The mechanism of chemical delivery to the vomeronasal organs in squamate reptiles: a comparative morphological approach

Vomeronasal chemoreception, an important chemical sense in squamate reptiles (lizards and snakes), is mediated by paired vomeronasal organs (VNOs), which are only accessible via ducts opening through the palate anteriorly. We comparatively examined the morphology of the oral cavity in lizards with unforked tongues to elucidate the mechanism of stage I delivery (transport of chemical-laden fluid from the tongue tips to the VNO fenestrae) and to test the generality of the Gillingham and Clark (1981. Can J Zool 59:1651-1657) hypothesis (based on derived snakes), which suggests that the sublingual plicae act as the direct conveyors of chemicals to the VNOs. At rest, the foretongue lies within a chamber formed by the sublingual plicae ventrally and the palate dorsally, with little or no space around the anterior foretongue when the mouth is closed. There is a remarkable conformity between the shape of this chamber and the shape of the foretongue. We propose a hydraulic mechanism for stage I chemical transport in squamates: during mouth closure, the compliant tongue is compressed within this cavity and the floor of the mouth is elevated, expressing fluid from the sublingual glands within the plicae. Chemical-laden fluid covering the tongue tips is forced dorsally and posteriorly toward the VNO fenestrae. In effect, the tongue acts as a piston, pressurizing the fluid surrounding the foretongue so that chemical transport to the VNO ducts is effected almost instantaneously. Our findings falsify the Gillingham and Clark (1981. Can J Zool 59:1651-1657) hypothesis for lizards lacking forked, retractile tongues.     

Fine structure of the dorsal lingual epithelium of the little tern, Sterna albifrons Pallas (Aves, Lari).

The dorsal surface of the tongue of the little tern, Sterna albifrons, has a distinctive anterior region for five-sixths of its length and a terminal posterior region. The anterior region observed by scanning electron microscopy is distinguished along its forward half by a median line from which median papillae protrude. The hind half of the anterior region has a median sulcus without papillae. The deciduous epithelium on both sides of the median line and sulcus bears scattered epithelial protrusions. The posterior lingual region has neither median papillae nor deciduous epithelium. So-called giant conical papillae are located in a transverse row between anterior and posterior regions. Delicate microridges adorn the surfaces of all outer epithelial cells in both regions. Examination of the dorsal lingual epithelium by light and electron microscopy provides histologic and cytologic criteria for distinguishing anterior and posterior regions. Basal cells are nearly alike throughout the dorsal epithelium. Intermediate layer cells of the anterior region contain numerous tonofibrils in electron-dense bundles composed of 10 nm tonofilaments. The outer layer is composed of electron-dense, well-keratinized cells, and electron-lucent epithelial protrusions are present on the exposed surface of the outermost cells. Median papillae are composed of typical keratinized cells, which are nearly filled with keratin filaments. Intermediate layer cells in the posterior region of the tongue are nearly filled with unbundled tonofilaments. There is only a very thin outer keratinized layer in this region.     

An ultrastructural study of the dorsal lingual epithelium of the crab-eating frog,Rana cancrivora

The amphibian tongue contains two types of papilla which are believed to function in gustation and in the secretion of salivary fluid. Scanning electron microscopy reveals that columnar, filiform papillae are compactly distributed over nearly the entire dorsal surface of the tongue of the frog, Rana cancrivora, and fungiform papillae are scattered among the filiform papillae. Microridges and microvilli are distributed on the epithelial cell surface of the extensive area of the filiform papillae. Light microscopy shows that the apex of each filiform papilla is composed of stratified columnar and/or cuboidal epithelium and its base is composed of simple columnar epithelium. Transmission electron microscopy reveals that most of the epithelium of the filiform papillae is composed of cells that contain numerous round electron-dense granules 1–3 μm in diameter. Cellular interdigitation is well developed between adjacent cells. On the free-surface of epithelial cells, microridges or microvilli are frequently seen. Between these granular cells, a small number of ciliated cells, mitochondria-rich cells and electron-lucent cells are inserted. In some cases, electron-dense granules are present in the ciliated cells. At higher magnification, the electron-dense granules appear to be covered with patterns of spots and tubules. Overall, the morphology and ultrastructure of the lingual epithelium of the three species of Rana that have been studied are quite similar, but they can be easily distinguished from those of Bufo japonicus. Therefore, it appears that lingual morphology is phylogenetically constrained among members of the predominantly freshwater genus Rana to produce uniformity of papillary structure and this morphology persists in Rana cancrivora despite the distinct saline environment in which it lives. © 1993 Wiley-Liss, Inc.     

