Culture of endodermal stem/progenitor cells of the mouse tongue

The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFP(hi) cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.     

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.     

Histochemical and ultrastructural features of the lingual epithelium of the rat snake (Elaphe climacophora)

The histological, ultrastructural and histochemical characteristics of the lingual epithelium of the rat snake (Elaphe climacophora) were investigated by light and transmission electron microscopy. The cells in the beta-layer of the epithelium of the bifurcated apex were filled with beta-keratin fibers and an amorphous matrix. Round projections covering the surface of the epithelial cells, namely, microfacets which contained pale granules, were clearly visible on the outer faces of Oberh?utchen cells on the epithelium, and they were identified as fine granules filled with lipid. These granules might play an important role as a coating on the surface of the bifurcated lingual apex. The lipid on the surface of the lingual apex might also serve to trap and retain odorant molecules. Keratohyalin-like granules were distributed within the a-layer of the epithelium of the bifurcated apex of the tongue in the resting phase and cellular interdigitation was well developed in this region. Evidence of a shedding line was apparent under the light microscope in the cleft between outer and inner epithelial generations. The epithelial surface of the body of the tongue appeared suitable for retention of odorant and other molecules.     

Optical and scanning electron microscopy observation of the mucosa of human fetal tongue

The structural features of the human foetal tongue have been studied in foetuses from 8th to 20th week of pregnancy. The characteristics of the developing papillae as well as of epithelial and mesenchymal layers have been pointed out. An early differentiation of the mesenchymal tissue has been observed, concerning phenomena of cellular condensation and reticular fibers organization both in superficial and deep layers. The hypothesis of the existence of straight interactions between epithelium and mesenchyme also in the developing human tongue mucosa has been suggested. Also the observations at SEM demonstrate that from the 8th to the 20th week the epithelial surface of the tongue reaches a stable structural pattern. From 11th week a characteristic cellular polymorphism occurs: cells with microvilli that diminish progressively, ciliated cells that disappear almost completely at the 20th week and cells whose free surface show microplicae, definitive stage of the tongue cell evolution.     

Steroid receptors and proliferation in the human breast

Despite recent gains in our knowledge of the hormonal control of proliferation and differentiation in the rodent mammary gland, the factors regulating these processes in the human are poorly understood. We have developed a model in which intact normal human breast tissue is grafted subcutaneously into adult female athymic nude mice and treated with oestrogen (E) and/or progesterone (P) at human physiological serum levels. We have shown that (i) E and not P is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, (ii) E induces progesterone receptor (PR) expression and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E is required to stimulate proliferation. These data raised the question of whether one cell type demonstrated two different responses to the two different E concentrations or whether PR expression and proliferation occurred in separate cell populations. Using dual label immunofluorescence, we showed that steroid receptor expression and proliferation (Ki67 antigen) are detected in separate cell populations in normal human breast epithelium, and that cells expressing the oestrogen receptor-alpha (ERalpha) invariably contained the PR. We also reported that this separation between steroid receptor expression and proliferation observed in the normal human epithelium is disrupted at an early stage in breast tumourigenesis. One interpretation supported by our recent findings is that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as "steroid hormone sensors". Such hormone sensor cells might secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing E or P concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.     

Effect of sensory and sympathetic denervation of the frog tongue on the catecholamine containing cells of the taste buds]

The structure of catecholamine-containing dumb-bell shaped cells of the taste buds was studied by luminescent microscopy in the epithelial layer of the frog's tongue (Rana temporaria). On the unilateral section of the lingual nerve, a maintained adrenergic innervation of vessels and of the epithelium was observed, a decreased number of dumb-bell shaped cells in the taste bud, and their significant enlargement, and increased cathecholamine luminescence. With desympathization, no adrenergic nerves were observed on the vessels and the epithelium of the tongue. The size of the taste buds in desympathized cells of the tongue is sharply decreased and their number is increased. There is a tendency to grouping of the dumbbell shaped cells into 3--4 taste buds in one fungiform papillina. The experiments with sensory and sympathetic denervation of the frog tongue distinctly showed the trophic action of sensory and sympathetic nerves on the taste organ of the frog.     

Effects of atrial natriuretic peptide AP II on proliferation processes in the epithelial tissue of white rats]

The effect of atrial natriuretic peptides synthetic analog AP II on corneal, skin, tongue and duodenum epithelium proliferation have been studied on male rats. The epithelium mitotic activity and DNA synthesis were evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 micrograms/kg AP II. 10 micrograms/kg AP II was found to have different influence on organs epithelium: it decreased the mitotic activity of skin and corneal epithelium, but activated the proliferation processes in tongue and duodenum epithelium. 100 micrograms/kg AP II stimulated cell mitogenesis in all organs studied. According to data obtained atrial natriuretic peptides are able to participate in cell division regulation in vivo.     

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.     

Colonization of a portion of the bovine tongue by unusual filamentous bacteria.

Tongue samples from cattle on varied diets and ranging in age from 2 months to adult were studied by transmission and scanning electron microscopy to observe the in situ distribution and adhesion patterns of two readily identifiable genera of filamentous bacterial. The two, both members of the Simonsiellaceae, adhere to the epithelium by means of fibers which are produced on one side of the bacterial filaments and subsequently display a sidedness in their manner of adhesion to epithelial surfaces. Other bacterial populations found on the tongue were normally members of chains and seldom present as single cells. This suggests that filamentous or chain-forming bacteria may have a selective advantage over single bacteria in their ability to colonize and remain attached to the epithelium of the tongue.     

