Apoptosis in mouse taste buds after denervation

Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing. Received: 20 November 1995 / Accepted: 15 May 1996     

Expression of cyclin-dependent kinase inhibitors in taste buds of mouse and hamster

Taste buds are specialized epithelial cell clusters in the oral squamous cell epithelium. Although taste buds have been reported to renew rapidly, the mechanism of cell cycle control in these specialized structures remains unresolved. To clarify the cell cycle status and role of cyclin-dependent kinase inhibitors (CDKI) for cell cycle control in the taste buds, we analyzed cell proliferation activity using bromodeoxyuridine (BrdU) and Ki-67 immunostainings and the expression of the Cip/Kip family of CDKI (p21Cip1, p27Kip1, and p57Kip2) in the circumvallate papillae of mouse and hamster. BrdU-positive cells were detected in the basal layer of the oral epithelium. In the taste buds, Ki-67-positive cells were seen in the basal area, with only a very few positive cells in the taste buds. Both p21Cip1 and p27Kip1 positive cells were seen in the suprabasal layer of the non-gustatory oral epithelium. In the taste buds, stronger p27Kip1 staining was detected than in the non-gustatory epithelium. Western blotting analysis revealed that p27Kip1 was abundant in the mucosal tissues from circumvallate papillae. Thus, our study suggests that the taste bud cells except for basal cells are post-mitotic cells and that the cell cycle arrest associated with taste bud cell differentiation could be regulated predominantly by p27Kip1.     

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.     

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.     

Preparation and isolation of a taste bud-derived fraction from bovine circumvallate papillae.

A crude extract from taste buds was prepared from circumvallate papillae of bovine tongues by a procedure consisting of freezing, coring, excision, treatment with hypotonic buffer, nitrogen pressurization and selective homogenization. Examination of taste buds by light and electron microscopy before and after this procedure indicated that some of the contents of the buds, mainly from the more apical portions, was extruded following the procedure; anatomical changes could not be observed in the epithelial tissue immediately surrounding the taste buds.     

Keratin filaments of epithelial and taste-bud cells in the circumvallate papillae of adult and developing mice

Summary Keratin filaments of epithelial- and taste-bud cells in the circumvallate papillae of adult and developing mice were studied by immunocytochemistry using monoclonal antikeratin antibodies (PKK2 and PKK3) and by conventional electron microscopy. Elongated cells (type-I,-II, and-III cells) of the taste buds were stained by PKK3 antibody, which reacts with 45-kdalton keratin, whereas basal cells of the taste buds and surrounding epithelial cells showed negative staining with PKK3. Such PKK3-reactive cells occurred at 0 day after birth, when taste-buds first appeared in the dorsal surface epithelium of the papillae. Thus 45-kdalton keratin seems to be an excellent immunocytochemical marker for identifying taste-bud cells. Epithelial cells in all layers of the trench wall and basal layer cells of the dorsal surface contained densely aggregated bundles of keratin filaments that reacted with PKK2 antibody, but not with PKK3. In contrast, taste-bud cells and spinous and granular layer cells of the dorsal surface possessed loose aggregated bundles of filaments that reacted with PKK3, but not with PKK2. These results suggest that the aggregation and distribution pattern of keratin filaments may reflect differences in the keratin subtypes that comprise these filaments.     

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.     

Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue

The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at - 70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.     