Light and scanning microscopy of the tongue and its gustatory organs in the common toad, Bufo bufo (L.).

Unlike frogs and European tree frogs, the common toad Bufo bufo possesses a tongue lacking filiform papillae on its dorsal surface. Instead, the mucosal epithelium forms irregular, high folds, dividing the surface of the tongue into numerous separate compartments. At the bottom of those compartments occur openings of tubular glands and singular ciliated cells. In a strongly distended tongue the folds of mucosa disappear, and the regions of glandular orifices assume a shape of pocket-like recesses. The taste discs with an average diameter of 120 micrograms are localized on the top of markedly shortened fungiform papillae. The superficial layer of taste discs contains a single type of glandular supporting cells, which in part of discs show features suggesting their gradual dezintegration, probably related to the process of cellular turnover in gustatory organs.     

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.     

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.     

Light and transmission electron microscopy of the dorsal lingual epithelium of Pelusios castaneus (Pleurodira, Chelidae) with special respect to its feeding mechanics

The ultrastructure of the dorsal lingual epithelium of the semi-aquatic West African mud turtle, Pelusios castaneus, is described. Our goal is to give additional information to previous studies of this species such as feeding pattern analysis and gross morphology. Tissue specimens were fixed in modified Karnovsky solution followed by osmium tetroxide, embedded in epoxy resin and observed using light and transmission electron microscopy. The dorsal tongue surface is covered with moderate papillae, which are coated by a stratified epithelium overlying a connective tissue core. Two epithelial regions can be differentiated, although differences are not very obvious: the apical area, where granular cells are more abundant than mucus cells, and the lateral area, where cell distribution is opposite. Within the epithelium, different layers are discernable on the basis of the cells' organelles, corresponding with a process of cell maturation and formation of different granules. These results together with data of previous studies of this species show that the ultrastructure of the lingual epithelium is similar to other turtles adapted to semi-aquatic environments; functional and morphological data indicate a generalist, being well but not highly adapted to feeding in an aquatic environment.     

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.     

Morphology of the tongue of Vermilingua (Xenarthra: Pilosa) and evolutionary considerations

The tongue of anteaters (Xenarthra, Pilosa, Vermilingua) is a highly specialized for myrmecophagy. Here, we describe the topography and histology of the tongue, and compare it to that of other xenarthrans and other myrmecophagous eutherian mammals. The tongue of Vermilingua is long and slender, with an apical protuberance, which differs between Myrmecophagidae and Cyclopes didactylus . In the former, the rostral region is conical, and in the latter, it is dorsoventrally compressed, as observed in sloths. The tongue of Vermilingua has filiform and circumvallate papillae on the surface; foliate and fungiform papillae are absent. The filiform papillae of Myrmecophaga tridactyla are simple all over the tongue, differing from Tamandua tetradactyla and Cyclopes didactylus , which present composed filiform papillae in the rostral and middle regions. Histologically, the tongue has a peculiar organization of muscular and neurovascular tissues, differing from the usual mammalian pattern. However, the tongue structure is less divergent in Cyclopes . The presence of two circumvallate papillae is common to the three major clades of Xenarthra (Cingulata, Folivora and Vermilingua). In each group, the tongue may reflect functional features related to myrmecophagous (anteaters and some armadillos), omnivorous (remaining armadillos) and folivorous (sloths) feeding habits. The similarities between the tongues of Vermiligua and other non‐xenarthran eutherian myrmecophagous mammals are somewhat general and, under close inspection, superficial, being an example of different lineages achieving the same morphofunctional adaptations through distinct evolutionary pathways.     

Light and scanning microscopy of the taste organs and vascularization of the tongue of the spotted salamander, Salamandra salamandra (L.)

The mucosa of the spotted salamander tongue and its taste organs were investigated by means of light and scanning electron microscopy. The most striking feature of the salamander tongue is an almost complete lack of papillae which are replaced by long, radially disposed folds with linear arrays of taste organs along their ridges. In respect of morphology, the taste organs of the salamander occupy an intermediate position between the taste buds of Urodela and taste discs of Salientia. Scanning electron microscopic examination of microcorrosion casts of the blood vessels of the tongue has revealed that the structure of subepidermal capillary network reflects the topography of the tongue surface and the distribution of its taste organs. In the core regions of the folds the capillary loops accompanying gustatory receptors empty via their shorter, descending arms into the draining vessels, the initial segments of which retain a course parallel to that of the folds. In the few fungiform papillae the capillary vessels form single loops whose distal ends come to lie in the vicinity of taste discs.     

