High Sox2 expression predicts taste lineage competency of lingual progenitors in vitro.

Taste buds on the tongue contain taste receptor cells (TRCs) that detect sweet, sour, salty, umami and bitter stimuli. Like non-taste lingual epithelium, TRCs are renewed from basal keratinocytes, many of which express the transcription factor SOX2. Genetic lineage tracing has shown that SOX2+ lingual progenitors give rise to both taste and non-taste lingual epithelium in the posterior circumvallate taste papilla (CVP) of mice. However, SOX2 is variably expressed among CVP epithelial cells, suggesting that their progenitor potential may vary. Using transcriptome analysis and organoid technology, we show that cells expressing SOX2 at higher levels are taste-competent progenitors that give rise to organoids comprising both TRCs and lingual epithelium. Conversely, organoids derived from progenitors that express SOX2 at lower levels are composed entirely of non-taste cells. Hedgehog and WNT/ß-catenin are required for taste homeostasis in adult mice. However, manipulation of hedgehog signaling in organoids has no impact on TRC differentiation or progenitor proliferation. By contrast, WNT/ß-catenin promotes TRC differentiation in vitro in organoids derived from higher but not low SOX2+ expressing progenitors.

SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis.

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.

A Mechanistic Overview of Taste Bud Maintenance and Impairment in Cancer Therapies.

Since the early 20th century, progress in cancer therapies has significantly improved disease prognosis. Nonetheless, cancer treatments are often associated with side effects that can negatively affect patient well-being and disrupt the course of treatment. Among the main side effects, taste impairment is associated with depression, malnutrition, and morbid weight loss. Although relatively common, taste disruption associated with cancer therapies remains poorly understood. Here, we review the current knowledge related to the molecular mechanisms underlying taste maintenance and disruption in the context of cancer therapies.

Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.

The integrity of taste buds is intimately dependent on an intact gustatory innervation, yet the molecular nature of this dependency is unknown. Here, we show that differentiation of new taste bud cells, but not progenitor proliferation, is interrupted in mice treated with a hedgehog (Hh) pathway inhibitor (HPI), and that gustatory nerves are a source of sonic hedgehog (Shh) for taste bud renewal. Additionally, epithelial taste precursor cells express Shh transiently, and provide a local supply of Hh ligand that supports taste cell renewal. Taste buds are minimally affected when Shh is lost from either tissue source. However, when both the epithelial and neural supply of Shh are removed, taste buds largely disappear. We conclude Shh supplied by taste nerves and local taste epithelium act in concert to support continued taste bud differentiation. However, although neurally derived Shh is in part responsible for the dependence of taste cell renewal on gustatory innervation, neurotrophic support of taste buds likely involves a complex set of factors.

Identifying Treatments for Taste and Smell Disorders: Gaps and Opportunities.

The chemical senses of taste and smell play a vital role in conveying information about ourselves and our environment. Tastes and smells can warn against danger and also contribute to the daily enjoyment of food, friends and family, and our surroundings. Over 12% of the US population is estimated to experience taste and smell (chemosensory) dysfunction. Yet, despite this high prevalence, long-term, effective treatments for these disorders have been largely elusive. Clinical successes in other sensory systems, including hearing and vision, have led to new hope for developments in the treatment of chemosensory disorders. To accelerate cures, we convened the "Identifying Treatments for Taste and Smell Disorders" conference, bringing together basic and translational sensory scientists, health care professionals, and patients to identify gaps in our current understanding of chemosensory dysfunction and next steps in a broad-based research strategy. Their suggestions for high-yield next steps were focused in 3 areas: increasing awareness and research capacity (e.g., patient advocacy), developing and enhancing clinical measures of taste and smell, and supporting new avenues of research into cellular and therapeutic approaches (e.g., developing human chemosensory cell lines, stem cells, and gene therapy approaches). These long-term strategies led to specific suggestions for immediate research priorities that focus on expanding our understanding of specific responses of chemosensory cells and developing valuable assays to identify and document cell development, regeneration, and function. Addressing these high-priority areas should accelerate the development of novel and effective treatments for taste and smell disorders.

ß-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice.

Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional ß-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest ß-catenin plays a greater role in renewal of anterior versus posterior taste buds.

Progress and renewal in gustation: new insights into taste bud development.

The sense of taste, or gustation, is mediated by taste buds, which are housed in specialized taste papillae found in a stereotyped pattern on the surface of the tongue. Each bud, regardless of its location, is a collection of ∼100 cells that belong to at least five different functional classes, which transduce sweet, bitter, salt, sour and umami (the taste of glutamate) signals. Taste receptor cells harbor functional similarities to neurons but, like epithelial cells, are rapidly and continuously renewed throughout adult life. Here, I review recent advances in our understanding of how the pattern of taste buds is established in embryos and discuss the cellular and molecular mechanisms governing taste cell turnover. I also highlight how these findings aid our understanding of how and why many cancer therapies result in taste dysfunction.

