Ultrastructural study of the precursor to fungiform papillae prior to the arrival of sensory nerves in the fetal rat.

The structure of precursors to fungiform papillae without taste buds, prior to the arrival of sensory nerve fibers at the papillae, was examined in the fetal rat on embryonic day 13 (E13) and 16 (E16) by light and transmission electron microscopy in an attempt to clarify the mechanism of morphogenesis of these papillae. At E13, a row of rudiments of fungiform papillae was arranged along both sides of the median sulcus of the lingual dorsal surface, and each row consisted of about 10 rudiments. There was no apparent direct contact between papillae rudiments and sensory nerves at this time. Bilaterally towards the lateral side of the tongue, adjacent to these first rudiments of fungiform papillae, a series of cord-like invaginations of the dorsal epithelium of the tongue into the underlying connective tissue, representing additional papillary primordia parallel to the first row, was observed. The basal end of each invagination was enlarged as a round bulge, indented at its tip by a mound of fibroblasts protruding into the bulge. At E16 there was still no apparent direct contact between rudiments of fungiform papillae and sensory nerves. Each rudiment apically contained a spherical core of aggregating cells, which consisted of a dense assembly of large, oval cells unlike those in other areas of the lingual dorsal epithelium. The differentiation of these aggregated cells was unclear. The basal lamina was clearly recognizable between the epithelium of the rudiment of fungiform papillae and the underlying connective tissue. Spherical structures, which appeared to be sections of the cord-like invaginations of the lingual epithelium that appeared on E13, were observed within the connective tissue separated from the dorsal lingual epithelium. Transverse sections of such structures revealed four concentric layers of cells: a central core, an inner shell, an outer shell, and a layer of large cells. Bundles of fibers were arranged in the central core, and the diameters of bundles varied somewhat depending on the depth of the primordia within the connective tissue and their distance from the median sulcus. Ultrastructural features of cells in the outer shell differed significantly in rudiments close to the lingual epithelium as compared to those in deeper areas of connective tissue. Around the outer shell there was a large-cell layer consisting of one to three layers of radially elongated, oval cells that contained many variously sized, electron-dense, round granules. Large numbers of fibroblasts formed dense aggregates around each spherical rudiment, and were separated by the basal lamina from the large-cell epithelial layer. Progressing from deep-lying levels of the rudiments of the papillae to levels close to the lingual surface epithelium, the central core, inner shell, and outer shell gradually disappeared from the invaginated papillary cords.     

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.     

Early morphogenesis of ciliated cells in human oral cavity

Ciliated cells were found in the epithelium of the oral cavity of human embryos and fetuses starting from the seventh week of prenatal development. At the early stages of prenatal development (until the 13th week), cells with cilia cover most of the dorsal surface of the tongue and the soft palate, whereas they are found only near the gland ducts in the circumvallate and foliate lingual papillae after 17 weeks of development. The ultrastructure of the axoneme of cilia corresponds to the structure of motile cilia and is represented by nine microtubule doublets that surround the central pair of microtubule singlets. An immunohistochemical study performed on weeks 10–12 of development identified nerve endings associated with the ciliated cells. Until the 14th week of development, the cytoplasm of ciliated cells is immunopositive for NSE. The spatial distribution of ciliated cells in the tongue epithelium until the 13th week of development is not related to the morphogenesis of lingual papillae, and their role in the human oral cavity during the first trimester of pregnancy is unclear and requires further study.     

Do Unique Proteins Exist in Taste Buds?

Proteins in papillae on the bovine tongue were analyzed by semi-micro, polyacrylamide gel electrophoresis. All the proteins in the papillae with taste buds were observed to be common to proteins in the surrounding epithelium without taste buds. The protein band which was reported to form a weak complex with compounds called sweet by man was also found in all parts of the tongue epithelium. The receptor molecules for chemical stimuli may be distributed in all the cells of the tongue epithelium or the content of receptor molecules in taste bud papillae may be extremely low.     

Stereo architecture of the interface of the epithelial cell layer and connective tissue core of the foliate papilla in the rabbit tongue.