Scanning electron microscopic study on the structure of the lingual papillae of the Saanen goat

The morphology of lingual papillae of the ten male mature Saanen goats (11 months old, approximately 42 kg in weight and of a known pedigree) was examined by scanning electron microscopy. Tissues were taken from the dorsal and ventral surfaces of the apex, body and root of the tongue, and were prepared accordingly and observed under the scanning electron microscope. On the dorsal and ventro-lateral surfaces of the lingual mucosa, three types of mechanical papillae (filiform, lenticular, and conical) and two types of gustatory papillae (vallate and fungiform) were observed. The structure and density of the filiform papillae differentiated on the anterior, posterior and ventro-lateral aspects of the tongue. Two types of lenticular papillae, both possessing a prominent surrounding papillary groove, were determined. The pyramidal-shaped type I lenticular papilla had a pointed apex while the round-shaped type II lenticular papilla possessed a blunt apex. Certain number of the type I lenticular papillae had double apices. The larger conical papillae were hollow structures, differing structurally from the filiform papillae with their larger size, a tip without projections and lack of the secondary papillae. The vallate papillae were present on both rims of the torus linguae, were encircled by a prominent gustatory furrow which was also surrounded by a thick annular fold. The fungiform papillae were scattered among the filiform papillae in the anterior two-thirds of the dorsal and lateral surfaces, and each of them was highly protected by surrounding filiform papillae, yet encircled by a papillary groove. Our findings indicate that Saanen goat have profuse distribution of papillae on the tongue displaying morphological features characteristic of mechanical function.     

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.     

Comparative morphology of changeable skin papillae in octopus and cuttlefish

A major component of cephalopod adaptive camouflage behavior has rarely been studied: their ability to change the three‐dimensionality of their skin by morphing their malleable dermal papillae. Recent work has established that simple, conical papillae in cuttlefish (Sepia officinalis) function as muscular hydrostats; that is, the muscles that extend a papilla also provide its structural support. We used brightfield and scanning electron microscopy to investigate and compare the functional morphology of nine types of papillae of different shapes, sizes and complexity in six species: S. officinalis small dorsal papillae, Octopus vulgaris small dorsal and ventral eye papillae, Macrotritopus defilippi dorsal eye papillae, Abdopus aculeatus major mantle papillae, O. bimaculoides arm, minor mantle, and dorsal eye papillae, and S. apama face ridge papillae. Most papillae have two sets of muscles responsible for extension: circular dermal erector muscles arranged in a concentric pattern to lift the papilla away from the body surface and horizontal dermal erector muscles to pull the papilla's perimeter toward its core and determine shape. A third set of muscles, retractors, appears to be responsible for pulling a papilla's apex down toward the body surface while stretching out its base. Connective tissue infiltrated with mucopolysaccharides assists with structural support. S. apama face ridge papillae are different: the contraction of erector muscles perpendicular to the ridge causes overlying tissues to buckle. In this case, mucopolysaccharide‐rich connective tissue provides structural support. These six species possess changeable papillae that are diverse in size and shape, yet with one exception they share somewhat similar functional morphologies. Future research on papilla morphology, biomechanics and neural control in the many unexamined species of octopus and cuttlefish may uncover new principles of actuation in soft, flexible tissue. J. Morphol. 275:371–390, 2014. © 2013 Wiley Periodicals, Inc.     

Morphology of the tongue dorsal surface in white sea bream (Diplodus sargus sargus)

In the present study, the tongue dorsal surface of the white sea bream Diplodus sargus sargus (Linnaeus, 1758) (teleosts, Sparidae) was studied, to give a support to the knowledge of the anatomical characteristics of the oral cavity of a species with a future potential use in aquaculture. The feeding habits in captivity are one of the critical steps in the species of commercial interest, strictly related to the morphological characteristics of the oropharyngeal cavity. This study was undertaken, using light and scanning electron microscopy, to analyse the morphology of the dorsal tongue surface to show whether relationships are present between the anatomy and the nutritional habits and choices of this farmed species. Three zones, an apex, a body and a root, can be clearly distinguished, with a pouch partially covering the apex. In the pouch, cylindroid and mucosal ridges were observed. Along the whole tongue surface, from the apex to the root, on the medial and lateral parts, many papillae are present, with a fungiform, cylindroid and cone‐like aspect. Taste buds are showed on the dorsal surface of some papillae.     

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.     

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.     

Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.