RENEWAL OF TASTE BUD CELLS IN RAT CIRCUMVALLATE PAPILLAE

The life span of taste bud cells in rat circumvallate papillae was measured by autoradiography after labeling them with a pulse of [³H]thymidine. Specimens of circumvallate papillae were taken daily 1·5-18·5 days after the isotope was administered; thereafter, specimens were taken on alternate days until 25·5 days. For each time interval, the number of labeled cell nuclei was counted in 200-450 taste buds and plotted as the ratio of labeled cells/taste bud v. time after injection of [³H]TdR. In all, 6958 taste buds were counted. The total number of labeled cells (dark plus light) per taste bud reached peaks at 6·5, 13·5 and 20·5 days. The curve for the number of labeled dark cells/bud had essentially the same shape as that for total cells. The number of labeled light cells/bud reached a modest peak at 6·5 days and slowly declined to a plateau for the remainder of the experiment. The data show that an average of 2 days elapsed after injection before labeled dark cells entered the bud and they spent an average of 7 days in the non-proliferating taste bud compartment; thus, the life span of the dark cell was 9 days. The life span of the light cell was difficult to estimate quantitatively, but this cell type was labeled at a much slower rate than dark cells and is assumed to have a significantly longer tenure in the taste bud.     

Co-expression pattern of Shh with Prox1 and that of Nkx2.2 with Mash1 in mouse taste bud

In mammals, taste buds are maintained by continuous turnover of cells, even in adulthood. Cell proliferation and differentiation continue to produce taste cells, which express various genes related to taste reception. We found the co-expression of Sonic hedgehog (Shh) with Prox1 and that of Nkx2.2 with Mash1 in adult mouse taste buds. Whereas Prox1was expressed strongly in cells in the basal region of mouse taste buds where Shh was co-expressed, it was expressed weakly in almost all taste bud cells lacking Shh expression. At 0.5 day after birth, when taste cells have not yet differentiated, the expressions of Shh and Prox1 completely overlapped in the epithelium of circumvallate papillae. Nkx2.2 was observed in cells expressing Mash1, but not in cells expressing genes related to taste reception, such as gustducin and T1R3. Almost all fusiform cells expressing Mash1 co-expressed Nkx2.2, while the majority of round cells expressing Mash1 in the basal region of taste buds lacked Nkx2.2 expression.     

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

Calcitonin gene-related peptide-like and neuron-specific enolase-like immunoreactivity (CGRP-IR and NSE-IR) were surveyed immunohistochemically in the fungi-form, foliate and circumvallate papillae in rats. A dense CGRP-IR network (subgemmal and extragemmal) in the taste papillae is linked to the presence of taste buds, even though CGRP-IR fibers are rarely present in the taste buds. Three typical fiber populations were detected with these two markers. (a) A population of coarse NSE-IR intragemmal fibers characterized by thick neural swellings, never expressing CGRP-immunoreactivity. (b) A population of thin varicose intragemmal NSE/CGRP-IR fibers. (c) A population of subgemmal and extragemmal NSE-/CGRP-IR fibers that partly penetrated the epithelium. The common distribution of CGRP-IR and NSE-IR fibers at the base of taste buds, their differential distribution and morphology within taste buds, added to their restricted nature (gustatory or somatosensory) suggest that a population of CGRP-IR fibers undergoes a target-induced inhibition of its CGRP phenotype while entering the taste buds. The combined use of NSE and CGRP allowed a better characterization of nerve fibers within and between all three types of taste papillae. NSE was also a very good marker for a subtype of taste bud cells in the foliate and in the circumvallate papillae, but no such cells could be observed in the fungiform papillae.     

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.     

Comparative lectin histochemistry on taste buds in foliate, circumvallate and fungiform papillae of the rabbit tongue.

Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungi-form taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.     

Scanning electron microscopic and histochemical studies of foliate papillae in the rabbit, rat and mouse

The foliate papillae of the rabbit, rat and mouse were studied by scanning electron microscopy and histochemistry. The papillae consisted of folds and grooves located on the posterolateral margin of the tongue in front of the circumvallate papillae. The numbers of folds and taste buds varied among the three animals species. Scanning electron microscopy showed that in longitudinal sections the taste buds were oval in shape and their pores were surrounded by microvilli. The reaction product of alkaline phosphatase could only be demonstrated in the superficial epithelium of the rabbit as well as in the mouse foliate papillae, but it also diffused into the taste buds in the rat. The intensity and distribution of the reactions of adenosine triphosphatase, acetylcholinesterase and butyrylcholinesterase were identical to those reported by other investigators in spite of differences in animal species and histochemical techniques employed.