The three-dimensional relationship between the epithelial cell layer and the underlying connective tissue core (CTC) of the foliate papilla of the rabbit tongue was studied by scanning electron microscopy after removal of the epithelial cell layer. The foliate papillae were fixed in Karnovsky's fixative, and the epithelial cell layers were exposed to long-term hydrochloric acid treatment (3.5 N HCl for 2-3 weeks at room temperature). The foliate papillae consisted of ridges and grooves located on the posterolateral margin of the tongue. They appeared as linear projections or ridges of lingual mucosa roughly perpendicular to the longitudinal axis of the tongue. These projections or ridges were parallel to one another and separated by grooves. After removal of the epithelium, two kinds of CTC folds appeared: one was the septal fold of CTC which runs in the central portion under each linear projection or ridge, and the other consisted of two sheets of groove side folds of CTC which run along both sides of the former and face the groove side epithelium. It was revealed that there are two sheets of septal epithelial processes, and each of them fits in between each septal fold and groove side fold of CTC. Numerous taste buds were located in the groove side epithelia, and their pores faced the surface of the groove. On the hollow surfaces that appeared on the surface of the groove side fold of CTC after removal of the epithelial cells with taste buds, nerve-terminal-like structures were encountered. Some openings of the ducts of small lingual glands were arranged linearly on the underside of the basal portion of each groove side epithelium.     

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.     

Fine structure of the dorsal lingual epithelium of the Japanese monkey Macaca fuscata fuscata.

Filiform papillae, which were densely distributed all over the dorsal surface of the lingual body, were crown-shaped, with a central, circular area that sloped in the anterior direction and several branches that surrounded it in a semicircle from the back of the central area. Dome-shaped, fungiform papillae were scattered among these filiform papillae. At the posterior end of the lingual body, there were four circumvallate papillae. Prominent microridges and elevated intercellular borders were widely distributed in the central area of the filiform papillae and the interpapillar region. On the surface of the branches of the filiform papillae, microridges were rarely seen. On the surface of the fungiform papillae, indistinct microridges were observed. Histologically, the dorsal lingual epithelium revealed three different regions: the epithelium on the anterior side of the filiform papillae, the epithelium on the posterior side of the filiform papillae and the interpapillar epithelium. Whereas the basal and suprabasal cells are similar throughout, differences characterize the intermediate and surface layers. Keratohyalin granules appear predominantly in the intermediate layer in the epithelium on the anterior side of filiform papillae. In the epithelium on the posterior side of the filiform papillae, no keratohyalin granules occur and, instead, tonofibrils are prominent. The cells become significantly flattened. In the interpapillar epithelium, no keratohyalin granules are visible, and the tonofilaments occupy almost the entire cytoplasm of most cells in the intermediate and surface layers. The cells are larger in volume in these layers.     

Morphogenesis of rat lingual filiform papillae.

The morphogenesis of filiform papillae on rat tongue was investigated with the electron microscope. Tongue rudiments were first seen on the 12th day of gestation. At 15-17 days, dermal papillae had formed and were arranged in hexagonal array on the dorsal lingual surface. Capping each dermal papilla was a two-layered epithelium that protruded slightly above the lingual surface, thus forming the early filiform papilla. In the next stage of development, at 18-19 days of gestation, the epithelium lining the papilla had differentiated into two cell populations, one producing hard keratin, the other producing soft keratin. Some of the keratinized epithelial cells assumed a position at an acute angle to the tongue surface and extended deep into the epithelium. In the next stage, 20-21 days, a cleft appeared within these angularly oriented cells. This resulted in the division of the epithelium into keraatin-lined individual filiform papillae. Finally, the individual papillae increased in size to the adult form.     

Changes in neurotrophin-3 messenger RNA expression patterns in the prenatal rat tongue suggest guidance of developing somatosensory nerves to their final targets

While brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) has been localized in the developing gustatory epithelium, little information is available about neurotrophin-3 (NT-3) mRNA expression pattern in the prenatal developing gustatory and lingual epithelium. In the present study, using in situ hybridization histochemistry, we report on NT-3 mRNA expression in the tongue of rats. At embryonic day (E) 13–17, NT-3 mRNA was expressed subepithelially in the periphery of the developing tongue, as well as among developing muscle. At E19, there was a shift in the expression of NT-3 mRNA. It was then expressed in the surface epithelium of the developing tongue in the developing filiform papillae and, in higher concentrations, in top-surface and fringe epithelium of the developing circumvallate papillae, and top- and lateral-surface epithelium of the developing fungiform papillae. NT-3 mRNA expression in areas rich in somatosensory innervation of the tongue, as well as its specific expression in defined regions compared with BDNF, and the decreased labeling noted from prenatal and early postnatal animals to adults indicate a specific role for NT-3 in the development of lingual somatosensory innervation, as well as for maintenance of this innervation.     

