Immunocytochemistry of keratins in the tongue mucosa of rats]

C12 and E2 monoclonal antibodies to keratins stained taste bud cells of foliate papillae as well as the cells of the associated glands. H4 monoclonal antibody to keratin reacted with the surrounding epithelial cells and did not react with the taste bud cells. The results show that the keratin subtype differs between taste bud and surrounding epithelial cells. The similar keratin composition of taste bud and associated gland cells confirm the view on the existence of the same type of cells in these two structures.     

Glycoconjugates and keratin 18 define subsets of taste cells

Summary Sections of neonatal, normal adult and denervated adult rat tongue were examined with lectin histochemistry. Attention was focused upon intragemmal cells (cells within the taste bud) and the surrounding perigemmal cells. Informative staining patterns were observed with four of 12 lectins:Ulex europaeus (UEA-I),Bauhinia purpurea (BPA),Helix pomatia (HPA) andLotus tetragonolobus (LTA) agglutinins. In normal adult tongues, BPA bound to those lingual epithelial cells lacking contact with the basal lamina. After they formed, vallate taste buds were laterally surrounded by distinctive BPA-positive cells. HPA reacted selectively with 28% and LTA with 23% of the intragemmal cells in vallate/foliate taste buds. In double-stained taste buds there was, a statistically significant overlap of LTA-positive cells and keratin 18-positive cells. The overlap between HPA binding and keratin 18 was more marked: double-stained cells comprized 67% of all stained cells. During taste bud development in neonates keratin 18 synthesis preceded HPA binding. In contrast, during the replacement of adult taste cells, keratin 18 synthesis and HPA binding were generally concurrent. Keratin 18 and HPA probably identify the same subset of older taste receptor cells. HPA may bind to glycoconjugates on the surface of keratin 18-positive cells. In denervated adult tongue the loss of all UEA-I-positive or BPA-positive perigemmal cells suggests that perigemmal as well as intragemmal cells are nerve-dependent.     

Keratin 19-like immunoreactivity in receptor cells of mammalian taste buds

Three monoclonal antibodies, 4.62, LPZK and 170.2.14, were usedto evaluate keratin 19-like immunoreactivity in gustatory epithelia.Keratin 19-like immunoreactivity was restricted to the intragemmalcells for all types of mammalian taste buds examined. Thesetaste buds included fungiform, foliate and vallate taste budsin rat, gerbil and rabbit, and nasopalatine, epiglottal andpalatine taste buds in rat. There was no keratin 19-like immunoreactivityin basal cells or in perigemmal cells lateral to the immunoreactivetaste receptor cells. Denervation of the rat vallate papillaeliminated all taste buds, as well as all immunoreactive tastecells. That the immunoreactive material in the taste cells waskeratin 19 was supported by the comparable staining of rat tastebuds with each of three monoclonal antibodies specific for keratin19. Furthermore, as predicted, these antibodies selectivelystained luminal cells of ral bile ducts, bladder, salivary ducts,trachea, ureter and uterus. It was concluded that monoclonalantibodies against keratin 19 can usefully distinguish intragemmaltaste receptor cells from keratinocytes, and from the perigemmaland basal cells of gustatory epithelia. Anti-keratin 19 antibodiesmay serve to identify differentiated taste cells in gustatoryepithelia undergoing taste bud development, renewal, degenerationor regeneration.     

RENEWAL OF CELLS WITHIN TASTE BUDS

Colchicine blocks mitotic division of the epithelial cells surrounding the taste bud of the rat tongue. Response to chemical stimulation decreases 50 per cent 3 hours after colchicine injection as measured by recording the electrical activity from the taste nerve bundle. Radioautography, using tritiated thymidine, shows that those epithelial cells surrounding the taste bud divide and that some of the daughter cells enter the taste bud and slowly move toward the center. The life span of the average cell is about 250 ± 50 hours, although some cells have a much shorter and others a much longer life span. These studies suggest that the cells within the taste bud, as well as the nerves, undergo considerable change with time. Corresponding changes in function are considered.     

Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.     

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.     

Distribution of vimentin in the developing chick taste bud during the perihatching period.

