Linda A. Barlow

Wnt-β-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-β-catenin signaling is activated in developing fungiform placodes and taste bud cells. A dominant stabilizing mutation of epithelial β-catenin causes massive overproduction of enlarged fungiform papillae and taste buds. Likewise, genetic deletion of epithelial β-catenin or inhibition of Wnt-β-catenin signaling by ectopic dickkopf1 (Dkk1) blocks initiation of fungiform papilla morphogenesis. Ectopic papillae are innervated in the stabilizing β-catenin mutant, whereas ectopic Dkk1 causes absence of lingual epithelial innervation. Thus, Wnt-β-catenin signaling is critical for fungiform papilla and taste bud development. Altered regulation of this pathway may underlie evolutionary changes in taste papilla patterning.

Fate mapping of mammalian embryonic taste bud progenitors

Mammalian taste buds have properties of both epithelial and neuronal cells, and are thus developmentally intriguing. Taste buds differentiate at birth within epithelial appendages, termed taste papillae, which arise at mid-gestation as epithelial thickenings or placodes. However, the embryonic relationship between placodes, papillae and adult taste buds has not been defined. Here, using an inducible Cre-lox fate mapping approach with the ShhcreERT2 mouse line, we demonstrate that Shh-expressing embryonic taste placodes are taste bud progenitors, which give rise to at least two different adult taste cell types, but do not contribute to taste papillae. Strikingly, placodally descendant taste cells disappear early in adult life. As placodally derived taste cells are lost, we used Wnt1Cre mice to show that the neural crest does not supply cells to taste buds, either embryonically or postnatally, thus ruling out a mesenchymal contribution to taste buds. Finally, using Bdnf null mice, which lose neurons that innervate taste buds, we demonstrate that Shh-expressing taste bud progenitors are specified and produce differentiated taste cells normally, in the absence of gustatory nerve contact. This resolution of a direct relationship between embryonic taste placodes with adult taste buds, which is independent of mesenchymal contribution and nerve contact, allows us to better define the early development of this important sensory system. These studies further suggest that mammalian taste bud development is very distinct from that of other epithelial appendages.

Notch‐associated gene expression in embryonic and adult taste papillae and taste buds suggests a role in taste cell lineage decisions

The Notch signaling pathway is involved in cell fate decisions during development. To explore the role of this signaling cascade in the taste system, we investigated the expression patterns of Notch signaling genes in fetal and adult mouse tongues using in situ hybridization. Three of the four murine Notch receptors, their ligands, Delta‐like 1 (Dll‐1), Jagged1, and Jagged2, as well as three transcription factors, Hes1, Hes6, and Mash1, are expressed in the embryonic taste epithelium. Expression is first detected in the circumvallate papilla at embryonic day E14.5, when Notch1, Jagged1, and Jagged2 are expressed broadly in the papilla and general lingual epithelium. In contrast, Mash1 and Hes6 are restricted to only a few epithelial cells in the apical region of the developing papilla. By E18.5, many of the genes now exhibit a bimodal expression pattern in the papillary epithelium: apically and dorsally they are expressed in sparse clusters of cells, while more ventrally expression typically occurs throughout the lower regions of the trenches. The extent of papilla innervation was compared with Mash1 and Hes6 expression. At E14.5, when Hes6 and Mash1 are already expressed in small numbers of epithelial cells, PGP9.5 immunoreactive fibers have not yet invaded the epithelium, consistent with the specification of taste bud primordia prior to nerve contact. All of the genes examined (except Notch2) are also expressed in subsets of cells within circumvallate taste buds in adult mice, although Notch1 is restricted to basal cells adjacent to taste buds. The onset of embryonic Notch associated gene expression after the morphological differentiation of the circumvallate papilla argues that this signaling cascade may specify taste receptor cell lineages within an already specified taste papilla. Similarly, Notch gene expression in adult taste buds suggests continued roles in cell lineage determination and cell turnover. J. Comp. Neurol. 464:49–61, 2003.

Amphibians provide new insights into taste-bud development

Until recently, the predominant model of taste-bud development was one of neural induction: ingrowing sensory fibers were thought to induce taste-bud differentiation late in embryonic development. Recent experimental studies, however, show that the development of taste buds is independent of their innervation. In amphibian embryos, the ability to generate taste buds is an intrinsic feature of the oropharyngeal epithelium long before the region becomes innervated. These studies indicate that patterning of the oropharyngeal epithelium occurs during gastrulation, and suggest that taste buds or their progenitors play the dominant role in the development of their own innervation.

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.

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.

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 ShhCreERT2 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. Developmental Dynamics 243:1286–1297, 2014.

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.

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. Summary: This Review article discusses how taste buds are established during development, highlighting the cellular and molecular mechanisms governing taste cell formation and turnover.

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

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