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Peripheral taste neurons exhibit functional, genetic, and morphological diversity, yet understanding how or if these attributes combine into taste neuron types remains unclear. In this study, we used male and female mice to relate taste bud innervation patterns to the function of a subset of proenkephalin-expressing (Penk+) taste neurons. We found that taste arbors (the portion of the axon within the taste bud) stemming from Penk+ neurons displayed diverse branching patterns and lacked stereotypical endings. The range in complexity observed for individual taste arbors from Penk+ neurons mirrored the entire population, suggesting that taste arbor morphologies are not primarily regulated by the neuron type. Notably, the distinguishing feature of arbors from Penk+ neurons was their propensity to come within 110 nm (in apposition with) different types of taste-transducing cells within the taste bud. This finding is contrary to the expectation of genetically defined taste neuron types that functionally represent a single stimulus. Consistently, further investigation of Penk+ neuron function revealed that they are more likely to respond to innately aversive stimuli-sour, bitter, and high salt concentrations-as compared with the full taste population. Penk+ neurons are less likely to respond to nonaversive stimuli-sucrose, umami, and low salt-compared with the full population. Our data support the presence of a genetically defined neuron type in the geniculate ganglion that is responsive to innately aversive stimuli. This implies that genetic expression might categorize peripheral taste neurons into hedonic groups, rather than simply identifying neurons that respond to a single stimulus., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Sour taste is detected by type III taste receptor cells that generate membrane depolarization with action potentials in response to HCl applied to the apical membranes. The shape of action potentials in type III cells exhibits larger afterhyperpolarization due to activation of transient A-type voltage-gated K + currents. Although action potentials play an important role in neurotransmitter release, the electrophysiological features of A-type K + currents in taste buds remain unclear. Here, we examined the electrophysiological properties of A-type K + currents in mouse fungiform taste bud cells using in-situ whole-cell patch clamping. Type III cells were identified with SNAP-25 immunoreactivity and/or electrophysiological features of voltage-gated currents. Type III cells expressed A-type K + currents which were completely inhibited by 10 mM TEA, whereas IP 3 R3-immunoreactive type II cells did not. The half-maximal activation and steady-state inactivation of A-type K + currents were 17.9 ± 4.5 (n = 17) and - 11.0 ± 5.7 (n = 17) mV, respectively, which are similar to the features of Kv3.3 and Kv3.4 channels (transient and high voltage-activated K + channels). The recovery from inactivation was well fitted with a double exponential equation; the fast and slow time constants were 6.4 ± 0.6 ms and 0.76 ± 0.26 s (n = 6), respectively. RT-PCR experiments suggest that Kv3.3 and Kv3.4 mRNAs were detected at the taste bud level, but not at single-cell levels. As the phosphorylation of Kv3.3 and Kv3.4 channels generally leads to the modulation of cell excitability, neuromodulator-mediated A-type K + channel phosphorylation likely affects the signal transduction of taste., (© 2024. The Author(s).)

Mammalian taste buds are highly regenerative and can restore themselves after normal wear and tear of the lingual epithelium or following physical and chemical insults, including burns, chemotherapy, and nerve injury. This is due to the continual proliferation, differentiation, and maturation of taste progenitor cells, which then must reconnect with peripheral gustatory neurons to relay taste signals to the brain. The turnover and re-establishment of peripheral taste synapses are vital to maintain this complex sensory system. Over the past several decades, the signal transduction and neurotransmitter release mechanisms within taste cells have been well delineated. However, the complex dynamics between synaptic partners in the tongue (taste cell and gustatory neuron) are only partially understood. In this review, we highlight recent findings that have improved our understanding of the mechanisms governing connectivity and signaling within the taste bud and the still-unresolved questions regarding the complex interactions between taste cells and gustatory neurons., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Little is known about the molecular mechanisms underlying drug-induced taste disorders, which can cause malnutrition and reduce quality of life. One of taste disorders is known adverse effects of bisphosphonates, which are administered as anti-osteoporotic drugs. Therefore, the present study evaluated the effects of risedronate (a bisphosphonate) on taste bud cells. Expression analyses revealed that farnesyl diphosphate synthase (FDPS, a key enzyme in the mevalonate pathway) was present in a subset of mouse taste bud and tongue epithelial cells, especially type III sour-sensitive taste cells. Other mevalonate pathway-associated molecules were also detected in mouse taste buds. Behavioral analyses revealed that mice administered risedronate exhibited a significantly enhanced aversion to HCl but not for other basic taste solutions, whereas the taste nerve responses were not affected by risedronate. Additionally, the taste buds of mice administered risedronate exhibited significantly lower mRNA expression of desmoglein-2, an integral component of desmosomes. Taken together, these findings suggest that risedronate may interact directly with FDPS to inhibit the mevalonate pathway in taste bud and tongue epithelial cells, thereby affecting the expression of desmoglein-2 related with epithelial barrier function, which may lead to alterations in behavioral responses to HCl via somatosensory nerves., (© 2022. The Author(s).)