The tissue environment within which taste bud cells develop has not been wholly elaborated. Previous studies of taste bud development in vertebrates, including the avian chick, have suggested that taste bud cells could arise from one, or several tissue sources (e.g. crest-mesenchyme, local ectoderm or endoderm). Thus, molecular markers which are present in gemmal as well as interfacing (peribud epithelium; mesenchyme-epithelium) regions, and their degree of expression during stages of taste bud development, are of special interest. The intermediate filament protein, vimentin, occurs in mesenchymal and mesodermally-derived (e.g. endothelial, fibroblast) cells as well as highly proliferating epithelium (e.g. tumors). The present study in chick gustatory tissue utilized antibodies against vimentin and the avidin-biotin-peroxidase technique to evaluate vimentin immunoreactivity (IR) within a timeframe which includes: 1) early stages of the taste bud primordium [embryonic days (E)17-E18)]; 2) the beginning of an accelerated bud cell proliferation at the time of initial, taste bud pore opening [around E19]; 3) attaining the adult complement of taste buds [around posthatch (H) day 1], and 4) completed organogenesis (H 17). During this time span, vimentin-IR was characterized in a region including and sometimes bridging taste bud and subepithelial connective tissue, whereas non-gustatory surrounding epithelium and salivary glands were vimentin-immuno-negative. Intragemmally, the proportion of vimentin-IR cells as related to total taste bud cells peaked at E19. These results indicate that vimentin expression, in part, is related to the onset of taste bud cell proliferation and suggest that mesenchyme could be one source of taste bud cells. Secondly, fibronectin, an extracellular matrix component of the epithelial basement membrane interface with mesenchyme, was expressed at or near the apical surfaces of taste bud cells projecting into the bud lumen, and in the basal gemmal region suggesting the possible role of fibronectin as a chemotactic anchor for differentiating and migrating taste bud receptor cells. Lastly, neuron-specific enolase-IR indicates that axonal varicosities are already present intragemmally at E17-E18, that is, during the incipient period of identifiable taste bud primordia.     

The effect of temperature on the turnover of taste bud cells in catfish.

Renewal of taste bud cells on the barbels of channel catfish was studied. Groups of catfish, held in and acclimitized to 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C dechlorinated tap water were injected with [3H]thymidine (3.0 muCi/g body weight intraperitoneally). Barbels were sampled at various times after injection and prepared for light microscope autoradiography. Results show that epithelial cells surrounding the taste buds divide and some of their daughter cells migrate into the taste buds. The time at which 50% of the labelled cells have degenerated is taken as the average turnover time or average life span of the taste bud cells. The average life span as well as the time spent inside the taste buds is highly temperature-dependent. At 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C the average life span is on the order of 40, 30, 15 and 12 days respectively. Further studies indicate that both light and dark staining cells of the taste bud were labelled.     

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland

Abstract. The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells.
We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically - detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.     

Induction of apoptosis by colchicine in taste bud and epithelial cells of the mouse circumvallate papillae

Apoptotic cells in the taste buds and epithelia of mouse circumvallate papillae after colchicine treatment were examined by the methods of in situ DNA nick-end labeling, immunocytochemistry, and electron microscopy. After colchicine treatment, numerous positive cells appeared in the taste buds by DNA nick-end labeling, and some epithelial cells in the basal and suprabasal layers in and around the circumvallate papillae also revealed positive staining. Condensed and fragmented nuclei with a high density were occasionally found in the taste bud cells and in the basal and suprabasal layer epithelial cells by electron-microscopic observation. An immunocytochemical reaction for tubulin revealed weak staining in taste bud cells, because of the depolymerization of microtubules, and a decrease of the microtubules in the taste bud cells was observed by electron microscopy. These results indicate that colchicine treatment of mice induces the apoptosis of taste bud and epithelial cells in the circumvallate papillae and dorsal epithelial cells around the circumvallate papillae.     

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.     

