Anterior and Posterior Tongue Regions and Taste Papillae: Distinct Roles and Regulatory Mechanisms with an Emphasis on Hedgehog Signaling and Antagonism.

Sensory receptors across the entire tongue are engaged during eating. However, the tongue has distinctive regions with taste (fungiform and circumvallate) and non-taste (filiform) organs that are composed of specialized epithelia, connective tissues, and innervation. The tissue regions and papillae are adapted in form and function for taste and somatosensation associated with eating. It follows that homeostasis and regeneration of distinctive papillae and taste buds with particular functional roles require tailored molecular pathways. Nonetheless, in the chemosensory field, generalizations are often made between mechanisms that regulate anterior tongue fungiform and posterior circumvallate taste papillae, without a clear distinction that highlights the singular taste cell types and receptors in the papillae. We compare and contrast signaling regulation in the tongue and emphasize the Hedgehog pathway and antagonists as prime examples of signaling differences in anterior and posterior taste and non-taste papillae. Only with more attention to the roles and regulatory signals for different taste cells in distinct tongue regions can optimal treatments for taste dysfunctions be designed. In summary, if tissues are studied from one tongue region only, with associated specialized gustatory and non-gustatory organs, an incomplete and potentially misleading picture will emerge of how lingual sensory systems are involved in eating and altered in disease.

Probing the multimodal fungiform papilla: complex peripheral nerve endings of chorda tympani taste and mechanosensitive fibers before and after Hedgehog pathway inhibition.

The fungiform papilla (FP) is a gustatory and somatosensory structure incorporating chorda tympani (CT) nerve fibers that innervate taste buds (TB) and also contain somatosensory endings for touch and temperature. Hedgehog (HH) pathway inhibition eliminates TB, but CT innervation remains in the FP. Importantly, after HH inhibition, CT neurophysiological responses to taste stimuli are eliminated, but tactile responses remain. To examine CT fibers that respond to tactile stimuli in the absence of TB, we used Phox2b-Cre; Rosa26LSL-TdTomato reporter mice to selectively label CT fibers with TdTomato. Normally CT fibers project in a compact bundle directly into TB, but after HH pathway inhibition, CT fibers reorganize and expand just under the FP epithelium where TB were. This widened expanse of CT fibers coexpresses Synapsin-1, ß-tubulin, S100, and neurofilaments. Further, GAP43 expression in these fibers suggests they are actively remodeling. Interestingly, CT fibers have complex terminals within the apical FP epithelium and in perigemmal locations in the FP apex. These extragemmal fibers remain after HH pathway inhibition. To identify tactile end organs in FP, we used a K20 antibody to label Merkel cells. In control mice, K20 was expressed in TB cells and at the base of epithelial ridges outside of FP. After HH pathway inhibition, K20 + cells remained in epithelial ridges but were eliminated in the apical FP without TB. These data suggest that the complex, extragemmal nerve endings within and disbursed under the apical FP are the mechanosensitive nerve endings of the CT that remain after HH pathway inhibition.

Recovery of taste organs and sensory function after severe loss from Hedgehog/Smoothened inhibition with cancer drug sonidegib.

Striking taste disturbances are reported in cancer patients treated with Hedgehog (HH)-pathway inhibitor drugs, including sonidegib (LDE225), which block the HH pathway effector Smoothened (SMO). We tested the potential for molecular, cellular, and functional recovery in mice from the severe disruption of taste-organ biology and taste sensation that follows HH/SMO signaling inhibition. Sonidegib treatment led to rapid loss of taste buds (TB) in both fungiform and circumvallate papillae, including disruption of TB progenitor-cell proliferation and differentiation. Effects were selective, sparing nontaste papillae. To confirm that taste-organ effects of sonidegib treatment result from HH/SMO signaling inhibition, we studied mice with conditional global or epithelium-specific Smo deletions and observed similar effects. During sonidegib treatment, chorda tympani nerve responses to lingual chemical stimulation were maintained at 10 d but were eliminated after 16 d, associated with nearly complete TB loss. Notably, responses to tactile or cold stimulus modalities were retained. Further, innervation, which was maintained in the papilla core throughout treatment, was not sufficient to sustain TB during HH/SMO inhibition. Importantly, treatment cessation led to rapid and complete restoration of taste responses within 14 d associated with morphologic recovery in about 55% of TB. However, although taste nerve responses were sustained, TB were not restored in all fungiform papillae even with prolonged recovery for several months. This study establishes a physiologic, selective requirement for HH/SMO signaling in taste homeostasis that includes potential for sensory restoration and can explain the temporal recovery after taste dysgeusia in patients treated with HH/SMO inhibitors.

