Changing patterns of cytokeratins and vimentin in the early chick embryo

The distribution of cytokeratins and vimentin intermediate filaments in the first 48 h of chick development has been determined using immunofluorescent labelling. During formation of the germ layers, cytokeratin expression is associated with the appearance of an integral epithelium (ectoderm), whereas vimentin expression is associated with cells that detach and migrate from this epithelium to form endoderm and mesoderm. Subsequently, vimentin persists in the endoderm and mesoderm and the tissues derived therefrom, such as the somites and developing heart, throughout the period of study. The appearance of cytokeratins at later stages of development occurs in some epithelia such as the ectoderm, endoderm, lateral plate and epimyocardium but not others including the neural plate, neural tube and somites. Expression of cytokeratins in endoderm and mesenchymal tissues occurs in tandem with vimentin. In conclusion, vimentin expression is related to its distribution in the epiblast before germ layer formation. Its initial appearance may be related to the motile behaviour of cells about to ingress through the primitive streak. The appearance of cytokeratin filaments, however, does not reflect germ layer derivation but rather the need for an epithelial sheet.     

Immunohistochemistry of the cytoskeleton in the excurrent ducts of the testis in birds of the Galloanserae monophyly

The presence, location and degree of immunoexpression of various microfilament (MF) and intermediate filament (IF) systems (actin, cytokeratins, desmin, vimentin) were studied in the excurrent ducts of the testis in sexually mature and active galliform (Japanese quail, domestic fowl, turkey) and anseriform (duck) birds. These proteins were variably expressed between the epithelia and periductal tissue (periductal smooth muscle cell layer and interductal connective tissue) types and between species. Variable heterogeneous co-expression of filament systems was also found in the various duct epithelia and periductal tissue types: co-expression of filament systems was the rule rather than the exception. In the duck, neither vimentin nor cytokeratin was present in any of the tissues, whereas actin and desmin (absent in the rete testis) were co-expressed in the efferent ducts and epididymal duct unit (comprising the ductus conjugens, ductus epididymidis and ductus deferens). Actin, desmin and vimentin were generally co-expressed in the rete testis, efferent ducts and epididymal duct unit of the quail, domestic fowl and turkey, with vimentin being more strongly immunoreactive than actin and desmin in the epididymal duct unit, but more weakly immunoexpressed in the efferent ducts. Cytokeratin was present and co-expressed with actin, desmin and vimentin in the rete testis, efferent ducts and epididymal duct unit of the domestic fowl and turkey, but not in the quail and duck. The periductal smooth muscle cell layer and interductal tissue co-expressed actin, desmin and vimentin variably in all birds. Luminal spermatozoa of both the turkey and duck were immunonegative for all protein systems, whereas those of the quail and domestic fowl co-expressed actin, desmin and vimentin moderately or strongly. The tissues of the reproductive tract of male birds thus contain cytoskeletal protein systems that are variably but mostly co-expressed and whose contractile ability appears necessary and sufficient for transportation through the various excurrent ducts of the voluminous testicular fluid and its high sperm content, characteristic features of male avian reproduction.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos.

We have isolated two mouse genes, Mox-1 and Mox-2 that, by sequence, genomic structure and expression pattern, define a novel homeobox gene family probably involved in mesodermal regionalization and somitic differentiation. Mox-1 is genetically linked to the keratin and Hox-2 genes of chromosome 11, while Mox-2 maps to chromosome 12. At primitive streak stages (approximately 7.0 days post coitum), Mox-1 is expressed in mesoderm lying posterior of the future primordial head and heart. It is not expressed in neural tissue, ectoderm, or endoderm. Mox-1 expression may therefore define an extensive 'posterior' domain of embryonic mesoderm before, or at the earliest stages of, patterning of the mesoderm and neuroectoderm by the Hox cluster genes. Between 7.5 and 9.5 days post coitum, Mox-1 is expressed in presomitic mesoderm, epithelial and differentiating somites (dermatome, myotome and sclerotome) and in lateral plate mesoderm. In the body of midgestation embryos, Mox-1 signal is restricted to loose undifferentiated mesenchyme. Mox-1 signal is also prominent over the mesenchyme of the heart cushions and truncus arteriosus, which arises from epithelial-mesenchymal transformation and over a limited number of craniofacial foci of neural crest-derived mesenchyme that are associated with muscle attachment sites. The expression profile of Mox-2 is similar to, but different from, that of Mox-1. For example, Mox-2 is apparently not expressed before somites form, is then expressed over the entire epithelial somite, but during somitic differentiation, Mox-2 signal rapidly becomes restricted to sclerotomal derivatives. The expression patterns of these genes suggest regulatory roles for Mox-1 and Mox-2 in the initial anterior-posterior regionalization of vertebrate embryonic mesoderm and, in addition, in somite specification and differentiation.     

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Origin of cutaneous smooth muscles in birds (author's transl)

The origin of smooth muscles in the skin of bird embryos has been analyzed in heterospecific quail/chick recombinants. The somitic mesoderm of the wing level of 2-day chick embryos was replaced by homotopic or heterotopic somitic mesoderm from quail embryos. The cellular constitution of tissues was observed in twelve recombinant embryos at 17 or 18 days of incubation. Results show that feather smooth muscles and vascular smooth muscles have the same origin as the cutaneous mesenchyme in which they differentiate. They are of somatopleural origin in the wing integument and of somitic (dermatomal) origin in the dorsal integument. This study further reveals that the muscular and connective tissue wall of blood vessels does not have the same embryonic origin as the endothelium. It is suggested that the latter originates from the primitive aorta.     

