The Formation of Endoderm-Derived Taste Sensory Organs Requires a Pax9-Dependent Expansion of Embryonic Taste Bud Progenitor Cells

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.     

Sonic hedgehog exerts distinct, stage-specific effects on tongue and taste papilla development

Taste papillae are ectodermal specializations that serve to house and distribute the taste buds and their renewing cell populations in specific locations on the tongue. We previously showed that Sonic hedgehog (Shh) has a major role in regulating the number and spatial pattern of fungiform taste papillae on embryonic rat tongue, during a specific period of papilla formation from the prepapilla placode. Now we have immunolocalized the Shh protein and the Patched receptor protein (Ptc), and have tested potential roles for Shh in formation of the tongue, emergence of papilla placodes, development of papilla number and size, and maintenance of papillae after morphogenesis is advanced. Cultures of entire embryonic mandible or tongues from gestational days 12 to 18 [gestational or embryonic days (E)12-E18] were used, in which tongues and papillae develop with native spatial, temporal, and molecular characteristics. The Shh signaling pathway was disrupted with addition of cyclopamine, jervine, or the 5E1 blocking antibody. Shh and Ptc proteins are diffuse in prelingual tissue and early tongue swellings, and are progressively restricted to papilla placodes and then to regions of developing papillae. Ptc encircles the dense Shh immunoproduct in papillae at various stages. When the Shh signal is disrupted in cultures of E12 mandible, tongue formation is completely prevented. At later stages of tongue culture initiation, Shh signal disruption alters development of tongue shape (E13) and results in a repatterned fungiform papilla distribution that does not respect normally papilla-free tongue regions (E13-E14). Only a few hours of Shh signal disruption can irreversibly alter number and location of fungiform papillae on anterior tongue and elicit papilla formation on the intermolar eminence. However, once papillae are well formed (E16-E18), Shh apparently does not have a clear role in papilla maintenance, nor does the tongue retain competency to add fungiform papillae in atypical locations. Our data not only provide evidence for inductive and morphogenetic roles for Shh in tongue and fungiform papilla formation, but also suggest that Shh functions to maintain the interpapilla space and papilla-free lingual regions. We propose a model for Shh function at high concentration to form and maintain papillae and, at low concentration, to activate between-papilla genes that maintain a papilla-free epithelium.     

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.     

Shh and ROCK1 modulate the dynamic epithelial morphogenesis in circumvallate papilla development

In rodents, a circumvallate papilla (CVP) develops with dynamic changes in epithelial morphogenesis during early tongue development. Molecular and cellular studies of CVP development revealed that there would be two different mechanisms in the apex and the trench wall forming regions with specific expression patterns of Wnt11 and Shh. Molecular interactions were examined using in vitro organ culture with over-expression of Shh, important signalling molecules and various inhibitors revealed that there are two significant different mechanisms in CVP formation by Wnt11 and Shh expressions. Wnt, a well known key molecule to initiate taste papillae, would govern Rho activation and cytoskeleton formation in the apex epithelium of CVP. In contrast, Shh regulates the cell proliferation to differentiate taste buds and to invaginate the epithelium for development of von Ebner's gland (VEG). Based on these results, we suggest that these different molecular signalling cascades of Wnt11 and Shh would play crucial roles in specific morphogenesis and pattern formation of CVP during early mouse embryo development.     

Taste bud density in circumvallate and fungiform papillae of the bovine tongue

Taste bud quantitation may provide useful parameters for interspecies comparisons of the gustatory system. The present study is a morphometric analysis of bovine taste papillae. Circumvallate and fungiform papillae from six bovine tongues were serially sectioned and, following staining, analyzed. Circumvallate papillae were found to have a mean volume of 3.66 +/- 2.82 mm3, a mean number of taste buds per papilla of 445 +/- 279, and a mean taste bud density of 155 +/- 112 buds/mm3. Values for lateral fungiform papillae for the same three parameters were 0.384 +/- 0.184 mm3, 13.2 +/- 13.4, and 40.8 +/- 46.6 buds/mm3, respectively. Values for dorsal fungiform papillae were 0.438 +/- 0.246 mm3, 4.39 +/- 4.78, and 14.0 +/- 17.1 buds/mm3, respectively. Circumvallate papillae were found to have a significantly greater volume, number of taste buds per papilla, and taste bud density than either type of fungiform papilla. These data should serve as background for biochemical, endocrinological, or neurological studies involving the bovine tongue.     

Blood vascular architecture of the rat lingual papillae with special reference to their relations to the connective tissue papillae and surface structures: a light and scanning electron microscope study

Subepithelial blood vessels of the rat lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat, four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla; a basket- or petal-like network in the fungiform papilla; a ring-like network in the foliate papilla, and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds.     

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.     

哺乳动物舌面味蕾的发育

根据近年来有关大鼠、小鼠味觉发育方面的大量研究,对哺乳动物味蕾(taste buds)发育的情况进行了综述和讨论.哺乳动物舌面上的味蕾分布在菌状乳头(fungiform papillae,FF)、叶状乳头(foliate papillae,FL)、轮廓状乳头(circumvallate papillae,CV)之中,味蕾细胞(taste bud cells)不断地进行着周期性的更新,味蕾的形态、数量和功能随动物随年龄而变化.有关味孔头的研究表明,味乳头(gustatory papillae)在味蕾形成和维持味蕾存在及正常发育方面有着独特的功能.味乳头和味蕾的发育过程与细胞信号分子(signaling molecules)、味觉神经(gustatory nerve fibers)等许多因素有着密切的关系,其中有些作用机理至今尚无定论.     

Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.     

Disruption of sonic hedgehog signaling alters growth and patterning of lingual taste papillae

Taste buds on the anterior part of the tongue develop in conjunction with epithelial-mesenchymal specializations in the form of gustatory (taste) papillae. Sonic hedgehog (Shh) and Bone Morphogenetic Protein 4 (BMP4) are expressed in developing taste papillae, but the roles of these signaling molecules in specification of taste bud progenitors and in papillary morphogenesis are unclear. We show here that BMP4 is not expressed in the early tongue, but is precisely coexpressed with Shh in papillary placodes, which serve as a signaling center for both gustatory and papillary development. To elucidate the role of Shh, we used an in vitro model of mouse fungiform papillary development to determine the effects of two functional inhibitors of Shh signaling: anti-Shh (5E1) antibody and cyclopamine. Cultured E11.5 tongue explants express Shh and BMP4(LacZ) in a pattern similar to that of intact embryos, localizing to developing papillary placodes after 2 days in culture. Tongues cultured with 5E1 antibody continue to express these genes in papillary patterns but develop more papillae that are larger and closer together than in controls. Tongues cultured with cyclopamine have a dose-dependent expansion of Shh and BMP4(LacZ) expression domains. Both antibody-treated and cyclopamine-treated tongue explants also are smaller than controls. Taken together, these results suggest that, although Shh is not involved in the initial specification of papillary placodes, Shh does play two key roles during pmcry development: (1) as a morphogen that directs cells toward a nonpapillary fate, and (2) as a mitogen, causing expansion of the interplacodal epithelium and underlying mesenchyme.     

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.     

Time course of morphological alterations of fungiform papillae and taste buds following chorda tympani transection in neonatal rats

The time course of structural changes in fungiform papillae was analyzed in rats that received unilateral chorda tympani nerve transection at 10 days of age. Morphological differences between intact and denervated sides of the tongue were first observed at 8 days postsection, with an increase in the number of fungiform papillae that did not have a pore. In addition, the first papilla with a filiform-like appearance was noted on the denervated side at 8 days postsectioning. By 11 days after surgery, the total number of papillae and the number of papillae with a pore were significantly lower on the transected side of the tongue as compared to the intact side. At 50 days postsection, there was an average of 70.5 fungiform papillae on the intact side and a mean of only 20.8 fungiform papillae the denervated side. Of those few remaining papillae on the cut side, an average of 13.5 papillae were categorized as filiform-like, while no filiform-like papillae occurred on the intact side. Significant reduction in taste bud volume was noted at 4 days posttransection and further decrements in taste bud volume were noted at 8 and 30 days postsection. Electron microscopy of the lingual branch of the trigeminal nerve from adult rats that received neonatal chorda tympani transection showed normal numbers of both myelinated and unmyelinated fibers. Thus, in addition to the well-characterized dependence of taste bud maintenance on the chorda tympani nerve, the present study shows an additional role of the chorda tympani nerve in papilla maintenance during early postnatal development.     

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.     

Regeneration ability of fungiform papillae and taste-buds in rats

We have earlier shown that the taste-bud-bearing fungiform papillaeform a stable pattern on the tongue of rats. In this study theeffect of removal of the fungiform papillae in rats was investigated.The fungiform papillae on a 10 x 5-mm area on one side of thetongue were removed after mapping of both sides under an operatingmicroscope. Serial sections of five rat tongues within 1 dayof biopsy showed that all but one papilla were gone. After 4,6 and 12 months an average of seven papillae with taste-budswere found in the operated area, compared to 20, 26 and 23 inthe controls. Comparison of tongue maps before and after theseperiods showed that papillae had not migrated from areas outsidethe area of the biopsies. To test the assumption that the extentof biopsy determined the amount of regeneration, only the upperpart of the papillae with their taste buds were removed in 15rats. Complete regeneration of papillae and taste buds was obtainedwithin 14 days. The function of the regenerated taste buds wastested by bilateral recording from the chorda tympani propernerves. No difference in response amplitudes was observed betweenthe sides. When, however, the whole papilla including its basewas removed, neither the papilla nor the taste-bud regenerated.The results show that the ability of the fungiform papilla andthe taste-bud to regenerate after removal of the papilla isrelated to the extent of the biopsy. If the entire papilla includingits base is removed, it will not regenerate. If only the upperpart is removed, complete regeneration of both papilla and itstaste-bud will occur.     

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.     

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.     

Pharmaco-histochemical studies on a specific monoamine in the gustatory epithelia of the rabbit

Summary The foliate, vallate and fungiform papillae of the rabbit's tongue were studied fluorescence-histochemically under normal and experimental conditions. In normal animals a yellow fluorescence suggesting the presence of a serotonin-like monoamine was demonstrated only in taste bud cells of the foliate papilla, though its intensity was very weak. The fluorescence disappeared completely following reserpine treatment, while it was significantly enhanced by the treatment with nialamide. The fluorescence of taste bud cells could be clearly distinguished from that of catecholamines by the treatment with -MMT followed by nialamide. When 5-HTP, 5-HT and 5,6-DHT were administered separately, each of these drugs was selectively taken up in taste bud cells of the foliate and vallate papillae, but no fluorescent cells were observed in the fungiform papilla.From the present results, it seems reasonable to conclude that the fluorigenic amine of taste bud cells may be 5-HT (serotonin), or at least an indoleamine derivative. Also, it is suggested that the taste bud of the vallate papilla contains a cell type which can potentially synthesize a biogenic amine in situ, or is actually synthesizing it in a very small amount just like in the case of the taste bud of the foliate one.