Hedgehog Signaling Regulates Taste Organs and Oral Sensation: Distinctive Roles in the Epithelium, Stroma, and Innervation.

The Hedgehog (Hh) pathway has regulatory roles in maintaining and restoring lingual taste organs, the papillae and taste buds, and taste sensation. Taste buds and taste nerve responses are eliminated if Hh signaling is genetically suppressed or pharmacologically inhibited, but regeneration can occur if signaling is reactivated within the lingual epithelium. Whereas Hh pathway disruption alters taste sensation, tactile and cold responses remain intact, indicating that Hh signaling is modality-specific in regulation of tongue sensation. However, although Hh regulation is essential in taste, the basic biology of pathway controls is not fully understood. With recent demonstrations that sonic hedgehog (Shh) is within both taste buds and the innervating ganglion neurons/nerve fibers, it is compelling to consider Hh signaling throughout the tongue and taste organ cell and tissue compartments. Distinctive signaling centers and niches are reviewed in taste papilla epithelium, taste buds, basal lamina, fibroblasts and lamellipodia, lingual nerves, and sensory ganglia. Several new roles for the innervation in lingual Hh signaling are proposed. Hh signaling within the lingual epithelium and an intact innervation each is necessary, but only together are sufficient to sustain and restore taste buds. Importantly, patients who use Hh pathway inhibiting drugs confront an altered chemosensory world with loss of taste buds and taste responses, intact lingual touch and cold sensation, and taste recovery after drug discontinuation.

Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans.

The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre-operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non-invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non-invasively.

Comparison of fungiform taste-bud distribution among age groups using confocal laser scanning microscopy in vivo in combination with gustatory function.

The aim of this study was to compare the distribution of taste buds in fungiform papillae (FP) and gustatory function between young and elderly age groups. Confocal laser scanning microscopy was used because it allows many FP to be observed non-invasively in a short period of time. The age of participants (n = 211) varied from 20 to 83 yr. The tip and midlateral region of the tongue were observed. Taste buds in an average of 10 FP in each area were counted. A total of 2,350 FP at the tongue tip and 2,592 FP in the midlateral region could be observed. The average number of taste buds was similar among all age groups both at the tongue tip and in the midlateral region. The taste function, measured by electrogustometry, among participants 20-29 yr of age was significantly lower than that in the other age groups; however, there was no difference among any other age groups in taste function. These results indicate that the peripheral gustatory system is well maintained anatomically and functionally in elderly people.

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance.

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.

Comparison of nestin-expressing multipotent stem cells in the tongue fungiform papilla and vibrissa hair follicle.

We have previously reported that hair follicles contain multipotent stem cells, which express nestin and participate in follicle growth at anagen as well as in the extension of the follicle sensory nerve. The nestin-driven green fluorescent protein (ND-GFP) transgenic mouse labels all nestin-expressing cells with GFP. The hair follicle nestin-GFP cells can differentiate into neurons, Schwann cells, and other cell types. In this study, we describe nestin-expressing multipotent stem cells in the fungiform papilla in the tongue. The nestin-expressing multipotent stem cells in the fungiform papilla are located around a peripheral sensory nerve immediately below the taste bud and co-express the neural crest cell marker p75(NTR) . The fungiform papilla cells formed spheres in suspension culture in DMEM-F12 medium supplemented with basic fibroblast growth factor (bFGF). The spheres consisted of nestin-expressing cells that co-expressed the neural crest marker p75(NTR) and which developed expression of the stem cell marker CD34. P75(NTR), CD34 and nestin co-expression suggested that nestin-expressing cells comprising the fungiform papilla spheres were in a relatively undifferentiated state. The nestin-expressing cells of these spheres acquired the following markers: ß III tubulin typical of nerve cells; GFAP typical of glial cells; K15 typical of keratinocytes; and smooth-muscle antigen (SMA), after transfer to RPMI 1640 medium with 10% fetal bovine serum (FBS), suggesting they differentiated into multiple cell types. The results of the current study indicate nestin-expressing fungiform papilla cells and the nestin-expressing hair follicle stem cells have common features of cell morphology and ability to differentiate into multiple cell types, suggesting their remarkable similarity.