In mammalian taste buds, Type I cells comprise half of all cells. These are termed "glial-like" based on morphologic and molecular features, but there are limited studies describing their function. We tested whether Type I cells sense chemosensory activation of adjacent chemosensory (i.e., Types II and III) taste bud cells, similar to synaptic glia. Using Gad2 ;;GCaMP3 mice of both sexes, we confirmed by immunostaining that, within taste buds, GCaMP expression is predominantly in Type I cells (with no Type II and ≈28% Type III cells expressing weakly). In dissociated taste buds, GCaMP+ Type I cells responded to bath-applied ATP (10-100 μm) but not to 5-HT (transmitters released by Type II or III cells, respectively). Type I cells also did not respond to taste stimuli (5 μm cycloheximide, 1 mm denatonium). In lingual slice preparations also, Type I cells responded to bath-applied ATP (10-100 μm). However, when taste buds in the slice were stimulated with bitter tastants (cycloheximide, denatonium, quinine), Type I cells responded robustly. Taste-evoked responses of Type I cells in the slice preparation were significantly reduced by desensitizing purinoceptors or by purinoceptor antagonists (suramin, PPADS), and were essentially eliminated by blocking synaptic ATP release (carbenoxolone) or degrading extracellular ATP (apyrase). Thus, taste-evoked release of afferent ATP from type II chemosensory cells, in addition to exciting gustatory afferent fibers, also activates glial-like Type I taste cells. We speculate that Type I cells sense chemosensory activation and that they participate in synaptic signaling, similarly to glial cells at CNS tripartite synapses. SIGNIFICANCE STATEMENT Most studies of taste buds view the chemosensitive excitable cells that express taste receptors as the sole mediators of taste detection and transmission to the CNS. Type I "glial-like" cells, with their ensheathing morphology, are mostly viewed as responsible for clearing neurotransmitters and as the "glue" holding the taste bud together. In the present study, we demonstrate that, when intact taste buds respond to their natural stimuli, Type I cells sense the activation of the chemosensory cells by detecting the afferent transmitter. Because Type I cells synthesize GABA, a known gliotransmitter, and cognate receptors are present on both presynaptic and postsynaptic elements, Type I cells may participate in GABAergic synaptic transmission in the manner of astrocytes at tripartite synapses., (Copyright © 2021 the authors.)

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier Inc.)

Action potentials play an important role in neurotransmitter release in response to taste. Here, I have investigated voltage-gated Na + channels, a primary component of action potentials, in respective cell types of mouse fungiform taste bud cells (TBCs) with in situ whole-cell clamping and single-cell RT-PCR techniques. The cell types of TBCs electrophysiologically examined were determined immunohistochemically using the type III inositol 1,4,5-triphoshate receptor as a type II cell marker and synaptosomal-associated protein 25 as a type III cell marker. I show that type II cells, type III cells, and TBCs not immunoreactive to these markers (likely type I cells) generate voltage-gated Na + currents. The recovery following inactivation of these currents was well fitted with double exponential curves. The time constants in type III cells (~20 ms and ~ 1 s) were significantly slower than respective time constants in other cell types. RT-PCR analysis indicated the expression of Nav1.3, Nav1.5, Nav1.6, and β1 subunit mRNAs in TBCs. Pharmacological inhibition and single-cell RT-PCR studies demonstrated that type II and type III cells principally express tetrodotoxin (TTX)-sensitive Nav1.3 channels and that ~ 30% of type I cells express TTX-resistant Nav1.5 channels. The auxiliary β1 subunit that modulates gating kinetics was rarely detected in TBCs. As the β1 subunit co-expressed with an α subunit is known to accelerate the recovery from inactivation, it is likely that voltage-gated Na + channels in TBCs may function without β subunits. Slow recovery from inactivation, especially in type III cells, may limit high-frequency firing in response to taste substances.

Embryonic taste bud primordia are specified as taste placodes on the tongue surface and differentiate into the first taste receptor cells (TRCs) at birth. Throughout adult life, TRCs are continually regenerated from epithelial progenitors. Sonic hedgehog (SHH) signaling regulates TRC development and renewal, repressing taste fate embryonically, but promoting TRC differentiation in adults. Here, using mouse models, we show TRC renewal initiates at birth and coincides with onset of SHHs pro-taste function. Using transcriptional profiling to explore molecular regulators of renewal, we identified Foxa1 and Foxa2 as potential SHH target genes in lingual progenitors at birth and show that SHH overexpression in vivo alters FoxA1 and FoxA2 expression relevant to taste buds. We further bioinformatically identify genes relevant to cell adhesion and cell locomotion likely regulated by FOXA1;FOXA2 and show that expression of these candidates is also altered by forced SHH expression. We present a new model where SHH promotes TRC differentiation by regulating changes in epithelial cell adhesion and migration., Competing Interests: EG, EL, LS, GT, DG, TF, JS, KJ, LB No competing interests declared, (© 2021, Golden et al.)

Circumvallate papilla (CVP) is a distinctively structured with dome-shaped apex, and the surrounding trench which contains over two hundred taste buds on the lateral walls. Although CVP was extensively studied to determine the regulatory mechanisms during organogenesis, it still remains to be elucidated the principle mechanisms of signaling regulations on morphogenesis including taste buds formation. The key role of Yes-associated protein (YAP) in the regulation of organ size and cell proliferation in vertebrates is well understood, but little is known about the role of this signaling pathway in CVP development. We aimed to determine the putative roles of YAP signaling in the epithelial patterning during CVP morphogenesis. To evaluate the precise localization patterns of YAP and other related signaling molecules, including β-catenin, Ki67, cytokeratins, and PGP9.5, in CVP tissue, histology and immunohistochemistry were employed at E16 and adult mice. Our results suggested that there are specific localization patterns of YAP and Wnt signaling molecules in developing and adult CVP. These concrete localization patterns would provide putative involvements of YAP and Wnt signaling for proper epithelial cell differentiation including the formation and maintenance of taste buds.

Taste substances are detected by taste receptor cells in the taste buds in the oral epithelium. Individual taste receptor cells contribute to evoking one of the five taste qualities: sweet, umami, bitter, sour, and salty (sodium). They are continuously replaced every few weeks by new ones generated from local epithelial stem cells. A POU transcription factor, Pou2f3 (also known as Skn-1a), regulates the generation and differentiation of sweet, umami, and bitter cells. However, the molecular mechanisms underlying terminal differentiation into these Pou2f3-dependent taste receptor cells remain unknown. To identify the candidate molecules that regulate the differentiation of these taste receptor cells, we searched for taste receptor type-specific transcription factors using RNA-sequence data of sweet and bitter cells. No transcription factor gene showing higher expression in sweet cells than in bitter cells was found. Eyes absent 1 (Eya1) was identified as the only transcription factor gene showing higher expression in bitter cells than in sweet cells. In situ hybridization revealed that Eya1 was predominantly expressed in bitter cells and also in the putative immature/differentiating taste bud cells in circumvallate and fungiform papillae and soft palate. Eya1 is a candidate molecule that regulates the generation and differentiation of bitter cells.

