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     

Biogenic Amines in the Taste Organ

The presence and content of biogenic amines in taste disk-bearingfungiform papillae of the frog, Rana esculenta, the only availablemodel of an isolated taste organ, were verified by means ofHPLC. Fungiform papillae were found to contain measurable amountsof serotonin, epinephrine and norepinephrine. The amounts ofserotonin and epinephrine were significantly higher in fungiformpapillae than in the general mucosa of the tongue. Moreover,the epinephrine content of fungiform papillae was found to differacross the tongue, in accordance with previous physiologicalstudies showing an inhomogeneous response of different tongueregions to taste stimuli. Ultrastructural and histochemicalinvestigations confirmed the presence of catecholamine and serotonin.The latter was found to be contained mainly in the basal cellsof the frog taste disk. These results extend previous qualitativedata on the presence of biogenic amines in taste chemoreceptors.Chem. Senses 20: 329–335, 1995.     

Scanning electron microscopy of developing papillae on the tongue of human embryos and fetuses

The appearance and differentiation of papillae on dorsal andlateral surfaces of human embryonic and fetal tongues, at variousdevelopmental ages, were studied by scanning electron microscopy.Formaldehyde and phosphate buffer fixation provided satisfactorypreservation. At 8–9 weeks, the anterior two-thirds ofthe tongue showed no obvious signs of papillae. In contrast,just anterior to the sulcus terminalis rounded elevations wereseen, suggesting initial signs of circumvallate papillae. At10–13 weeks, the distribution and shape of elevationson the anterior two-thirds of the tongue indicated the beginningof fungiform papillae. Openings located on the dorsal surfaceof many of these fungiforms contained an amorphous central structureprojecting out of the papilla. First signs of foliate papillaewere seen at 10 weeks. At 15–18 weeks, fungiform and filiformpapillae were recognized, although sometimes their borders wereobscured by scaling epithelial cells. At 23–26 weeks,all papillae exhibited their adult form. ^*Presented, in part, at the VIth International Symposium onOlfaction and Taste, Gif-sur-Yvette, Paris, France, 15–17thJuly, 1977.     

Quantification of fungiform papillae and taste pores in living human subjects

A method developed to quantify taste buds in living human subjectsto study the relationship between taste sensitivity and tastebud distribution was used to count the taste buds in 10 humansubjects; fungiform papillae were mapped in 12 subjects. Tastebuds were identified by staining taste pores with methyleneblue, and images of the papillae and their taste pores wereobtained with videomicroscopy and an image processor. Fungiformpapillae showed a 3.3-fold range in density, from 22.1 to 73.6papillae/cm² with an average of 41.1 ± 16.8/cm² (s.d.,n = 2). There was a 14-fold range in taste pore density, from36 to 511 pores/cm² among subjects, with an average of 193 ±133/cm² (s.d., n = 10). Fungiform papillae contained from 0to 22 taste pores, with an average per subject of 3.75 ±1.4 taste pores/papilla (s.d., n = 10). We hypothesize thatsome differences in human taste sensitivity may be related tothese variations in taste bud density.     

Labeling afferent nerves in the tongue by peripheral transganglionic transport of HRP and WGA-HRP in the rat

Peripheral transganglionic transport of horseradish pcroxidase(HRP) and wheat germ agglutinin–horseradish peroxidaseconjugate (WGA–HRP) was used to label afferent fibersin the taste buds and lingual epithelium of the rat. Microinjectionsof the tracer were made in the brain stem central projectionarea of the afferent nerves to the tongue. Optimal labelingof nerve endings in the tongue was obtained when 2 µlof 20% HRP was injected into the brain stem and postinjectionsurvival times of 24–36 h were used. The distributionof single nerves was studied by using this tracing procedurein combination with strategic transections of the various afferentnerves supplying the tongue. Labeled nerve fibers from the combinedchorda tympani–lingual nerve were found in the epitheliumand in taste buds in the fungiform and anterior foliate papillaeof the anterior 3/4 of the tongue. Labeled nerve fibers in theepithelium of the anterior 2/3 of the tongue but none in tastebuds were found when the lingual nerve alone was studied, althoughnumerous perigeminal fibers were found. The glossopharyngealnerve was found to innervate die posterior 1/4 of the tongueepithelium including the taste buds of the circumvallate papillae.The glossopharyngeal nerve on one side was found to innervatethe taste buds on both sides of the midline. The results showthat this tracing procedure can be a useful supplement to othermethods for studying afferent nerves in the tongue.     

Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning: fungiform papillae double in number and form in novel locations in dorsal lingual epithelium

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.     

Bone morphogenetic proteins and noggin: inhibiting and inducing fungiform taste papilla development

Fungiform papillae are epithelial specializations that develop in a linear pattern on the anterior mammalian tongue and differentiate to eventually contain taste buds. Little is known about morphogenetic and pattern regulation of these crucial taste organs. We used embryonic rat tongue, organ cultures to test roles for bone morphogenetic proteins, BMP2, 4 and 7, and antagonists noggin and follistatin, in development of papillae from a stage before morphological initiation (E13) or from a stage after the pre-papilla placodes have formed (E14). BMPs and noggin proteins become progressively restricted to papilla locations during tongue development. In E13 cultures, exogenous BMPs or noggin induce increased numbers of fungiform papillae, in a concentration-dependent manner, compared to standard tongue cultures; BMPs, but not noggin, lead to a decreased tongue size at this stage. In E14 cultures, however, exogenous BMP2, 4 or 7 each inhibits papilla formation so that there is a decrease in papilla number. Noggin substantially increases number of papillae in E14 cultures. Using beads for a highly localized protein delivery, papillae are inhibited in the surround of BMP-soaked beads and induced in large clusters around noggin-soaked beads. Follistatin, presented in culture medium or by bead, does not alter papilla formation or number. In all fungiform papillae that form under various culture conditions, the molecular marker, sonic hedgehog, is within each papilla. However, the BMP inhibitory effect on papillae is not prevented by disrupting sonic hedgehog signaling through addition of cyclopamine to cultures. BMPs and noggin alter cell proliferation in tongue epithelium in opposite ways, demonstrated with Ki67 immunostaining. We propose that the BMPs and noggin, colocalized within papilla placodes and the fungiform papillae per se, have opposing inhibitory and activating or inducing roles in papilla development in linear patterns. We present a model for these effects.     

Epithelial overexpression of BDNF or NT4 disrupts targeting of taste neurons that innervate the anterior tongue

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are essential for the survival of geniculate ganglion neurons, which provide the sensory afferents for taste buds of the anterior tongue and palate. To determine how these target-derived growth factors regulate gustatory development, the taste system was examined in transgenic mice that overexpress BDNF (BDNF-OE) or NT4 (NT4-OE) in basal epithelial cells of the tongue. Overexpression of BDNF or NT4 caused a 93 and 140% increase, respectively, in the number of geniculate ganglion neurons. Surprisingly, both transgenic lines had severe reduction in fungiform papillae and taste bud number, primarily in the dorsal midregion and ventral tip of the tongue. No alterations were observed in taste buds of circumvallate or incisal papillae. Fungiform papillae were initially present on tongues of newborn BDNF-OE animals, but many were small, poorly innervated, and lost postnatally. To explain the loss of nerve innervation to fungiform papillae, the facial nerve of developing animals was labeled with the lipophilic tracer DiI. In contrast to control mice, in which taste neurons innervated only fungiform papillae, taste neurons in BDNF-OE and NT4-OE mice innervated few fungiform papillae. Instead, some fibers approached but did not penetrate the epithelium and aberrant innervation to filiform papillae was observed. In addition, some papillae that formed in transgenic mice had two taste buds (instead of one) and were frequently arranged in clusters of two or three papillae. These results indicate that target-derived BDNF and NT4 are not only survival factors for geniculate ganglion neurons, but also have important roles in regulating the development and spatial patterning of fungiform papilla and targeting of taste neurons to these sensory structures.     

