Laryngeal chemosensory clusters

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

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

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

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

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

Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in pig taste papillae.

Immunoreactivity to neuron-specific enolase (NSE), a specific neuronal marker, and calcitonin gene-related peptide (CGRP) was localized in lingual taste papillae in the pigs. Sequential staining for NSE and CGRP by an elution technique allowed the identification of neuronal subpopulations. NSE-staining revealed a large neuronal network within the subepithelial layer of all taste papillae. NSE-positive fibers then penetrated the epithelium as isolated fibers, primarily in the foliate and circumvallate papillae, or as brush-shaped units formed by a multitude of fibers, especially in the fungiform papillae and in the apical epithelium of the circumvallate papilla. Taste buds of any type of taste papillae were found to express a dense subgemmal/intragemmal NSE-positive neuronal network. CGRP-positive nerve fibers were numerous in the subepithelial layer of all three types of taste papillae. In the foliate and circumvallate papillae, these fibers penetrated the epithelium to form extragemmal and intragemmal fibers, while in the fungiforms, they concentrated almost exclusively in the taste buds as intragemmal nerve fibers. Intragemmal NSE- and CGRP-positive fiber populations were not readily distinguishable by typical neural swellings as previously observed in the rat. The NSE-positive neuronal extragemmal brushes never expressed any CGRP-like immunoreactivity. Even more surprising, fungiform taste buds, whether richly innervated by or devoid of NSE-positive intragemmal fibers, always harboured numerous intragemmal CGRP-positive fibers. Consequently, NSE is not a general neuronal marker in porcine taste papillae. Our observations also suggest that subgemmal/intragemmal NSE-positive fibers are actively involved in synaptogenesis within taste buds. NSE-positive taste bud cells were found in all three types of taste papillae. CGRP-positive taste bud cells were never observed.     

Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity

Taste represents a major form of sensory input in the animal kingdom. In mammals, taste perception begins with the recognition of tastant molecules by unknown membrane receptors localized on the apical surface of receptor cells of the tongue and palate epithelium. We report the cloning and characterization of two novel seven-transmembrane domain proteins expressed in topographically distinct subpopulations of taste receptor cells and taste buds. These proteins are specifically localized to the taste pore and are members of a new group of G protein-coupled receptors distantly related to putative mammalian pheromone receptors. We propose that these genes encode taste receptors.     

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

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

Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.     

Taste placodes are primary targets of geniculate but not trigeminal sensory axons in mouse developing tongue

Tongue embryonic taste buds begin to differentiate before the onset of gustatory papilla formation in murine. In light of this previous finding, we sought to reexamine the developing sensory innervation as it extends toward the lingual epithelium between E 11.5 and 14.5. Nerve tracings with fluorescent lipophilic dyes followed by confocal microscope examination were used to study the terminal branching of chorda tympani and lingual nerves. At E11.5, we confirmed that the chorda tympani nerve provided for most of the nerve branching in the tongue swellings. At E12.5, we show that the lingual nerve contribution to the overall innervation of the lingual swellings increased to the extent that its ramifications matched those of the chorda tympani nerve. At E13.0, the chorda tympani nerve terminal arborizations appeared more complex than those of the lingual nerve. While the chorda tympani nerve terminal branching appeared close to the lingual epithelium that of the trigeminal nerve remained rather confined to the subepithelial mesenchymal tissue. At E13.5, chorda tympani nerve terminals projected specifically to an ordered set of loci on the tongue dorsum corresponding to the epithelial placodes. In contrast, the lingual nerve terminals remained subepithelial with no branches directed towards the placodes. At E14.5, chorda tympani nerve filopodia first entered the apical epithelium of the developing fungiform papilla. The results suggest that there may be no significant delay between the differentiation of embryonic taste buds and their initial innervation.     

Innervation of developing human taste buds. An immunohistochemical study

 Morphological changes in developing human gustatory papillae during the 6th to the 23rd postovulatory week have been studied. The general innervation pattern of taste papillae and taste bud primordia was revealed immunohistochemically using antibodies against protein gene product 9.5 (PGP9.5), neurofilament H (NFH), neurofilament L (NFL), neurone-specific enolase (NSE), and tubulin. The autonomic and somatosensory nerve supply has been investigated using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), the neuronal form of nitric oxide synthase (n-NOS), and, enzyme histochemically, NADPH-diaphorase. Nerve fibers approach the basal membrane of the lingual epithelium around the 7th postovulatory week and invade the epithelium of papilla-like structures at the 8th week, but some also penetrate the basal membrane of the non-papillary epithelium. They are in close contact with slender epithelial cells that are considered to be the taste bud’s progenitor cells. Early human taste buds situated at the anterior part of the tongue do not necessarily require a dermal (later fungiform) papilla. The NADPH-diaphorase reaction revealed positive results in dermal nerve fibers, but the immunohistochemical reaction against n-NOS was negative. Immunohistochemical detection of neuropeptides and vasoactive substances rendered negative results for developmental stages of 7–18 postovulatory weeks. By the 18th week, only SP was detected in dermal papillae, but not in the vicinity of taste buds’ primordia. Thus, autonomic and somatosensory nerves seem not to play a key role in formation and maintenance of early human taste buds. Accepted: 31 July 1997     