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreER T2 /tdT mouse, GFP + neural fold transplantation to GFP - chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreER T2 specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP + /GFP - chicken chimera model, GFP + cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells) (postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated the cell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that Mash1-expressing cells in Ascl1 CreERT2 ::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic L -amino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most of gustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase C β2 (PLCβ2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.

Taste bud cells regenerate throughout life. Taste bud maintenance depends on continuous replacement of senescent taste cells with new ones generated by adult taste stem cells. More than a century ago it was shown that taste buds degenerate after their innervating nerves are transected and that they are not restored until after reinnervation by distant gustatory ganglion neurons. Thus, neuronal input, likely via neuron-supplied factors, is required for generation of differentiated taste cells and taste bud maintenance. However, the identity of such a neuron-supplied niche factor(s) remains unclear. Here, by mining a published RNA-sequencing dataset of geniculate ganglion neurons and by in situ hybridization, we demonstrate that R-spondin-2, the ligand of Lgr5 and its homologs Lgr4/6 and stem-cell-expressed E3 ligases Rnf43/Znrf3, is expressed in nodose-petrosal and geniculate ganglion neurons. Using the glossopharyngeal nerve transection model, we show that systemic delivery of R-spondin via adenovirus can promote generation of differentiated taste cells despite denervation. Thus, exogenous R-spondin can substitute for neuronal input for taste bud cell replenishment and taste bud maintenance. Using taste organoid cultures, we show that R-spondin is required for generation of differentiated taste cells and that, in the absence of R-spondin in culture medium, taste bud cells are not generated ex vivo. Thus, we propose that R-spondin-2 may be the long-sought neuronal factor that acts on taste stem cells for maintaining taste tissue homeostasis., Competing Interests: The authors declare no competing interest.

Sense of taste is central to evaluate food before digestion. Taste stem cells undergo constant differentiation throughout the life. However, the mechanism underlying the generation of taste receptor cells is still not clear. Here, we cultured taste organoids from either Lgr5+ or Lgr5-cells, and found the preferential generation of Car4+ and Gustducin + taste receptor cells in organoids derived from Lgr5+ cells in circumvallate, foliate or fungiform papillae. Taste organoids derived from Lgr5+ cells in circumvallate papillae of neonatal mice showed stronger capacity to generate taste receptor cells compared to the organoids from Lgr5+ cells of the adult circumvallate papillae. Massive transcriptional differences were found in multiple signaling pathways including taste transduction between organoids derived from circumvallate papillae of adult and neonatal mice. Inhibiting the Notch signaling pathway by LY411575 enhanced taste receptor cell generation in organoids from circumvallate papillae and modulated multiple signaling pathways. Thus, we concluded that receptor cell generation in taste organoids was age-related and regulated via multiple signaling pathways., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The present study aims to compare the morphology of the oropharyngeal roof of young and adult domestic pigeon (Columba livia domestica) by gross observation, morphometric measurements, and scanning electron microscopy (SEM). The oropharyngeal roof was divided into the palate and pharyngeal roof. The palate was narrow triangular in shape and concave along its length. It could be divided into a rostral part contained three longitudinal palatine ridges and a caudal part contained the choanal slit. The choanal slit consisted of narrow rostral and wide caudal parts. The edges of the narrow part were encircled by small caudomedially directed papillae. On the contrary, the edges of the wide part of slit were free from papillae. By SEM, the palatal mucosa in young pigeon showed primordia of small papillae which increased in number and size forming a longitudinal row of papillae parallel to the edges of the rostral narrow part of slit in adult pigeon. The surface of the pharyngeal roof appeared smooth in young pigeon, while in adult pigeon, it showed dome-shaped elevations. The infundibular cleft had smooth edges. The caudal part of the pharyngeal roof formed an elevated transverse mucosal fold on which a transverse row of conical-shaped papillae was present. In conclusion, our results documented the presence of some differences between the oropharyngeal roof of young and adult pigeon, which suggest a high degree of functional adaptation in adult pigeon to their diet compared to young pigeon. Such adaptations might increase the efficiency of food prehension in adult pigeon. The present study compared the morphology of the oropharyngeal roof of young and adult domestic pigeon by gross observation, morphometry, and scanning electron microscopy. The morphometrical data showed higher values in adult pigeon compared to young pigeon. The palatal mucosa and the pharyngeal roof of adult pigeon showed papillae and elevations that were not present in young pigeon. Our results suggest a high degree of functional adaptation in adult pigeon to their diet compared to young pigeon. Such adaptations might increase the efficiency of food prehension in adult pigeon., (© 2020 Wiley Periodicals, Inc.)

The current study was done to provide comprehensive information on the anatomical features of the lips and cheeks of the goat by gross examination and morphometric analysis in addition to scanning electron microscope (SEM). Samples from 12 normal healthy adult goat's heads of both sexes were collected directly after slaughtering. The lips and cheeks were dissected, and specimens were collected for both light and SEM. The lips of goat were soft and mobile. The free border of both lips was characterized rostrally by the presence of labial projections. The number, size, and arrangement of labial projections differed in the upper and lower lips. On the other hand, the buccal papillae were arranged into 6-8 longitudinal rows parallel to the cheek teeth. The length of these papillae decreased caudally while they were absent on the most caudal part of the cheek. Presence of several types and shapes of labial projections and papillae, and buccal papillae suggest a high degree of mechanical adaptation of the lips and cheeks of the goat. This study provides baseline data for clinical studies. This study is the first report to shed light on the morphology of the lips and cheeks of the goat by gross and scanning electron microscopy., (© 2020 Wiley Periodicals, Inc.)

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCβ signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCβ3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCβ3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression., Competing Interests: The authors have declared that no competing interests exist.