Morphogenesis of fungiform papillae in developing miniature pigs.

Objective: Fungiform papillae contain taste buds and play a critical role in mastication and the gustatory system. In this study, we report a series of sequential observations of organogenesis of fungiform papillae in miniature pigs, as well as changes in the expression of BMP2, BMP4, Wnt5a, Sox2, and Notch1 signaling pathway components. Design: In this study, we investigated the spatiotemporal expression patterns of BMP, Wnt, Sox2 and Notch in the fungiform papillae of miniature pigs at the bud stage (E40), cap stage (E50) and bell stage (E60). Pregnant miniature pigs were obtained, and the samples were processed for histological staining. Immunohistochemistry and real-time PCR were used to detect the mRNA and protein expression levels of BMP2, BMP4, Wnt5a, Sox2, and Notch1. Results: At E40, fungiform papillae were present on the anterior two-thirds of the tongue in a specific array and pattern. The fungiform papillae were enlarged and basically developed at E50 and were largest at the earlier stage (E60). Most of the BMP2 was concentrated in the epithelial layer and the connective tissue core of the fungal papilloma and gradually accumulated from E40-E60. BMP-4 was weakly expressed in the fungiform papillae epithelia, but BMP-4-positive cells were also observed in the developing tongue muscle at E50 and E60. Wnt5a-positive cells were observed in the fungiform papillae epithelia and developing tongue muscle at all three time points. Sox2-positive cells were observed only in fungiform papillae epithelial cells, and Notch1-positive cells could not be detected. Conclusions: This study provides primary data regarding the morphogenesis and expression of developmental signals in the fungiform papillae of miniature pigs, establishing a foundation for further research in both this model and humans.

Anatomy and development of the human taste system.

The sense of taste relies on well-defined neuroanatomical structures, namely, the taste buds and afferent nerve fibers. Taste buds are clusters of 50-100 neuroepithelial cells located throughout the oral cavity, including the epiglottis and larynx. They are responsible for the initial transduction process that ultimately results in the perception of bitter, sour, salty, sweet, and umami (savory) sensations. They service as the initial sentinel for a sensory system critical in evolution for distinguishing "dangerous" food components, often perceived as bitter or unpleasant, from "useful" ones, often perceived as pleasant, salty, or sweet. This chapter describes the anatomy and development of the human peripheral taste system and provides historical context for what is presently known about this element of this important sensory system. Its main focus is on the fundamental question of how tastants are perceived-a question that has been of philosophical and scientific interest for more than two millennia. Descriptions of lingual and extralingual taste buds, their blood and nerve supplies, and the associated salivary glands are provided, including details of their microstructure and transduction mechanisms.

Association of tongue brushing with the number of fungiform taste buds and taste perception: A preliminary study using confocal laser scanning microscopy in combination with a filter-paper disc method.

