no effect on the staining. Therefore, the Ca-ATPase staining of plasma membranes at mM concentrations of calcium is thought to correspond to one or more ecto-Ca-ATPase activities with unknown functions. Roles related to increased energy requirements or to the possible function of ATP as a neurotransmitter or -modulator are proposed.     

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 ± 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 ± 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP₃R₃)_, α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP₃R₃, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP₃R₃, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ～3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10–12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.     

Lgr5 Identifies Progenitor Cells Capable of Taste Bud Regeneration after Injury

Taste buds are composed of a variety of taste receptor cell types that develop from tongue epithelium and are regularly replenished under normal homeostatic conditions as well as after injury. The characteristics of cells that give rise to regenerating taste buds are poorly understood. Recent studies have suggested that Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) identifies taste bud stem cells that contribute to homeostatic regeneration in adult circumvallate and foliate taste papillae, which are located in the posterior region of the tongue. Taste papillae in the adult anterior region of the tongue do not express Lgr5. Here, we confirm and extend these studies by demonstrating that Lgr5 cells give rise to both anterior and posterior taste buds during development, and are capable of regenerating posterior taste buds after injury induced by glossopharyngeal nerve transection.     

Identification of electrophysiologically distinct cell subpopulations in Necturus taste buds

We used the patch clamp technique to record from taste cells in thin transverse slices of lingual epithelium from Necturus maculosus. In this preparation, the epithelial polarity and the cellular organization of the taste buds, as well as the interrelationships among cells within the taste bud, were preserved. Whole-cell recording, combined with cell identification using Lucifer yellow, allowed us to identify distinct subpopulations of taste cells based on their electrophysiological properties. Receptor cells could be divided in two groups: one group was characterized by the presence of voltage-gated Na+, K+, and Ca2+ currents; the other group was characterized by the presence of K+ currents only. Therefore, receptor cells in the first group would be expected to be capable of generating action potentials, whereas receptor cells in the second group would not. Basal taste cells could also be divided into two different groups. Some basal cells possessed voltage-gated Na+, K+, and Ca2+ conductances, whereas other basal cells only had K+ conductance. In addition to single taste cells, we were able to identify electrically coupled taste cells. We monitored cell- cell coupling by measuring membrane capacitance and by observing Lucifer yellow dye coupling. Electrical coupling in pairs of dye- coupled taste receptor cells was strong, as indicated by experiments with the uncoupling agent 1-octanol. Electrically coupled receptor cells possessed voltage-gated currents, including Na+ and K+ currents. The electrophysiological differentiation among taste cells presumably is related to functional diversifications, such as different chemosensitivities.