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na + sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na + influx without changing the intracellular Ca 2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na + entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca 2+ signals. This work also reveals ENaC-independent salt attraction., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Among the 5 taste qualities, salt is the least understood. The receptors, their expression pattern in taste cells, and the transduction mechanisms for salt taste are still unclear. Previous studies have suggested that low concentrations of NaCl are detected by the amiloride-sensitive epithelial Na+ channel (ENaC), which in other systems requires assembly of 3 homologous subunits (α, β, and γ) to form a functional channel. However, a new study from Lossow and colleagues, published in this issue of Chemical Senses, challenges that hypothesis by examining expression levels of the 3 ENaC subunits in individual taste cells using gene-targeted mice in combination with immunohistochemistry and in situ hybridization. Results show a lack of colocalization of ENaC subunits in taste cells as well as expression of subunits in taste cells that show no amiloride sensitivity. These new results question the molecular identity of the amiloride-sensitive Na+ conductance in taste cells., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A mouse single taste bud contains 10-100 taste bud cells (TBCs) in which the elongated TBCs are classified into 3 cell types (types I-III) equipped with different taste receptors. Accordingly, differences in the cell numbers and ratios of respective cell types per taste bud may affect taste-nerve responsiveness. Here, we examined the numbers of each immunoreactive cell for the type II (sweet, bitter, or umami receptor cells) and type III (sour and/or salt receptor cells) markers per taste bud in the circumvallate and foliate papillae and compared these numerical features of TBCs per taste bud to those in fungiform papilla and soft palate, which we previously reported. In circumvallate and foliate taste buds, the numbers of TBCs and immunoreactive cells per taste bud increased as a linear function of the maximal cross-sectional taste bud area. Type II cells made up approximately 25% of TBCs irrespective of the regions from which the TBCs arose. In contrast, type III cells in circumvallate and foliate taste buds made up approximately 11% of TBCs, which represented almost 2 times higher than what was observed in the fungiform and soft palate taste buds. The densities (number of immunoreactive cells per taste bud divided by the maximal cross-sectional area of the taste bud) of types II and III cells per taste bud are significantly higher in the circumvallate papillae than in the other regions. The effects of these region-dependent differences on the taste response of the taste bud are discussed., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, β-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and β-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, β-, and γ-subunits and ask for a careful investigation of the channel composition., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study was conducted with the tongue samples of different life stages of hilsa, that is, adult Marine hilsa, adult Riverine hilsa, and Riverine juvenile hilsa, respectively. Three types of taste buds (Types I, II, and III based on their elevation from the epithelium at different levels) of the tongue, which may be to ensure full utilization of the gustatory ability of the fish were rocorded. Presence of specific taste buds indicate that the fish hilsa dwells in turbid waters with a possible preference toward diatom like planktonic food source. Enhanced expression of taste receptors (T1R1 and T1R3) and associated stimulatory G-proteins subunits on tongue also indicate occurrence of amino acid like substances that guided sensory cues for feeding by this fish. A firm regularity or stringency of the free surface of the epithelial cells may be attributed to compactly arranged microridges. These structures protect against physical abrasions potentially caused during food manoeuvring and swallowing. In our present observations, the surface architectures of the tongue of hilsa are discussed within the background of migratory adaptation of the species in the context of feeding and habitat preferences., (© 2020 Wiley Periodicals, Inc.)

The tayra (Eira barbara) is a mammal belonging to the Mustelidae family that occurs in all Brazilian biomes. The present work aimed to describe the morphology of the tongue of these specimens highlighting their structures and particularities that will serve as a subsidy to elucidate the anatomy of the same and for comparative studies among other species of domestic and wild animals. Five adult male specimens of E. barbara were studied, which were fixed using 10% aqueous formaldehyde solution. The tongue was removed by opening the oral cavity and separating the maxillary/mandible bone complex. Being in possession of the material, photodocumentations and collection of the fragments were made for the proper preparation of histological slides and Scanning Electron Microscopy (SEM). The lingual papillae found in tayra were mechanical: filiform and conical; and gustative: fungiform and circumvallated. Histologically, the papillae are constituted by keratinized stratified epithelium and in the innermost region, it was composed of tissue connective dense unshaped followed by a layer of muscle bundles of skeletal striated. In the region of the root of the tongue of E. barbara, there were a set of small mixed salivary glands (serous and mucous) and the punctual presence of gustatory corpuscles at the level of epithelium. The morphological description of the E. barbara tongue revealed similarity to that described in literature for other domestic and wild mammals. However, the particularity of the absence of foliate papilla and the quantitative of four papillae circumvallate in the region of the root of the tongue of this species., (© 2020 Blackwell Verlag GmbH.)

The gustation system for sweeteners is well-known to be regulated by nutritional and metabolic conditions, but there is no or little information on the underlying mechanism. Here, we examined whether elevation of the blood glucose level was involved in alteration of the expression of sweet taste receptors in circumvallate papillae (CP) and sweet taste sensitivity in male Sprague-Dawley rats. Rats under 4 h-fed conditions following 18 h-fasting exhibited elevated blood glucose levels and decreased pancreatic T1R3 expression, compared to rats after 18 h-fasting treatment, and they exhibited increased protein expression of sweet taste receptors T1R2 and T1R3 in CP. Under streptozotocin (STZ)-induced diabetes mellites (DM) conditions, the protein expression levels of T1R2 and T1R3 in CP were higher than those under control conditions, and these DM rats exhibited increased lick ratios in a low sucrose concentration range in a brief access test with a mixture of sucrose and quinine hydrochloride (QHCl). These findings indicate that the elevation of blood glucose level is a regulator for an increase in sweet taste receptor protein expression in rat CP, and such alteration in STZ-induced DM rats is involved in enhancement of their sweet taste sensitivity.