Morphometry and cellular dynamics of denervated fungiform taste buds in the hamster

For most species and gustatory papillae denervation resultsin a virtual disappearance of taste buds. This is not the casefor hamster fungiform papillae, which contain taste buds thatsurvive denervation. To characterize these taste buds, in thisstudy, counts and measurements were made of all buds on theanterior 3 mm of the hamster tongue at 36 or 91 days after resectingthe chorda/lingual nerve. Taste bud numbers were, at both timeperiods, unaffected by denervation. However, bud dimensionswere affected with denervated buds 25–30% smaller thancontrol ones. Counts of taste bud cells indicated that decreasesin bud size may result from shrinkage, but not a loss of cells.Tritiated thymidine autoradiography was used to evaluate whetherdenervation influences the mitotic activity or the migratorypattern of bud cells. For every animal, the average number oflabelled cells per bud was slightly lower on the denervatedthan the control side of the tongue. However, when labelledcell positions were evaluated at 0.25, 3 and 6 days after thymidine,the distances from the sides of the bud increased at increasingtimes after injection for both the innervated and the denervatedbuds. Stem cells were located laterally or basally in the bud.Labelled cells that migrated into the centers of the buds werefew and seen only at 6 days post-injection time in both controland experimental buds. The moderate effects of denervation ontaste bud sizes and mitotic activities may indicate a generalizedatrophy. Remarkably intact were taste bud numbers and the migratorypatterns of cells, features of anterior tongue taste buds inthe hamster that are relatively invulnerable to resection ofthe chorda /lingual nerve.     

Developmental time course of peripheral cross‐modal sensory interaction of the trigeminal and gustatory systems

Few sensory modalities appear to engage in cross‐modal interactions within the peripheral nervous system, making the integrated relationship between the peripheral gustatory and trigeminal systems an ideal model for investigating cross‐sensory support. The present study examined taste system anatomy following unilateral transection of the trigeminal lingual nerve (LX) while leaving the gustatory chorda tympani intact. At 10, 25, or 65 days of age, rats underwent LX with outcomes assessed following various survival times. Fungiform papillae were classified by morphological feature using surface analysis. Taste bud volumes were calculated from histological sections of the anterior tongue. Differences in papillae morphology were evident by 2 days post‐transection of P10 rats and by 8 days post in P25 rats. When transected at P65, animals never exhibited statistically significant morphological changes. After LX at P10, fewer taste buds were present on the transected side following 16 and 24 days survival time and remaining taste buds were smaller than on the intact side. In P25 and P65 animals, taste bud volumes were reduced on the denervated side by 8 and 16 days postsurgery, respectively. By 50 days post‐transection, taste buds of P10 animals had not recovered in size; however, all observed changes in papillae morphology and taste buds subsided in P25 and P65 rats. Results indicate that LX impacts taste receptor cells and alters epithelial morphology of fungiform papillae, particularly during early development. These findings highlight dual roles for the lingual nerve in the maintenance of both gustatory and non‐gustatory tissues on the anterior tongue. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 626–641, 2016     

Lgr5 Identifies Progenitor Cells Capable of Taste Bud Regeneration after Injury

Taste buds are composed of a variety of taste receptor cell types that develop from tongue epithelium and are regularly replenished under normal homeostatic conditions as well as after injury. The characteristics of cells that give rise to regenerating taste buds are poorly understood. Recent studies have suggested that Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) identifies taste bud stem cells that contribute to homeostatic regeneration in adult circumvallate and foliate taste papillae, which are located in the posterior region of the tongue. Taste papillae in the adult anterior region of the tongue do not express Lgr5. Here, we confirm and extend these studies by demonstrating that Lgr5 cells give rise to both anterior and posterior taste buds during development, and are capable of regenerating posterior taste buds after injury induced by glossopharyngeal nerve transection.     

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.     