THE EFFECT OF TEMPERATURE ON THE TURNOVER OF TASTE BUD CELLS IN CATFISH

Renewal of taste bud cells on the barbels of channel catfish was studied. Groups of catfish, held in and acclimitized to 14°C, 18°C, 22°C and 30°C dechlorinated tap water were injected with [³H]thymidine (3.0 μCi/g body weight intraperitoneally). Barbels were sampled at various times after injection and prepared for light microscope autoradiography. Results show that epithelial cells surrounding the taste buds divide and some of their daughter cells migrate into the taste buds. The time at which 50% of the labelled cells have degenerated is taken as the average turnover time or average life span of the taste bud cells. The average life span as well as the time spent inside the taste buds is highly temperature-dependent. At 14°C, 18°C, 22°C and 30°C the average life span is on the order of 40, 30, 15 and 12 days respectively. Further studies indicate that both light and dark staining cells of the taste bud were labelled.     

Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated.

Certain cultured epithelial cells contain separate vimentin and keratin-type intermediate filament networks. The intracellular injection of monoclonal antibodies directed against either vimentin or keratin filaments into PtK2 cultured epithelial cells specifically disrupted the organization of both filament types. Neither antibody had any effect when injected into cells which, while containing vimentin or keratin filaments, lacked the specific filament type which that antibody recognized. These experiments suggest that keratin and vimentin filament networks are associated in some way with one another.     

Ulex Europaeus Agglutinin-1 Is a Reliable Taste Bud Marker for In Situ Hybridization Analyses

Taste signals are received by taste buds. To better understand the taste reception system, expression patterns of taste-related molecules are determined by in situ hybridization (ISH) analyses at the histological level. Nevertheless, even though ISH is essential for determining mRNA expression, few taste bud markers can be applied together with ISH. Ulex europaeus agglutinin-1 (UEA-1) appears to be a reliable murine taste bud marker based on immunohistochemistry (IHC) analyses. However, there is no evidence as to whether UEA-1 can be used for ISH. Thus, the present study evaluated UEA-1 using various histochemical methods, especially ISH. When lectin staining was performed after ISH procedures, UEA-1 clearly labeled taste cellular membranes and distinctly indicated boundaries between taste buds and the surrounding epithelial cells. Additionally, UEA-1 was determined as a taste bud marker not only when used in single-colored ISH but also when employed with double-labeled ISH or during simultaneous detection using IHC and ISH methods. These results suggest that UEA-1 is a useful marker when conducting analyses based on ISH methods. To clarify UEA-1 staining details, multi-fluorescent IHC (together with UEA-1 staining) was examined, resulting in more than 99% of cells being labeled by UEA-1 and overlapping with KCNQ1-expressing cells.     

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.     

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.     

The distribution and origin of keratin 20-containing taste buds in rat and human

Sections of tissues containing lingual and extra-lingual taste buds were evaluated with monoclonal antibodies against cytokeratins. In the caudal third of the rat's tongue, keratin 20 immunoreactivity was restricted to taste buds, whereas keratins 7, 8, 18, and 19 were expressed in vallate and foliate taste buds and in cells of salivary ducts that merge with these taste epithelia. Hence, antibodies against keratin 20 most clearly distinguished differentiated taste cells from all other cells. In rat epiglottis, taste buds and isolated bipolar cells were keratin-20-positive. In rat nasopalatine papilla and palate, antibodies against keratin 20 identified Merkel cells, none of which was near to the keratin-20-negative taste buds. Nor were Merkel cells present at epiglottal taste buds or the keratin-20-negative fungiform taste buds or elsewhere in rat tongue. Hence, Merkel cells make no contribution to rat fungiform, epiglottal, nasopalatine, or palatal taste buds. Human and rat keratin-20-positive tissues are reported to be endodermal derivatives with the exception of Merkel cells and luminal urothelial cells. In rats the distribution of keratin-20-positive taste buds was in full agreement with the classical view that the posterior third of the tongue is derived from endoderm (keratin-20-positive taste buds), whereas the anterior two-thirds of the tongue is derived from stomadeal ectoderm (keratin-20-negative taste buds). The equally intense keratin 20 immunoreactivity of human fungiform and vallate taste buds violates this traditional rostro-caudal segregation and suggests that endodermally derived tissues may be present in the tip of the human tongue.