Considering that nutrients are required in health and diseases, the detection and ingestion of food to meet the requirements is attributable to the sense of taste. Altered taste sensations lead to a decreased appetite, which is usually one of the frequent causes of malnutrition in patients with diseases. Ongoing taste research has identified a variety of drug pathways that cause changes in taste perceptions in cancer, increasing our understanding of taste disturbances attributable to aberrant mechanisms of taste sensation. The evidence discussed in this review, which addresses the implications of innate immune responses in the modulation of taste functions, focuses on the adverse effects on taste transmission from taste buds by immune modulators responsible for alterations in the perceived intensity of some taste modalities. Another factor, damage to taste progenitor cells that directly results in local effects on taste buds, must also be considered in relation to taste disturbances in patients with cancer. Recent discoveries discussed have provided new insights into the pathophysiology of taste dysfunctions associated with the specific treatments. SIGNIFICANCE STATEMENT: The paradigm that taste signals transmitted to the brain are determined only by tastant-mediated activation via taste receptors has been challenged by the immune modification of taste transmission through drugs during the processing of gustatory information in taste buds. This article reports the findings in a model system (mouse taste buds) that explain the basis for the taste dysfunctions in patients with cancer that has long been observed but never understood., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Head and neck cancer patients receiving conventional repeated, low dose radiotherapy (fractionated IR) suffer from taste dysfunction that can persist for months and often years after treatment. To understand the mechanisms underlying functional taste loss, we established a fractionated IR mouse model to characterize how taste buds are affected. Following fractionated IR, we found as in our previous study using single dose IR, taste progenitor proliferation was reduced and progenitor cell number declined, leading to interruption in the supply of new taste receptor cells to taste buds. However, in contrast to a single dose of IR, we did not encounter increased progenitor cell death in response to fractionated IR. Instead, fractionated IR induced death of cells within taste buds. Overall, taste buds were smaller and fewer following fractionated IR, and contained fewer differentiated cells. In response to fractionated IR, expression of Wnt pathway genes, Ctnnb1, Tcf7, Lef1 and Lgr5 were reduced concomitantly with reduced progenitor proliferation. However, recovery of Wnt signaling post-IR lagged behind proliferative recovery. Overall, our data suggest carefully timed, local activation of Wnt/β-catenin signaling may mitigate radiation injury and/or speed recovery of taste cell renewal following fractionated IR.

In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every ∼20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The metabolic hormone adiponectin is secreted into the circulation by adipocytes and mediates key biological functions, including insulin sensitivity, adipocyte development, and fatty acid oxidation. Adiponectin is also abundant in saliva, where its functions are poorly understood. Here we report that murine taste receptor cells (TRCs) express specific adiponectin receptors and may be a target for salivary adiponectin. This is supported by the presence of all three known adiponectin receptors in transcriptomic data obtained by RNA-seq analysis of purified circumvallate (CV) taste buds. As well, immunohistochemical analysis of murine CV papillae showed that two adiponectin receptors, ADIPOR1 and T-cadherin, are localized to subsets of TRCs. Immunofluorescence for T-cadherin was primarily co-localized with the Type 2 TRC marker phospholipase C β2, suggesting that adiponectin signaling could impact sweet, bitter, or umami taste signaling. However, adiponectin null mice showed no differences in behavioral lick responsiveness compared with wild-type controls in brief-access lick testing. AAV-mediated overexpression of adiponectin in the salivary glands of adiponectin null mice did result in a small but significant increase in behavioral lick responsiveness to the fat emulsion Intralipid. Together, these results suggest that salivary adiponectin can affect TRC function, although its impact on taste responsiveness and peripheral taste coding remains unclear., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

WT1 is a transcriptional activator that controls the boundary between multipotency and differentiation. The transcriptional cofactor BASP1 binds to WT1, forming a transcriptional repressor complex that drives differentiation in cultured cells; however, this proposed mechanism has not been demonstrated in vivo. We used the peripheral taste system as a model to determine how BASP1 regulates the function of WT1. During development, WT1 is highly expressed in the developing taste cells while BASP1 is absent. By the end of development, BASP1 and WT1 are co-expressed in taste cells, where they both occupy the promoter of WT1 target genes. Using a conditional BASP1 mouse, we demonstrate that BASP1 is critical to maintain the differentiated state of adult taste cells and that loss of BASP1 expression significantly alters the composition and function of these cells. This includes the de-repression of WT1-dependent target genes from the Wnt and Shh pathways that are normally only transcriptionally activated by WT1 in the undifferentiated taste cells. Our results uncover a central role for the WT1-BASP1 complex in maintaining cell differentiation in vivo., (© 2019 Gao et al.)

In this study, it was aimed to perform light microscopy and scanning electron microscopy (SEM) investigation of the tongue and papilla belonging to two wolves. Light microscopy and SEM images of tongues were taken. It was observed that papillae filiformes concentrated in apex of these investigated tongues. In addition to, these papillae were observed in the whole tongues. It was also determined that papillae fungiformes were distributed rarely in between papillae filiformes. Papillae foliatae were placed in the lateral side of tongues. There were two papillae vallatae in the median part of tongues. Papillae vallatae was determined in radix linguae. It was observed that papillae vallatae formed circular extensions in many different dimensions and a hole was present in a circular structure in the center. Papillae conicae were seen on dorsal surface of radix linguae. Papillae foliatae were seen four laminal structure and placed on the each lateral side of tongues. To the best of our knowledge, this is the first study that presents of light microscopy and SEM findings related with the tongues of wolves., (© 2018 Wiley Periodicals, Inc.)

Taste buds are comprised of taste cells, which are classified into types I to IV. Transient receptor potential (TRP) channels play a significant role in taste perception. TRP vanilloid 4 (TRPV4) is a non-selective cation channel that responds to mechanical, thermal, and chemical stimuli. The present study aimed to define the function and expression of TRPV4 in taste buds using Trpv4-deficient mice. In circumvallate papillae, TRPV4 colocalized with a type IV cell and epithelial cell marker but not type I, II, or III markers. Behavioural studies showed that Trpv4 deficiency reduced sensitivity to sourness but not to sweet, umami, salty, and bitter tastes. Trpv4 deficiency significantly reduced the expression of type III cells compared with that in wild type (WT) mice in vivo and in taste bud organoid experiments. Trpv4 deficiency also significantly reduced Ki67-positive cells and β-catenin expression compared with those in WT circumvallate papillae. Together, the present results suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice.