Colonization of a portion of the bovine tongue by unusual filamentous bacteria.

Tongue samples from cattle on varied diets and ranging in age from 2 months to adult were studied by transmission and scanning electron microscopy to observe the in situ distribution and adhesion patterns of two readily identifiable genera of filamentous bacterial. The two, both members of the Simonsiellaceae, adhere to the epithelium by means of fibers which are produced on one side of the bacterial filaments and subsequently display a sidedness in their manner of adhesion to epithelial surfaces. Other bacterial populations found on the tongue were normally members of chains and seldom present as single cells. This suggests that filamentous or chain-forming bacteria may have a selective advantage over single bacteria in their ability to colonize and remain attached to the epithelium of the tongue.     

In vitro growth of lingual epithelial basal cells as affected by an extract of the nodose ganglion

Nodose ganglion extract has a stimulating effect on the in vitro proliferation of tongue epithelium basal cells. Neurotrophic control of these target cells can be realized by mitogen-like chemical factors which are probably synthesized in sensory neurons, secreted from afferent nerve terminals and stimulate the proliferation of competent cells.     

Fibroblast and epidermal growth factors modulate proliferation and neural cell adhesion molecule expression in epithelial cells derived from the adult mouse tongue

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.     

Tissue kinetics in mouse tongue mucosa during daily fractionated radiotherapy

Abstract. The purpose of the present investigation was to quantify cell flux between the distinct layers of the epithelial lining of the ventral surface of mouse tongue during daily fractionated radiotherapy.
A fraction of DNA-synthesizing cells in control epithelium, or at various days through a course of daily fractionated radiotherapy with 3 or 4 Gy per day, was labelled with [³H]dT or BrdUrd, respectively. The labelling indices (LI) in the different epithelial layers were defined histologically after autoradiography, or immunohistochemistry, at intervals between 1 and 10 days after label administration.
In tongue epithelium of untreated mice, the minimum residence time of cells in the germinal layer is 2–3 days. Migration through the functional layers requires an additional 2–3 days before labelled cells are observed in the most superficial layer of nucleated cells. A plateau in LI is observed for several days post-labelling in control epithelium, indicating an equilibrium between loss and proliferation of labelled cells. During fractionated radiotherapy, the minimum time from division to occurrence of labelled cells in the stratum lucidum is less than 2 days, and hence significantly shorter than in control epithelium. In contrast to untreated epithelium, no plateau in the germinal layer LI is seen, indicating that frequently both labelled daughters from dividing labelled cells are being lost from this compartment.
In conclusion, the present data support a recently described model of radiation-induced accelerated repopulation in squamous epithelia, which postulates that the majority of damaged cells undergoes abortive'divisions resulting in two differentiating daughters.     

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.     

Reciprocal interactions of Fgf10/Fgfr2b modulate the mouse tongue epithelial differentiation

The molecular mechanisms for epithelial differentiation have been studied by observing skin development in embryogenesis, but the early signaling modulations involved in tongue epithelial differentiation are not completely understood. Based on the gene expression patterns of the Fgf signaling molecules and previous results from Fgf10 and Fgfr2b knockout mice, it was hypothesized that there would be fundamental signaling interactions through the epithelial Fgfr2b and its mesenchymal ligand Fgf10 to regulate tongue epithelium differentiation. To elucidate these reciprocal interactions in tongue epithelial differentiation, this study employed an in vitro tongue organ culture system with antisense-oligodeoxynucleotides (AS-ODNs) and recombinant protein-soaked bead implantation for the loss-of-function and gain-of-function studies. Functional analysis of Fgf signaling revealed precise reciprocal interactions, which showed that mesenchymal Fgf10 rather than Fgf7 modulates tongue epithelial differentiation via Fgfr2b in a temporal- and spatial-specific manner.     

The influence of cholic acid and cholesterol on cell proliferation in the gallbladder mucosa of the mouse

The effect of a cholesterol-cholic acid diet on cell proliferation in the gallbladder epithelial cells of the mouse was investigated systematically. As early as 48 h after the commencement of the diet an outburst of proliferative activity in the gallbladder epithelium was observed. This accelerated proliferation was accompanied by an increased exfoliation of epithelial cells into the gallbladder lumen. In the further course of the experimental diet the 3H-labelling index fell but remained significantly higher than in the control group. The epithelium displayed villus-like folds, and the gallbladder wall became noticeably thicker. The cholesterol-cholic acid diet stimulated proliferation not only in the gallbladder epithelial cells but also in fibroblasts and hepatocytes.     

New information on the location of Capillaria procyonis (Nematoda: Trichuroidea) in the tongue of a raccoon (Procyon lotor)

Histologic examination of the tongue of one juvenile male raccoon demonstrated the presence of adults and unembryonated eggs of the nematode Capillaria procyonis within parasite-induced tunnels in the stratified squamous epithelium. There was no inflammatory infiltrate within the epithelium or the subjacent lamina propria. The shape of tunnels approximated that of the resident nematodes and were lined by flattened and attenuated epithelial cells. Therefore, the site of infection of C. procyonis in the raccoon includes the oral, lingual and pharyngeal epithelium in addition to the esophagus.     

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.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.