Morphology, and muscle- and papilla-volume ratios, of the tongue of Laudakia stellio (Agamidae, Squamata): a histological and stereological study

We examined the histological structure of the tongue of Laudakia stellio, the starred agama lizard (Agamidae, Squamata), under light microscopy. We also investigated the muscle and papilla volume ratios, with volumes of each aspect of interest estimated according to the Cavalieri method. The macroscopically short, thick and muscle-rich front tip of the tongue of L. stellio does not show any bifurcation, and under light microscopy, the oval-shaped papilla-free front tip was seen to be covered by keratinized stratified epithelium. The dorsal and ventral parts were different, with the former partially covered by keratinized stratified epithelium and rich in secretory glands and secretory cells. The ventral part, which contained keratinized stratified cells, had a flat surface with no papillae. The dorsal surface of the anterior and posterior parts contained fungiform papillae, with the apical parts of these papillae containing minimal keratin; the interpapillar space was covered by keratin-free squamous stratified epithelium. The middle section of the tongue contained cylindrical-type papillae, with serous and mucous secretory glands and ducts at their base. Finally, the frontal and middle parts of the ventral and dorsal surfaces did not contain any taste buds, although there were some in the hind part of the dorsal surface. As morphometric estimates of volumes of the muscles and papillae, the mean volume ratios (relative to total tongue volume)+/-standard deviation were 0.66+/-0.03 and 0.33+/-0.03, with mean coefficients of error of 0.02 and 0.03, respectively.     

Cytokeratin expression in human tongue epithelium.

The epithelium of the human tongue shows diverse morphological variations from one site to another and even within the epithelium of the same papilla. This complexity has led to confusion regarding tongue epithelium as being orthokeratinized, parakeratinized, or nonkeratinized. Cytokeratins have been shown to characterize different epithelia. The present paper describes cytokeratin expression by adult tongue epithelia and relates their distribution to morphology. Six healthy human tongue specimens were obtained after plastic surgery and cytokeratin expression was investigated immunohistochemically, using a panel of 15 antibodies for cytoskeletal proteins, and biochemically using two-dimensional gel electrophoresis. The results showed that the ventral and lateral surfaces of the tongue are related to the nonkeratinizing stratified squamous epithelia, esophageal type, whereas the dorsal surface showed mixed expression of cytokeratins. In the tip of filiform and on the surface of fungiform papillae, cytokeratins of terminal differentiation are expressed as skin type; and in the rest of the papillae as well as in interpapillary areas, the epithelium expresses esophageal type cytokeratins. Certain simple epithelial cytokeratins were found in taste buds. Cytokeratin 19 was also detected in the basal cell layer of all esophageal type epithelia in the tongue. The present results provide basis for studies on the biological events in epithelial differentiation during development and in pathology.     

Differential localization of distinct keratin mRNA-species in mouse tongue epithelium by in situ hybridization with specific cDNA probes

The tongue of the adult mouse is covered by a multilayered squamous epithelium which is continuous on the ventral surface, however interrupted on the dorsal surface by many filiform and few fungiform papillae. The filiform papillae themselves are subdivided into an anterior and posterior unit exhibiting different forms of keratinization. Thus, the entire epithelium shows a pronounced morphological diversity of well recognizable tissue units. We have used a highly sensitive in situ hybridization technique to investigate the differential expression of keratin mRNAs in the tongue epithelium. The hybridization probes used were cDNA restriction fragments complementary to the most specific 3'-regions of any given keratin mRNA. We could show that independent of the morphologically different tongue regions, all basal cells uniformly express the mRNA of a type I 52-kD keratin, typical also for basal cells of the epidermis. Immediately above the homogenous basal layer a vertically oriented specialization of the keratin expression occurs within the morphological tissue units. Thus the dorsal interpapillary and ventral epithelium express the mRNAs of a type II 57-kD and a type I 47-kD keratin pair. In contrast, in the anterior unit of the filiform papillae, only the 47-kD mRNA is present, indicating that this keratin may be coexpressed in tongue epithelium with different type II partners. In suprabasal cells of both, the fungiform papillae and the posterior unit of the filiform papillae, a mRNA of a type I 59-kD keratin could be detected; however, its type II 67-kD epidermal counterpart seems not to be present in these cells. Most surprisingly, in distinct cells of both types of papillae, a type I 50-kD keratin mRNA could be localized which usually is associated with epidermal hyperproliferation. In conclusion, the in situ hybridization technique applied has been proved to be a powerful method for detailed studies of differentiation processes, especially in morphologically complex epithelia.     