In the mammalian taste system, the taste receptor type 2 (T2R) family mediates bitter taste, and the taste receptor type 1 (T1R) family mediates sweet and umami tastes (the heterodimer of T1R2/T1R3 forms the sweet taste receptor, and the heterodimer of T1R1/T1R3 forms the umami taste receptor). In the chicken genome, bitter (T2R1, T2R2, and T2R7) and umami (T1R1 and T1R3) taste receptor genes have been found. However, the localization of these taste receptors in the taste buds of chickens has not been elucidated. In the present study, we demonstrated that the bitter taste receptor T2R7 and the umami taste receptor subunit T1R1 were expressed specifically in the taste buds of chickens labeled by Vimentin, a molecular marker for chicken taste buds. We analyzed the distributions of T2R7 and T1R1 on the oral epithelial sheets of chickens and among 3 different oral tissues of chickens: the palate, the base of the oral cavity, and the posterior tongue. We found that the distribution patterns and numbers were similar between taste bud clusters expressing these receptors and those expressing Vimentin. These results indicated broad distributions of T2R7 and T1R1 in the gustatory tissues of the chicken oral cavity. In addition, 3D-reconstructed images clearly revealed that high levels of T2R7 and T1R1 were expressed in Vimentin-negative taste bud cells. Taken together, the present results indicated the presence of bitter and umami sensing systems in the taste buds of chickens, and broad distribution of T2R7 and T1R1 in the chicken oral cavity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The CALHM1/CALHM3 channel in the basolateral membrane of polarized taste cells mediates neurotransmitter release. However, mechanisms regulating its localization remain unexplored. Here, we identified CALHM1/CALHM3 in the basolateral membrane of type II taste cells in discrete puncta localized close to afferent nerve fibers. As in taste cells, CALHM1/CALHM3 was present in the basolateral membrane of model epithelia, although it was distributed throughout the membrane and did not show accumulation in puncta. We identified canonical basolateral sorting signals in CALHM1 and CALHM3: tyrosine-based and dileucine motifs. However, basolateral sorting remained intact in mutated channels lacking those signals, suggesting that non-canonical signals reside elsewhere. Our study demonstrates intrinsic basolateral sorting of CALHM channels in polarized cells, and provides mechanistic insights.

Overconsumption of NaCl has been linked to increased hypertension-related morbidity. Compounds that can enhance NaCl responses in taste cells could help reduce human NaCl consumption without sacrificing perceived saltiness. Spilanthol is an unsaturated alkylamide isolated from the Jambu plant (Acmella oleracea) that can induce tingling, pungency, and numbing in the mouth. Structurally similar fatty acid amides, such as sanshool, elicit numbing and tingling sensations by inhibiting 2-pore-domain potassium leak channels on trigeminal sensory neurons. Even when insufficient to induce action potential firing, leak current inhibition causes depolarization and increased membrane resistance, which combine to make cells more sensitive to subsequent depolarizing stimuli, such as NaCl. Using calcium imaging, we tested whether spilanthol alters sensitivity to NaCl in isolated circumvallate taste bud cells and trigeminal sensory neurons of mice (Mus musculus). Micromolar spilanthol elicited little to no response in taste bud cells or trigeminal neurons. These same perithreshold concentrations of spilanthol significantly enhanced responses to NaCl (140 and 200 mM) in taste bud cells. Trigeminal neurons, however, exhibited response enhancement only at the highest concentrations of NaCl and spilanthol tested. Using a combination of potassium depolarization, immunohistochemistry, and Trpm5-GFP and Tas1r3-GFP mice to characterize taste bud cells by type, we found spilanthol enhancement of NaCl responses most prevalent in NaCl-responsive type III cells, and commonly observed in NaCl-responsive type II cells. Our results indicate that spilanthol enhances NaCl responses in taste bud cells and point to a family of compounds that may have utility as salty taste enhancers.

Cells can communicate with one another through physical connections and chemical signaling, activating various signaling pathways that can affect cellular functions and behaviors. In taste buds, taste cells transmit taste information to neurons via paracrine signaling. However, no previous studies have reported the in vitro co-culture of taste and neuronal cells, which allows us to monitor intercellular communications and better understand the mechanism of taste perception. Here, we introduce the first investigation on the proximate assembly and co-culture of taste cells and neurons to monitor the intercellular transmission of taste signals. Taste cells and neurons are placed closely using a pair of single-stranded oligonucleotides conjugated with polyethylene glycol and a phospholipid. Complementary oligonucleotide conjugates are anchored into the cellular membrane of neonatal taste cells and embryonic hippocampal neuronal cells, respectively, and then the cells are self-assembled into a functional multicellular unit for taste perception. Treatment of the assembled cells with a bitter tastant generates the sequential influx of calcium ions into the cytoplasm in taste cells and then in neuronal cells. Our work demonstrates that the cellular self-assembly is critical for efficient taste signal transduction, which can be used as a promising platform to construct cell-based biosensors for taste sensing.

