Keratin expression in rat intestinal crypt and villus cells. Analysis with a panel of monoclonal antibodies.

Seven monoclonal antibodies were prepared against cytoskeletal components of rat intestinal brush borders. In the following paper (Chandler, J. S., Calnek, D., and Quaroni, A., J. Biol. Chem. 266, 11932-11938), three of them were shown to be specific for, respectively, keratin 8 (RK4), keratin 19 (RK7), and a newly identified type I keratin (keratin 21) (RK5). With these antibodies we have investigated the changes in keratin gene expression accompanying intestinal cell differentiation. Keratin 21 was detected exclusively in differentiated villus cells and in goblet, enteroendocrine, and Paneth cells in the crypts; in the proliferative crypt cells keratin 19 was predominant. Analysis of keratins expressed by cultured rat crypt cells (IEC cells) confirmed the absence of keratin 21 in undifferentiated intestinal cells. Changes in keratin's expression similar to those observed with cell differentiation in the adult intestinal mucosa were also demonstrated during early fetal intestinal development: the stratified epithelium present at 15-16 days of gestation contained predominantly keratin 19 with only a small amount of keratin 8; keratin 21 was first detected at 18-19 days of gestation, concomitant with the appearance of a well formed brush border and an apical cytoplasmic terminal web. These results suggest that keratin tonofilaments may play a role in the morphological and structural alterations accompanying intestinal cell differentiation in vivo.     

Keratin 20 helps maintain intermediate filament organization in intestinal epithelia

Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80-->His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia.     

Keratin protein expression during the development of conducting airway epithelium in nonhuman primates

Keratin protein expression during the development of Rhesus monkey conducting airway epithelium was investigated by both biochemical and immunohistochemical methods. Keratin proteins were extracted from tracheal and intrapulmonary airway tissues of fetal (at 80- and 140-day gestational ages), neonatal, and adult animals. Using immunoblot analyses and immunohistochemistry with various monoclonal (AE1, AE3, AE8, 6.01 and 6.11) and monospecific antibodies (anti-50/55 and anti-40 kDa), the presence of keratins 5, 6, 8, 13, 14, and 19 in adult airway epithelium were demonstrated. Except for keratin 13 (51 kDa), the remaining keratins could be immunologically detected in fetal and neonatal tissues. To further understand the nature of the synthesis of keratin 13 during development, airway epithelial cells from different ages were isolated and cultured in vitro. Cultured cells were labeled with 35S-methionine, and the patterns of keratin protein were analyzed by one- and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results indicated that the cultured airway cells synthesized additional keratins including 7, 15, 16, 17, and 18. However, consistent with the in vivo finding, fetal cells synthesized less or no keratin 13. These in vivo and in vitro studies strongly suggest that the synthesis of the keratin 13 in monkey conducting airway epithelium is developmentally regulated.     

Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts

We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16,K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid-inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: "keratinizing" (gingiva, hard palate), "typical nonkeratinizing" (inner cheek, floor of mouth, ventral tongue) and "special non-keratinizing" (soft palate), all of which differed from the epidermal keratinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult "special nonkeratinizing" subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation.     

Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid

Rabbit tracheal epithelial (RbTE) cells in primary culture undergo at confluence a multistep program of squamous differentiation. This study examines the expression of keratins in RbTE cells in relation to this differentiation process. During the exponential growth phase RbTE cells are undifferentiated and express three major keratins, K5, K14, and K19, and two minor keratins, K6 and K16. Squamous differentiation is accompanied by increased expression of keratins K6, K16, and K19, and in particular of keratin K13, which reacts specifically with the monoclonal antibody AE8. These changes in keratin synthesis coincide with the commitment to terminal differentiation. Retinoic acid, an inhibitor of the expression of the squamous differentiated phenotype, inhibits the increase in the expression of K6, K16, and K13 and reduces the expression of K5 and K14; however, retinoic acid treatment results in increased levels of keratin K19 and K18. Retinoic acid inhibits the expression of K16 and K13 at concentrations as low as 10(-9)-10(-10) M. At least some of these changes in keratins appear to be related to alterations in the cellular levels of the respective mRNAs. Our results indicate that specific changes in keratin expression, in particular keratin K13, correlate with the onset of squamous differentiation in RbTE cells. Induction of the expression of keratin K13 may function as a marker of squamous differentiation in tracheobronchial epithelial cells.     

Development of fetal rat intestine in organ and monolayer culture

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.     

Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs.

The keratins 8 and 18 of simple epithelia differ from stratified epithelial keratins in tissue expression and regulation. To examine the specific properties of human keratin 8, we cloned and sequenced the cDNA from a placental mRNA expression library and defined the optimum state of such clones for expression in bacterial plasmid vectors. Using the polymerase chain reaction we identified and sequenced three introns and located the single active gene for keratin 8, out of a background of 9 to 24 pseudogenes, on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is thus far unique among the keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral keratin gene than the keratins of more highly differentiated epithelia.     

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.     

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.     

Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium. An immunohistomorphological study using monoclonal antibodies to keratins

Keratin expression in hamster tracheal epithelium was investigated during organ culture in serum-free, hormone-supplemented medium using monospecific monoclonal antibodies. Generally, tracheal basal cells expressed keratins detected by antibodies RCK102 and RCK103, while columnar epithelial cells were stained positively by RGE53, RCK103, RCK105 and HCK19. Metaplastic squamous cell foci reacted with antibodies RKSE60, RCK103 and HCK19. Early metaplastic alterations were more clearly RKSE60-positive than the mature lesions. In the vitamin A-depleted tracheas basal cells were clearly RCK102-positive. Superficial cells in the central part of areas of squamous metaplasia induced by cigarette smoke condensate expressed the basal cell keratins, and were negative for the columnar cell keratin 18 detected by the RGE53 antibody. This finding suggests that in cigarette smoke condensate-induced squamous metaplasia basal cells play an important role. The mucus-producing cells at the edges of metaplastic squamous cell foci expressed the keratins specific to columnar cells. Cigarette smoke condensate exposure accelerated epithelial keratinization compared to the vitamin A-depleted epithelium. It was concluded that not only small mucous granule cells, but also basal cells are involved in the development and maintenance of induced squamous metaplasia in tracheal epithelium. Furthermore, in vitro vitamin A-depleted epithelium did not coexpress vimentin in addition to the different keratins.     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

The human thymic microenvironment: Thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation

Normal T-cell development is dependent on interactions with the thymic microenvironment; thymic epithelial cells are thought to play a key role in the induction of thymocyte maturation, both through direct contact and, indirectly, via thymic hormone secretion. It has been postulated that thymic epithelial cells progress through an antigenically defined pathway of differentiation similar to that of epidermal keratinocytes. As keratins vary according to epithelial cell type and the stage of epithelial cell maturation, we used a panel of monoclonal antibodies against keratins to study specific types of keratin intermediate filaments within human thymic epithelium. The demonstration in human thymus of keratins previously shown to be associated with distinct stages of epidermal keratinocytic maturation would support the hypothesis that thymic epithelial cells undergo sequential stages of differentiation. Two-dimensional immunoblot analysis of cytoskeletal extracts from human thymus revealed that thymic epithelium contains the following keratins: 1-2, 5, 6, 7, 8, 10, 13, 14, 15, 16, and 17 (molecular masses, 65-67, 58, 56, 54, 52, 56.5, 51, 50, 50', 48, and 46 kilodaltons, respectively). Thus, in thymic epithelium, we found keratins previously observed in epidermal basal cells (5, 14, 15), as well as keratins specific for terminally differentiated keratinocytes in supra-basal epidermis (1-2, 10). Indirect immunofluorescence (IF) performed on fetal and postnatal human thymus demonstrated that keratin epitopes recognized by antibodies AE-3, 35 beta H11, and RTE-23 are present on epithelial cells of the subcapsular cortex, the cortex, the medulla, and Hassall's bodies. In contrast, antibodies AE-1 and RTE-22 reacted primarily with neuroendocrine thymic epithelium (subcapsular cortex, medulla, Hassall's bodies). The epithelial reactivity of antibody AE-2 was limited to epithelial cells in Hassall's bodies and did not appear until 16 weeks of fetal gestation i.e., when Hassall's bodies first formed. Two-dimensional gel analysis of thymic keratins demonstrated that antibody AE-2 identified only the keratins with molecular masses of 56.6 and 65-67 kilodaltons (10 and 1-2 respectively) in thymus. These data, together with the selective reactivity of AE-2 with Hassall's bodies in fluorescence assays, demonstrate the localization in Hassall's bodies of the high-molecular-weight keratins associated with the late stages of epidermal cell maturation. In summary, we demonstrated that human thymic epithelium contains specific keratins found in multiple epithelial types as well as keratins associated with both early and late stages of epidermal cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families.

Human epithelial cells cultured from stratified and simple squamous tissues all produce keratins of 40,000 to 58,000 daltons, but within this range the number and sizes vary with different epithelial cells. We have shown that this tissue-specific variation in the keratins is not due to posttranslational modification or processing, but rather to the differential expression of a family of heterogeneous but closely related mRNAs. All of these epithelial keratin mRNAs can be further grouped into two distinct subfamilies by their ability to hybridize with either of two cloned epidermal keratin cDNAs. All of the keratin mRNAs hybridize to one or the other, but not both, of the two cloned cDNAs. However, the mRNAs within each group hybridize with varying degrees of stringency, indicating that they are of similar but not identical sequence. Both types of keratin mRNAs are always expressed in every epithelial cell line studied, suggesting that filament assembly is dependent on the presence of both types of keratins. Within each of these two groups, the slight sequence differences in each class may reflect subtle tissue-specific variations in the structural and functional requirements of the epithelial cytoskeleton.     

Organization and characterization of the keratin cytoskeleton in the previtellogenic ovarian follicle of the lizard Podarcis sicula raf

The cytokeratin (CK) cytoskeleton, previously described by immunofluorescence in the ovarian follicle of Podarcis sicula, at the electron microscope results constituted by bundles of 10 nm thick intermediate filaments containing keratin. These bundles are better evident in the cytoplasm of the pyriform cell apex pointed toward the oocyte surface and mostly in the intercellular bridges connecting fully differentiated pyriform cells to the oocyte. During the differentiation of pyriform cells, the intermediate filament bundles first appear inside the intercellular bridge, when the small follicle cells progressively enlarge after their fusion with the oocyte and assume a morphology of "intermediate" cells. The present study also reports a comparative analysis by immunolabeling, SDS-PAGE, and immunoblotting with anticytokeratins CK8, CK18, and CK19 antibodies of both the ovarian follicle and the intestine of Podarcis sicula. These antibodies, specific to the keratins of monolayered intestinal cells, react also against those expressed in the oocytes of Xenopus laevis. This study shows the presence in the ovarian follicle of this reptile only of keratin forms of homologues to the CK8 and CK18 of mammals and the lack of CK19. The same analysis carried out utilizing AE1 and AE3 antibodies, which recognize most of the acidic and basic keratins in mammals, has shown additional forms of keratins specifically expressed in the ovarian follicle (50 kDa) and in both the examined tissues (49 and 60 kDa). The reported results indicate that in the ovarian follicle of this reptile, keratins have peculiar characteristics that can be explained by the unique structural function of the cytoskeleton in this system.     

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.     

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."     

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.