OBJECTIVE: The aim of this study was to investigate the association of tongue brushing with the number of fungiform taste buds and taste perception using a confocal laser scanning microscopy in combination with a filter-paper disc method (FPDM). METHODS: Twenty-four subjects with or without a habit of tongue brushing (11 males and 13 females, 20-46 years old) participated in this study. Nine of the 24 subjects had no habit of tongue brushing (Group 1, n=9). Fifteen subjects had a habit of tongue brushing, and the brushing regions of the tongue were as follows: central region (Group 2, n=7), or entire region (Group 3, n=8) of the tongue dorsum. Using confocal laser scanning microscopy, the average number of taste buds per fungiform papilla (FP) was counted. Taste perception was evaluated using an FPDM. These observations were performed in the midlateral region of the tongue since the distribution of fungiform papillae is large in the midlateral region compared to that in the central region. RESULTS: The subjects in Group 3 showed a significantly decreased number of fungiform taste buds compared to Group 1 and Group 2. Group 3 also showed significantly higher FPDM scores than the other two groups. CONCLUSIONS: Excessive tongue brushing of the entire tongue dorsum, including the midlateral region, may have an association with the decreased number of FP and taste buds and decreased taste sensation. To avoid these conditions, instituting proper tongue brushing methods, such as limiting it to the central region of the tongue and using a light touch, is suggested and is important for the subjects who are eager to participate in tongue brushing.

Gpr177-mediated Wnt Signaling is Required for Fungiform Placode Initiation.

Fungiform papillae are formed as patterned rows on the surface of the anterior tongue at early organogenesis and contain one taste bud in each papilla to form one of the important sensory organs. Despite the essential role of Wnt/ß-catenin signaling in controlling the development of fungiform taste papillae, the universal function of Wnt ligands in the initiation of the fungiform placode has not been completely elucidated. Here, by Shh (Cre) -mediated oral epithelial deletion of Wntless (Gpr177), a regulator essential for intracellular Wnt trafficking, we demonstrate that an overall function of Wnts is required for initiation of the fungiform placode. Multiple Wnts are expressed in the tongue epithelium at E11.5 before initiation of the fungiform placodes. Epithelial Gpr177 loss-of-function, associated with reduction of canonical Wnt signaling in lingual epithelium as exhibited by a loss of TopGal activity and Axin2 expression, results in the failure of fungiform placode initiation, as assessed by diminished expression of several taste placode molecular markers. Moreover, LiCl treatment of Gpr177 epithelial-deficient tongue explants at E11.5, but not at E12.5, restores tongue placode formation, demonstrating that Wnt ligands in the tongue surface prior to but not after fungiform placode initiation are responsible for fungiform papilla initiation. Epithelium-specific expression of an active ß-catenin in the Gpr177-deficient tongue leads to fungiform papillae generation, suggesting that an intra-epithelial response to Wnts is required for placode initiation. Together, these results suggest that Gpr177 controls epithelial initiation of the fungiform placode through signaling via epithelial Wnt ligands.

Regional differences in suprathreshold intensity for bitter and umami stimuli.

The sense of taste is often referred to as a 'nutritional gatekeeper', thought to have evolved to indicate energy sources and prevent ingestion of potential toxins. Fungiform papillae are structures on the anterior tongue in which taste buds are situated. They are concentrated at the tongue's tip and they can provide a useful estimate of overall taste bud density for taste research. Some reports suggest taste perception may differ subtly across tongue regions, irrespective of FP number. Other data show an association between taste intensity perception for the bitter compound 6-n-propylthiouracil (PROP) and FP density. However, contradictions exist in the literature, with more recent, larger studies suggesting little or no association between FP number and perceived taste intensity. Much research has examined the relation between FP density and PROP perception, while other tastes have been less thoroughly studied. Here, in a cohort of mainly Caucasian individuals, aged 18-45, recruited from the campus of a large rural university, we examined regional and whole-mouth taste intensities, and FP density using an updated method of a digital still photography method first described in 2005. We found regional differences in suprathreshold intensity. Although all taste sensations were experienced all over the tongue, once again disproving the mythical tongue map, we also observed bitter and umami taste perception to be significantly greater on the posterior tongue than on the anterior tongue. In contrast, there were no regional differences observed for sweet, salty or sour tastes. The relation of FP density to whole-mouth intensity of 6-n-propylthiouracil, and to the intensity of saltiness of NaCl, sweetness from sucrose or from Acesulfame-K, bitterness of quinine, or burning from capsaicin delivered to different regions of the tongue are also discussed.