This paper presents a dual functional extracellular recording biosensor based on a light-addressable potentiometric sensor (LAPS). The design and fabrication of this biosensor make it possible to record both extracellular membrane potential changes and ATP release from a single taste bud cell for the first time. For detecting ATP release, LAPS chip was functionalized with ATP-sensitive DNA aptamer by covalent immobilization. Taste bud cells isolated from rat were cultured on LAPS surface. When the desired single taste bud cell was illuminated by modulated light, ATP release from single taste bud cells can be measured by recording the shifts of bias voltage-photocurrent curves (I-V curves) when the LAPS chip is working in discrete mode. On the other hand, extracellular membrane potential changes can be monitored by recording the fluctuation of LAPS photocurrent when the LAPS chip is working in continuous mode. The results show this biosensor can effectively record the enhancive effect of the bitter substance and inhibitory effect of the carbenoxolone (CBX) on the extracellular membrane potential changes and ATP release of single taste bud cells. In addition, the inhibitory effect of CBX also confirms LAPS extracellular recordings are originated from bitter signal transduction. It is proved this biosensor is suitable for extracellular recording of ATP release and membrane potential changes of single taste bud cells. It is suggested this biosensor could be applied to investigating taste signal transduction at the single-cell level as well as applied to other types of cells which have similar functions to taste bud cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This research aimed to reveal the general morphology and topographic distribution of lingual papillae, epithelial characteristics, mucosal structure, and glands with their mucin content in the sheep tongue, with consideration of species-specific characteristics. The tongues of ten sheep were analyzed for this purpose. Filiform and fungiform papillae existed within the borders of the ventral surface of the lingual apex. The majority of the filiform papillae had multiple secondary projections. Fungiform papillae were also seen on the lingual torus among lenticular papillae, as well as 6 to 10 circumvallate papillae arranged on its caudal border. The species-specific details of the general anatomical structure of the tongue were determined and, in general, the papillary organization in the sheep was similar to goats, while the papillary organization also was similar to features with deer species, specifically the filiform papilla from the mechanical papillae and fungiform papilla from the gustatory papillae. Neutral and weak sulfated mucins and N-acetyl sialomucins were located in seromucous glands, salivary duct epithelium and von Ebner's glands. Carboxylated acid mucins and N-acetyl sialomucins were not present in seromucous and von Ebner's glands. In seromucous glands, MUC1, MUC5AC, and MUC6 localized only in epithelial cells of ducts, whereas MUC2 localized in both glandular and ductal epithelial cells. All MUCs were present in both von Ebner's glands and salivary ducts. We showed that this mucin composition, may serve as a physical barrier in the initial section of the digestive system. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Adult tongue epithelium is continuously renewed from epithelial progenitor cells, a process that requires hedgehog (HH) signaling. In mice, pharmacological inhibition of the HH pathway causes taste bud loss within a few weeks. Previously, we demonstrated that sonic hedgehog (SHH) overexpression in lingual progenitors induces ectopic taste buds with locally increased SOX2 expression, suggesting that taste bud differentiation depends on SOX2 downstream of HH. To test this, we inhibited HH signaling in mice and observed a rapid decline in Sox2 and SOX2-GFP expression in taste epithelium. Upon conditional deletion of Sox2 , differentiation of both taste and non-taste epithelial cells was blocked, and progenitor cell number increased. In contrast to basally restricted proliferation in controls, dividing cells were overabundant and spread to suprabasal epithelial layers in mutants. SOX2 loss in progenitors also led non-cell-autonomously to taste cell apoptosis, dramatically shortening taste cell lifespans. Finally, in tongues with conditional Sox2 deletion and SHH overexpression, ectopic and endogenous taste buds were not detectable; instead, progenitor hyperproliferation expanded throughout the lingual epithelium. In summary, we show that SOX2 functions downstream of HH signaling to regulate lingual epithelium homeostasis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-μm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca 2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mouse taste receptor cells survive from 3-24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors Gli2 and Gli3 in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that Gli3 is highly expressed in Tas1r3-expressing taste receptor cells and Lgr5+ taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that Gli3 was expressed in taste buds in all taste fields. Conditional knockout mice lacking Gli3 in the posterior tongue (Gli3CKO) had larger taste buds containing more taste cells than did control wild-type (Gli3WT) mice. In comparison to wild-type mice, Gli3CKO mice had more Lgr5+ and Tas1r3+ cells, but fewer type III cells. Similar changes were observed ex vivo in Gli3CKO taste organoids cultured from Lgr5+ taste stem cells. Further, the expression of several taste marker and Gli3 target genes was altered in Gli3CKO mice and/or organoids. Mirroring these changes, Gli3CKO mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that Gli3 is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially Tas1r3+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging.

How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog ( Shh ), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh -expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration., Competing Interests: The authors declare no conflict of interest.

Class B scavenger receptor family members, scavenger receptor B1 (SR-B1) and cluster of differentiation 36 (CD36), are broadly expressed cell-surface proteins, both of which are believed to serve as multifaceted players in lipid and lipoprotein metabolism in mammals. Because of its presence in the apical part of taste receptor cells within circumvallate taste buds and its ability to recognise long-chain fatty acids, CD36 has been believed to participate in the sensing of the lipid species within the oral cavity. However, there have been no attempts to address whether SR-B1 has such a role to date. In this study, by reverse transcription- polymerase chain reaction analysis, we detected SR-B1 mRNA in a total RNA sample isolated from the circumvallate papillae of mouse tongue. Immunohistochemical analysis of tongue sections from the animals revealed the expression of SR-B1 protein in a population of taste bud cells of circumvallate papillae. In addition, the pattern of staining in the papillae for SR-B1 agreed closely with that for CD36 in double immunostaining analysis. We performed a cell-free in-vitro assay utilising a peptide mimic of SR-B1 and provided evidence that the receptor could recognise certain of the unsaturated long-chain fatty acids such as oleic acid. Our present findings suggest an additional role for SR-B1 as a captor of specific fatty acids in the oral cavity of mammals and contribute to expanding our knowledge of the physiological function of the receptor.

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., Competing Interests: The authors declare no conflict of interest.