ß-Catenin signaling regulates temporally discrete phases of anterior taste bud development.

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of ß-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of ß-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of ß-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of ß-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, ß-catenin activation a day later within Shh(+) placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of ß-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of ß-catenin within Shh(+) precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by ß-catenin.

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

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

Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation.

Effect of Radiation on Sucrose Detection Thresholds of Mice.

Radiotherapy is one of the most common treatments for head and neck cancers, with an almost obligate side effect of altered taste (Conger AD. 1973. Loss and recovery of taste acuity in patients irradiated to the oral cavity. Radiat Res. 53:338-347.). In mice, targeted irradiation of the head and neck causes transient repression of proliferation of basal epithelial cells responsible for taste cell replacement, leading to a temporary depletion of taste sensory cells within taste buds, including Type II taste cells involved in detection of sweet stimuli (Nguyen HM, Reyland ME, Barlow LA. 2012. Mechanisms of taste bud cell loss after head and neck irradiation. J Neurosci. 32:3474-3484.). These findings suggest that irradiation may elevate sucrose detection thresholds, peaking at 7 days postirradiation when loss of Type II cells is greatest. To test this hypothesis, sucrose detection thresholds (concentration detected in 50% of presentations) were measured in mice for 15 days after treatment of: 1) irradiation while anesthetized, 2) anesthetic alone, or 3) saline. Mice were trained to distinguish water from several concentrations of sucrose. Mice were irradiated with one 8 Gy dose (RADSOURCE-2000 X-ray Irradiator) to the nose and mouth while under 2,2,2-tribromethanol anesthesia (Avertin). Unexpectedly, mice given anesthesia showed a small elevation in sucrose thresholds compared to saline-injected mice, but irradiated mice show significantly elevated sucrose thresholds compared to either control group, an effect that peaked at 6-8 days postirradiation. The timing of loss and recovery of sucrose sensitivity generally coincides with the reported maximal reduction and recovery of Type II taste cells (Nguyen HM, Reyland ME, Barlow LA. 2012. Mechanisms of taste bud cell loss after head and neck irradiation. J Neurosci. 32:3474-3484.). Thus, even a single dose of irradiation can significantly alter detection of carbohydrates, an important consideration for patients undergoing radiotherapy.

Wnt-beta-catenin signaling initiates taste papilla development.

Fungiform taste papillae form a regular array on the dorsal tongue. Taste buds arise from papilla epithelium and, unusually for epithelial derivatives, synapse with neurons, release neurotransmitters and generate receptor and action potentials. Despite the importance of taste as one of our five senses, genetic analyses of taste papilla and bud development are lacking. We demonstrate that Wnt-beta-catenin signaling is activated in developing fungiform placodes and taste bud cells. A dominant stabilizing mutation of epithelial beta-catenin causes massive overproduction of enlarged fungiform papillae and taste buds. Likewise, genetic deletion of epithelial beta-catenin or inhibition of Wnt-beta-catenin signaling by ectopic dickkopf1 (Dkk1) blocks initiation of fungiform papilla morphogenesis. Ectopic papillae are innervated in the stabilizing beta-catenin mutant, whereas ectopic Dkk1 causes absence of lingual epithelial innervation. Thus, Wnt-beta-catenin signaling is critical for fungiform papilla and taste bud development. Altered regulation of this pathway may underlie evolutionary changes in taste papilla patterning.

Developing a sense of taste.

Taste buds are found in a distributed array on the tongue surface, and are innervated by cranial nerves that convey taste information to the brain. For nearly a century, taste buds were thought to be induced by nerves late in embryonic development. However, this view has shifted dramatically. A host of studies now indicate that taste bud development is initiated and proceeds via processes that are nerve-independent, occur long before birth, and governed by cellular and molecular mechanisms intrinsic to the developing tongue. Here we review the state of our understanding of the molecular and cellular regulation of taste bud development, incorporating important new data obtained through the use of two powerful genetic systems, mouse and zebrafish.

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

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

FGF signaling regulates the number of posterior taste papillae by controlling progenitor field size.

The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1(-/-);Spry2(-/-) embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.

Mechanisms of taste bud cell loss after head and neck irradiation.