Mosaic analysis of the embryonic origin of taste buds

The embryonic origins of taste receptor cells have not beenestablished experimentally. Although related receptor cells(e.g. hair cells of the inner ear, lateral line receptors) areknown to arise from neurogenic ectoderm (e.g. neural crest orplacodes), taste buds are described as arising from local epithelialcells. Also unknown is whether or not each taste bud is a cloneof cells, i.e. arising from a single progenitor. To addressthese problems, mosaic and chimeric analyses of lingual epitheliumand taste buds have been undertaken. This paper describes thetheory of chimeric and mosaic cell lineage analyses, the advantagesand disadvantages, and the preliminary results obtained fromthe examination of the taste buds and lingual epithelium of:1) mosaic Xenopus, 2) chimeric mice and 3) X-inactivation mosaicmice.     

Cytokeratin 14 is expressed in immature cells in rat taste buds

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2′-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2–3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP₃R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.     

Fibroblast and epidermal growth factors modulate proliferation and neural cell adhesion molecule expression in epithelial cells derived from the adult mouse tongue

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.     

Changes of the lingual epithelium in Ambystoma mexicanum

Changes in the lingual epithelium during ontogenesis and after induced metamorphosis in Ambystoma mexicanum are described as observed by light microscopy and scanning electron microscopy. The epithelium of the tongue is always multilayered in the larva as well as in the adult. It consists of a stratum germinativum with little differentiated basal cells and a stratum superficiale (superficial layer) with specialized superficial cells and goblet cells. Usually, there are more than two layers because of a stratum intermedium consisting of replacement cells. The apical cell membrane of the superficial cells is perforated by fine pores. Its most typical feature are microridges. Maturing superficial cells possess microvilli. Goblet cells occur in early larvae primarily in the centre of the tongue. They spread throughout the dorsal face of the tongue as their numbers increase during ontogenesis. The small apices of the goblet cells are intercalated in the wedges between the superficial cells. Leydig cells are not found on the larval tongue but on that of adults. Due to metamorphosis, the epithelium of the tongue changes. It is furrowed in its anterior part. The furrows house the openings of the lingual glands. The surface is further modulated by ridges which are densely coated by microvilli and which bear the taste buds. The villi of the tongue which lack extrusion pores show cilia and microvilli but lack microridges. The Leydig cells disappear during metamorphosis. In addition to the two types of goblet cells found in different regions of the glandular tubules, goblet cells occur in the caudal part. They secrete directly into the cavity of the mouth. The posterior part is characterised by a dense coat of cilia.     

Building sensory receptors on the tongue

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

The role of sodium and potassium transport pathways in taste transduction: an overview

Recent studies have shown that taste sensations are mediatedby a multiplicity of transduction mechanisms. The taste of saltis produced in part by the entry of Na⁺ through channels inthe apical taste cell membrane. Na⁺ transport also mediatessweet perception in some species. The taste of KCI requiresentry of K⁺ through apical potassium channels. The productionof second messengers such as cAMP by taste stimuli or tastemodifiers can depolarize taste cells by inducing an enzymaticcascade that alters K⁺ permeability.     

Effect of sensory and sympathetic denervation of the frog tongue on the catecholamine containing cells of the taste buds]

The structure of catecholamine-containing dumb-bell shaped cells of the taste buds was studied by luminescent microscopy in the epithelial layer of the frog's tongue (Rana temporaria). On the unilateral section of the lingual nerve, a maintained adrenergic innervation of vessels and of the epithelium was observed, a decreased number of dumb-bell shaped cells in the taste bud, and their significant enlargement, and increased cathecholamine luminescence. With desympathization, no adrenergic nerves were observed on the vessels and the epithelium of the tongue. The size of the taste buds in desympathized cells of the tongue is sharply decreased and their number is increased. There is a tendency to grouping of the dumbbell shaped cells into 3--4 taste buds in one fungiform papillina. The experiments with sensory and sympathetic denervation of the frog tongue distinctly showed the trophic action of sensory and sympathetic nerves on the taste organ of the frog.     

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

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

Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

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

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