The relationship between fungiform papillae density and detection threshold for sucrose in the young males

The aim of this study was to investigate the relationship offungiform papillae density with taste detection thresholds forsucrose of young male adults. One hundred and eighty two subjectsaged 18–23 years (mean age: 21.9 ± 1.2 years) wereincluded. The densities of fungiform papillae were recordedwith the aid of the digital camera, and the taste detectionthresholds for sucrose were detected using a modified forced-choicetriangle test. The mean density of papillae within all 170 statisticparticipants was 92.43 ± 2.64/cm², for the 6-mm-diameterstained section of the tongue tip. The average detection thresholdwas 10.83 ± 0.24 mmol/l, and the highest and lowest detectionthresholds were 19.88 ± 1.31 and 5.85 ± 0.43 mmol/l,respectively. Also, an inverse correlation between the fungiformpapillae density and the detection threshold was observed.     

Spacing patterns on tongue surface-gustatory papilla

The dorsal surface of the mammalian tongue is covered with four kinds of papillae, fungiform, circumvallate, foliate and filiform papillae. With the exception of the filiform papillae, these types of papillae contain taste buds and are known as the gustatory papillae. The gustatory papillae are distributed over the tongue surface in a distinct spatial pattern. The circumvallate and foliate papillae are positioned in the central and lateral regions respectively and the fungiform papillae are distributed on the anterior part of the tongue in a stereotyped array. The patterned distribution and developmental processes of the fungiform papillae indicate some similarity between the fungiform papillae and the other epithelial appendages, including the teeth, feathers and hair. This is because 1) prior to the morphological changes, the signaling molecules are expressed in the fungiform papillae forming area with a stereotyped pattern; 2) the morphogenesis of the fungiform papillae showed specific structures in early development, such as epithelial thickening and mesenchymal condensation and 3) the fungiform papillae develop through reciprocal interactions between the epithelium and mesenchymal tissue. These results led us to examine whether or not the early organogenesis of the fungiform papillae is a good model system for understanding both the spacing pattern and the epithelial-mesenchymal interaction during embryogenesis.     

A scanning electron microscopic study of the dorsal surface of the human tongue

To study the dorsal surface of the human tongue using a scanning electron microscopy (SEM), tissue specimens were taken from the anterior part of the tongues of 15 individuals aged from 21- to 28-years-old. The formalin-fixed samples were processed routinely for SEM. With SEM the surface of the normal tongue mucosa was shown to be rather evenly covered by filiform papillae, with some fungiform papillae scattered among them. Filiform papillae consisted of two parts: the body and hairs. The mucosal surface of the body was smooth; the squamous epithelial cells were polygonal, and their boundaries were prominent. On the surface of the superficial epithelial cells were parallel or branching microplicae. Each filiform papilla had 6-10 hairs, which were scaled and covered by an extensive plaque of microorganism. The upper surface of the fungiform papillae was smooth; only a few desquamating cells were seen. The superficial cells had a pitted appearance and cell boundaries overlapped. Taste pores, up to 3 pores in a single papilla, were found on the upper surface. Desquamation was more pronounced on the base of the fungiform papillae than on the upper surface. In almost all fungiform papillae some hairs protruded from the base. Parallel microplicae were found on the surface of the superficial cells of the base. The structure and function of the human tongue, as well as the microplicae of its superficial cells, are compared to those of various species of animals.     

Taste Disks are Induced in the Lingual Epithelium of Salamanders during Metamorphosis

Morphological changes of oral cavity during metamorphosis withspecial reference to the taste organ were examined in Ezo salamanders(Hynobius retardatus) and axolotls (Ambystoma mexicanum), andcompared with those in bullfrogs (Rana catesbeiana). The non-distensibletongue of salamanders changed the structure progressively duringmetamorphosis: a small area of the rostrum protruded and developedcaudally with recession of the flat area of the tongue. Theprotrusion that developed on the tongue had numerous papillae,as seen in the frog tongue. The apical region of the papillaeoccasionally had a cell mass similar to the taste disk of frogs(termed a taste disk-like cell mass). On the flat area of thetongue, the barrel-shaped taste buds of larval salamanders weretransformed into taste buds with a wider receptor area. Thebarrel-shaped taste buds decreased progressively during metamorphosis,while taste disk-like cell masses increased. Neuronal labelingwith an antibody to neuron-specific enolase and fluorescentcarbocyanine dye showed that the taste disk-like cell massesin metamorphosed salamanders were innervated by the glossopharyngealnerve (nerve IX). Nerve IX responded to taste stimulation aswell as mechanical stimulation applied to the rostral tongue.During metamorphosis the salamanders undergo transformationand rearrangement of taste organs on the tongue possibly asan adaptation to the terrestrial environment. Chem. Senses 22:535–545, 1997.     