Taste loss in human patients following radiotherapy for head and neck cancer is a common and significant problem, but the cellular mechanisms underlying this loss are not understood. Taste stimuli are transduced by receptor cells within taste buds, and like epidermal cells, taste cells are regularly replaced throughout adult life. This renewal relies on progenitor cells adjacent to taste buds, which continually supply new cells to each bud. Here we treated adult mice with a single 8 Gy dose of x-ray irradiation to the head and neck, and analyzed taste epithelium at 1-21 d postirradiation (dpi). We found irradiation targets the taste progenitor cells, which undergo cell cycle arrest (1-3 dpi) and apoptosis (within 1 dpi). Taste progenitors resume proliferation at 5-7 dpi, with the proportion of cells in S and M phase exceeding control levels at 5-6 and 6 dpi, respectively, suggesting that proliferation is accelerated and/or synchronized following radiation damage. Using 5-bromo-2-deoxyuridine birthdating to identify newborn cells, we found that the decreased proliferation following irradiation reduces the influx of cells at 1-2 dpi, while the robust proliferation detected at 6 dpi accelerates entry of new cells into taste buds. In contrast, the number of differentiated taste cells was not significantly reduced until 7 dpi. These data suggest a model where continued natural taste cell death, paired with temporary interruption of cell replacement, underlies taste loss after irradiation.

Epibranchial placode-derived neurons produce BDNF required for early sensory neuron development.

In mice, BDNF provided by the developing taste epithelium is required for gustatory neuron survival following target innervation. However, we find that expression of BDNF, as detected by BDNF-driven ß-galactosidase, begins in the cranial ganglia before its expression in the central (hindbrain) or peripheral (taste papillae) targets of these sensory neurons, and before gustatory ganglion cells innervate either target. To test early BDNF function, we examined the ganglia of bdnf null mice before target innervation, and found that while initial neuron survival is unaltered, early neuron development is disrupted. In addition, fate mapping analysis in mice demonstrates that murine cranial ganglia arise from two embryonic populations, i.e., epibranchial placodes and neural crest, as has been described for these ganglia in non-mammalian vertebrates. Only placodal neurons produce BDNF, however, which indicates that prior to innervation, early ganglionic BDNF produced by placode-derived cells promotes gustatory neuron development.

The sense of taste: Development, regeneration, and dysfunction.

Gustation or the sense of taste is a primary sense, which functions as a gatekeeper for substances that enter the body. Animals, including humans, ingest foods that contain appetitive taste stimuli, including those that have sweet, moderately salty and umami (glutamate) components, and tend to avoid bitter-tasting items, as many bitter compounds are toxic. Taste is mediated by clusters of heterogeneous taste receptors cells (TRCs) organized as taste buds on the tongue, and these convey taste information from the oral cavity to higher order brain centers via the gustatory sensory neurons of the seventh and ninth cranial ganglia. One remarkable aspect of taste is that taste perception is mostly uninterrupted throughout life yet TRCs within buds are constantly renewed; every 1-2 months all taste cells have been steadily replaced. In the past decades we have learned a substantial amount about the cellular and molecular regulation of taste bud cell renewal, and how taste buds are initially established during embryogenesis. Here I review more recent findings pertaining to taste development and regeneration, as well as discuss potential mechanisms underlying taste dysfunction that often occurs with disease or its treatment. This article is categorized under: Infectious Diseases > Stem Cells and Development Cancer > Stem Cells and Development Neurological Diseases > Stem Cells and Development.

Taste bud cells of adult mice are responsive to Wnt/ß-catenin signaling: implications for the renewal of mature taste cells.

Wnt/ß-catenin signaling initiates taste papilla development in mouse embryos, however, its involvement in taste cell turnover in adult mice has not been explored. Here we used the BATGAL reporter mouse model, which carries an engineered allele in which the LacZ gene is expressed in the presence of activated ß-catenin, to determine the responsiveness of adult taste bud cells to canonical Wnt signaling. Double immunostaining with markers of differentiated taste cells revealed that a subset of Type I, II, and III taste cells express ß-galactosidase. Using in situ hybridization, we showed that ß-catenin activates the transcription of the LacZ gene mainly in intragemmal basal cells that are immature taste cells, identified by their expression of Sonic Hedgehog (Shh). Finally, we showed that ß-catenin activity is significantly reduced in taste buds of 25-week-old mice compared with 10-week-old animals. Our data suggest that Wnt/ß-catenin signaling may influence taste cell turnover by regulating cell differentiation. Reduced canonical Wnt signaling in older mice could explain in part the loss of taste sensitivity with aging, implicating a possible deficiency in the rate of taste cell renewal. More investigations are now necessary to understand if and how Wnt signaling regulates adult taste cell turnover.

Cellular Diversity and Regeneration in Taste Buds.

Taste buds are the sensory end organs for gustation, mediating sensations of salty, sour, bitter, sweet and umami as well as other possible modalities, e.g. fat and kokumi. Understanding of the structure and function of these sensory organs has increased greatly in the last decades with advances in ultrastructural methods, molecular genetics, and in vitro models. This review will focus on the cellular constituents of taste buds, and molecular regulation of taste bud cell renewal and differentiation.