Activation of Type III cells in mammalian taste buds is implicated in the transduction of acids (sour) and salty stimuli. Several lines of evidence suggest that function of Type III cells in the anterior taste fields may differ from that of Type III cells in posterior taste fields. Underlying anatomy to support this observation is, however, scant. Most existing immunohistochemical data characterizing this cell type focus on circumvallate taste buds in the posterior tongue. Equivalent data from anterior taste fields-fungiform papillae and soft palate-are lacking. Here, we compare Type III cells in four taste fields: fungiform, soft palate, circumvallate, and foliate in terms of reactivity to four canonical markers of Type III cells: polycystic kidney disease 2-like 1 (PKD2L1), synaptosomal associated protein 25 (SNAP25), serotonin (5-HT), and glutamate decarboxylase 67 (GAD67). Our findings indicate that while PKD2L1, 5-HT, and SNAP25 are highly coincident in posterior taste fields, they diverge in anterior taste fields. In particular, a subset of taste cells expresses PKD2L1 without the synaptic markers, and a subset of SNAP25 cells lacks expression of PKD2L1. In posterior taste fields, GAD67-positive cells are a subset of PKD2L1 expressing taste cells, but anterior taste fields also contain a significant population of GAD67-only expressing cells. These differences in expression patterns may underlie the observed functional differences between anterior and posterior taste fields., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Carbonic anhydrases form an enzyme family of 16 members, which reversibly catalyze the hydration of carbon dioxide to bicarbonate and protons. In lung, kidney, and brain, presence of carbonic anhydrases is associated with protons and bicarbonate transport in capillary endothelium of lung, reabsorption of bicarbonate in proximal renal tubules, and extracellular buffering. In contrast, their role in taste is less clear. Recently, carbonic anhydrase IV expression was detected in sour-sensing presynaptic taste cells and was associated with the taste of carbonation, yet the precise role and cell population remained uncertain. To examine the role of carbonic anhydrase 4-expressing cells in taste reception, we generated a mouse strain carrying a modified allele of the carbonic anhydrase 4 gene in which the coding region of the red fluorescent protein monomeric Cherry is attached to that of carbonic anhydrase 4 via an internal ribosome entry site. Monomeric Cherry fluorescence was detected in lingual papillae as well as taste buds of soft palate and naso-incisor duct. However, expression patterns on the tongue differ between posterior and fungiform papillae. Whereas monomeric Cherry auto-fluorescence was almost always co-localized with presynaptic cell markers aromatic L-amino-acid decarboxylase, synaptosomal-associated protein 25 or glutamic acid decarboxylase 67 in fungiform papillae and taste buds of palate and naso-incisor duct, monomeric Cherry-positive cells in posterior tongue papillae represent only a subpopulation of presynaptic cells. We conclude that this model is well suited for detailed investigation into the role of carbonic anhydrase in gustation and other processes., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many commonly prescribed chemotherapy drugs such as cyclophosphamide (CYP) have adverse side effects including disruptions in taste which can result in loss of appetite, malnutrition, poorer recovery and reduced quality of life. Previous studies in mice found evidence that CYP has a two-phase disturbance in taste behavior: a disturbance immediately following drug administration and a second which emerges several days later. In this study, we examined the processes by which CYP disturbs the taste system by examining the effects of the drug on taste buds and cells responsible for taste cell renewal using immunohistochemical assays. Data reported here suggest CYP has direct cytotoxic effects on lingual epithelium immediately following administration, causing an early loss of taste sensory cells. Types II and III cells in fungiform taste buds appear to be more susceptible to this effect than circumvallate cells. In addition, CYP disrupts the population of rapidly dividing cells in the basal layer of taste epithelium responsible for taste cell renewal, manifesting a disturbance days later. The loss of these cells temporarily retards the system's capacity to replace Type II and Type III taste sensory cells that survived the cytotoxic effects of CYP and died at the end of their natural lifespan. The timing of an immediate, direct loss of taste cells and a delayed, indirect loss without replacement of taste sensory cells are broadly congruent with previously published behavioral data reporting two periods of elevated detection thresholds for umami and sucrose stimuli. These findings suggest that chemotherapeutic disturbances in the peripheral mechanisms of the taste system may cause dietary challenges at a time when the cancer patient has significant need for well balanced, high energy nutritional intake.

Taste cells in taste buds are epithelial sensory cells. Old taste bud cells die and are replaced by new ones generated from taste stem cells. Identifying and characterizing adult taste stem cells is therefore important to understand how peripheral taste tissues are maintained. SOX2 is expressed in oral epithelium including gustatory papillae and has been proposed to be a marker of adult taste stem/progenitor cells. Nevertheless, this hypothesis has never been directly tested. Here, by single-color genetic lineage tracing using Sox2-CreERT2 strain, we reveal that all types of taste bud cells distributed throughout the oral epithelium are derived from stem cells that express SOX2. Short-term tracing shows that SOX2-positive taste stem cells actively supply taste bud cells. At the base of epithelium outside taste buds are distributed proliferation marker- and SOX2-positive cells. Consistently, taste stem cells identified by Lgr5 expression in the circumvallate papillae also express SOX2. Together, taste stem cells distributed in oral epithelia express SOX2., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RNA-Seq is a powerful tool in transcriptomic profiling of cells and tissues. We recently identified many more taste buds than previously appreciated in chickens using molecular markers to stain oral epithelial sheets of the palate, base of oral cavity, and posterior tongue. In this study, RNA-Seq was performed to understand the transcriptomic architecture of chicken gustatory tissues. Interestingly, taste sensation related genes and many more differentially expressed genes (DEGs) were found between the epithelium and mesenchyme in the base of oral cavity as compared to the palate and posterior tongue. Further RNA-Seq using specifically defined tissues of the base of oral cavity demonstrated that DEGs between gustatory (GE) and non-gustatory epithelium (NGE), and between GE and the underlying mesenchyme (GM) were enriched in multiple GO terms and KEGG pathways, including many biological processes. Well-known genes for taste sensation were highly expressed in the GE. Moreover, genes of signaling components important in organogenesis (Wnt, TGFβ/ BMP, FGF, Notch, SHH, Erbb) were differentially expressed between GE and GM. Combined with other features of chicken taste buds, e.g., uniquely patterned array and short turnover cycle, our data suggest that chicken gustatory tissue provides an ideal system for multidisciplinary studies, including organogenesis and regenerative medicine.

In mammals, taste buds typically contain 50-100 tightly packed taste-receptor cells (TRCs), representing all five basic qualities: sweet, sour, bitter, salty and umami. Notably, mature taste cells have life spans of only 5-20 days and, consequently, are constantly replenished by differentiation of taste stem cells. Given the importance of establishing and maintaining appropriate connectivity between TRCs and their partner ganglion neurons (that is, ensuring that a labelled line from sweet TRCs connects to sweet neurons, bitter TRCs to bitter neurons, sour to sour, and so on), we examined how new connections are specified to retain fidelity of signal transmission. Here we show that bitter and sweet TRCs provide instructive signals to bitter and sweet target neurons via different guidance molecules (SEMA3A and SEMA7A). We demonstrate that targeted expression of SEMA3A or SEMA7A in different classes of TRCs produces peripheral taste systems with miswired sweet or bitter cells. Indeed, we engineered mice with bitter neurons that now responded to sweet tastants, sweet neurons that responded to bitter or sweet neurons responding to sour stimuli. Together, these results uncover the basic logic of the wiring of the taste system at the periphery, and illustrate how a labelled-line sensory circuit preserves signalling integrity despite rapid and stochastic turnover of receptor cells.