Development of mechanical papillae of the tongue in the domestic goose (<Emphasis Type="Italic">Anser anser f. domestica</Emphasis>) during the embryonic period

Three types of mechanical papillae, i.e., conical, filiform, and hair-like papillae, are present on the tongue in the domestic goose. Within conical papillae, we distinguish three categories: large and small conical papillae on the body and conical papillae on the lingual prominence. The arrangement of mechanical papillae on the tongue in Anseriformes is connected functionally with different feeding mechanisms such as grazing and filter-feeding. The present work aims to determine whether morphology of three types of mechanical papillae in goose at the time of hatching is the same as in an adult bird and if the tongue is prepared to fulfill feeding function. Our results revealed that the primordia of the large conical papillae start to develop during the differentiation stage. The primordia of the small conical papillae and conical papillae of the lingual papillae start to develop during the growth stage. At the end of the growth stage, only large conical papillae, three pairs of small conical papillae, and conical papillae of the lingual prominence have similar arrangement as in an adult bird. The shape and arrangement of the remaining small conical papillae probably will be changed after hatching. During embryonic period, the filiform papillae and hair-like papillae are not formed. The embryonic epithelium that covered the mechanical papillae undergoes transformation leading to the formation of multilayered epithelium. During prehatching stage, epithelium becomes orthokeratinized epithelium. In conclusion, the tongue of the domestic goose after hatching is well prepared only for grazing. The filtration of food from water is limited due to the lack of filiform papillae.     

Morphological evidence of basal keratinocyte migration during the re-epithelialization process

The regeneration of wounded stratified epithelium is accomplished via the migration of keratinocytes from the margins of the wound. However, the process of keratinocyte migration on the wound surface and the role of epithelial stem cells during re-epithelialization remain to be elucidated. Therefore, we administered BrdU to embryonic mice and generated epithelial defects on the buccal mucosa of these mice at two weeks after birth, using CO₂ laser irradiation, with which we removed the entire thickness of the epithelium. In the unwounded epithelium, cytokeratin 14, p63, and BrdU were localized within the basal layer of the epithelium, but the majority of cells within the regenerated epithelium were immunopositive for these proteins. PCNA-negative and BrdU-positive basal keratinocytes, which evidence a slow cell cycle, were localized solely within the basal layer of the unwound epithelium facing the tips of dermal papillae. After laser irradiation, these basal keratinocytes facing the tips of the papillae evidenced positive immunoreactivity for PCNA, in addition to BrdU. These results indicate that epithelial stem cells of oral mucosa may be localized in the basal layer of the epithelium facing the tips of dermal papillae, and may migrate laterally with other basal keratinocytes in response to external stimuli. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Scanning-electron-microscopic study of the dorsal lingual surface of the squirrel monkey

The structure of the lingual papillae and the ultrastructure of the surface of the lingual dorsal epithelial cells of squirrel monkeys were observed by scanning electron microscopy. Filiform papillae were distributed over the entire dorsal surface of the tongue, except for the radix zone. Fungiform papillae were scattered among these filiform papillae. In the middle of the posterior end of the lingual body, a single vallate papilla was located. Higher magnification of the lingual dorsal epithelium revealed that prominent microridges and elevated intercellular borders occurred widely in the basofrontal area of the filiform papillae, interpapillar area and lingual radix zone. On the surface of the upper part of the filiform papillae, fine pits and hollows were observed. Indistinct microridges were distributed over the surface of the fungiform papillae.     

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.     

Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.