Chorda tympani nerve transection at different developmental ages produces differential effects on taste bud volume and papillae morphology in the rat

Chorda tympani nerve transection (CTX) results in morphological changes to fungiform papillae and associated taste buds. When transection occurs during neonatal development in the rat, the effects on fungiform taste bud and papillae structure are markedly more severe than observed following a comparable surgery in the adult rat. The present study examined the potential "sensitive period" for morphological modifications to tongue epithelium following CTX. Rats received unilateral transection at 65, 30, 25, 20, 15, 10, or 5 days of age. With each descending age at the time of transection, the effects on the structural integrity of fungiform papillae were more severe. Significant losses in total number of taste buds and filiform-like papillae were observed when transection occurred 5-30 days of age. Significant reduction in the number of taste pores was indicated at every age of transection. Another group of rats received chorda tympani transection at 10, 25, or 65 days of age to determine if the time course of taste bud degeneration differed depending on the age of the rat at the time of transection. Taste bud volumes differed significantly from intact sides of the tongue at 2, 8, and 50 days post-transection after CTX at 65 days of age. Volume measurements did not differ 2 days post-transection after CTX at 10 or 25 days of age, but were significantly reduced at the other time points. Findings demonstrate a transitional period throughout development wherein fungiform papillae are highly dependent upon the chorda tympani for maintenance of morphological integrity.     

Light- and Scanning-Electron-Microscopic Analyses of the Plica glossoepiglottica lateralis of the neonate and adultPan troglodytes troglodytes — Blumenbach (1799)

A unique type of papillae is evident in the plicae glossoepiglotticae laterales of the tongue ofPan troglodytes troglodytes. Areas of occurrence show flat lobes of varying length with elongated miniature processes on their free tips. The function of these papillae is presumably the convection of liquid food into the pharynx (along with the lateral folds). Furthermore, it is assumed that the papillae are sensory organs which relay taste, temperature, pain, and pressure, similar to the papillae filiformes of the tongue of other mammalia (Kunze 1969). Free nerve fibres and nerve endings were found in both the epithelium and the connective-tissue. Nerve structures resembling the “Organs of Meissner” were also discovered in the subepithelial connective-tissue. Goblet cells are found in the surface layers in the epithelium of the papillae of the newborn. These are absent in the papillae of the adultPan troglodytes troglodytes. The secretion of the goblet cells functions as a mechanical and chemical protectant for the papillae.     

Visualizing Taste Papillae In Vivo with Scanning Electron Microscopy of a High Resolution Cast

A method using polyvinylsiloxane (PVS), a high-resolution dentalimpression material, to obtain negative images of lingual surfacesis described. Epoxy-resin tongue replicas made from these impressionswere examined with scanning electron microscopy (SEM). Thismethod has been developed to visualize structural details ofthe tongue surface of living human beings and laboratory animals.The utility of the method is demonstrated with hamster tongues,which have well-defined fungiform papillae with single tastepores, and human tongues, which have more variable surface structures.Replicas made from PVS impressions of tongues of living hamsterswere compared with the same tongues after fixation. The replicascontained much of the detail present in fixed tongues. WithSEM, it was possible to identify individual fungiform papillae,which contained depressions with the size and the location ofhamster taste pores. Individual papillae could also be recognizedin human-tongue replicas, but taste pores could not be identifiedwith certainty. These replicas provide permanent, three-dimensionalrecords of tongue topography that could be used to documentchanges due to trauma, disease and aging.