Co-localization of vimentin and cytokeratins in M-cells of rabbit gut-associated lymphoid tissue (GALT)

Summary The occurrence of cytokeratins, vimentin, and desmin in the dome epithelia and adjacent non-dome epithelia in four locations of gut-associated lymphoid tissues (GALT) of adult and newborn rabbits (Peyer's patches, sacculus rotundus, caecal lymphoid patches and appendix) was studied with monoclonal antibodies, using the indirect immunoperoxidase technique. In all locations investigated in adult animals, antibodies specific for vimentin labelled (1) M-cells, which engulf intraepithelial lymphocytes, (2) columnar epithelial cells at the base of the domes lacking an apparent contact with lymphocytes (immature M-cells), and (3) flat cells, which lie in the lamina propria under the dome epithelium, and which line the basal lamina with thin cytoplasmic processes. In newborn rabbits, columnar epithelial cells resembling the immature M-cells of adults were selectively stained with vimentin antibodies. In M-cells, the strongest immunoreactivity was present in the perinuclear region and close to the pocket membrane, whereas the most apical and most basal parts of the cytoplasm showed no vimentin-immunoreactivity. Enterocytes in the dome epithelium and in the non-dome epithelium were vimentin-negative. M-cells and enterocytes bound antibodies against cytokeratin peptides 18 and 19 in adults and newborn animals. Compared with enterocytes, M-cells showed less intense staining for cytokeratins. Dome epithelia and no-dome epithelia did not contain desmin-immunoreactive cells. The results suggest that vimentin is a sensitive marker for M-cells in rabbit GALT.     

Differential Location of Different Types of Intermediate-Sized Filaments in Various Tissues of the Chicken Embryo

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

小鼠早期胚胎发育期间TGF—β免疫组织化学定位

The distribution of transforming growth factor beta-1 (TGF-beta-1) in the early developing mouse embryos between day 1 and day 12 of gestation was examined by immunohistochemical techniques. Polyclonal rabbit antiserum raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta-1 from human platelets was used. The following results were obtained: 1. Embryonic cells of early cleavage stages (2, 4 and 8 cells) and late morulae showed positive immunofluorescent reaction without any difference in staining intensity (Plate I, Figs. 1-4). 2. Marked staining of blastocysts in toto or sections with anti-TGF-beta-1 antibodies by either immunofluorescence or immunoperoxidase reaction was also observed. Inner cell mass (ICM) cells and trophoectoderm cells were both reacted, but more intense staining was found in primary endoderm cells differentiated from ICM cells adjacent to blastocoele (Plate II, Fig. 5). 3. Scattered granules stained strongly with immunoperoxidase reaction were present in embryonic ectoderm and visceral endoderm surrounding the forming mesoderm which was only slightly stained (Plate II, Fig. 6). 4. Intense immunoperoxidase staining was also present in mesoderm of visceral yolk sac of day 8 and day 10 embryos (Plate II, Fig. 7). 5. During the formation of somites, neural tube and limb bud, remarkable staining was found in mesenchyme, individual cells of somites, mucous layer of gut tubes, heart and limb buds (Plate III, Figs. 8-10). No significant staining was seen in neural cells per se except the inner surface of neural tube. The results of present studies indicate that abundant TGF-beta-1 is present in preimplantation mouse embryos including cleavage, morulae and blastocyst stages. In postimplantation embryos, TGF-beta-1 appears to play an important role in the differentiation of endoderm and mesoderm, particularly in the development of extraembryonic tissues, and in later morphogenetic and histogenetic events involving mainly mesoderm or mesenchyme cells.     

Local expression of cytokeratins 8, 17 and 18 in the mesenchyme and smooth muscles in the early stages of human organogenesis

Expression of cytokeratins 7, 8, 17, 18 in human embryos and fetuses of 6.5-13 weeks was studied using light and electron immunocytochemistry and immunoelectroblotting with the monoclonal antibodies. Cytokeratins 8 and 18 were expressed in 6.5-8 week old embryos not only in epithelium but also in mesenchyme of allantois, urogenital sinus, Wolffian and Mullerian ducts, mesentery, urinary bladder and certain regions of colon, rectum and atrium cordis walls. Furthermore, starting from the 10th week smooth-muscle cells of ring layer in caudal part of rectum bound antibodies against cytokeratin 17 in addition to those against cytokeratins 8 and 18. Corresponding mesenchymal and smooth-muscle cells of adult individuals did not react with either of them. Cytokeratins were still synthesized when mesenchymal cells of embryonic intestine wall were cultivated in vitro. Intermediate filaments of these cells contain cytokeratins 8 and 18, as demonstrated by electron immunocytochemistry and immunoelectroblotting. Thus, the expression of cytokeratins is not restricted to adult and embryonic epithelial tissues but is also characteristic of mesenchyme and smooth muscle differentiation in human embryos and fetuses.     

Heterogeneity in the immunolocalization of cytokeratin specific monoclonal antibodies in the rat eye: evaluation of unusual epithelial tissue entities

Summary The immunocytochemical localization of cytokeratin and vimentin in rat eye tissues was investigated using a panel of 39 monoclonal antibodies specific for single or multiple of cytokeratin polypeptides and one polyclonal anti CK20 antiserum. The retinal and the ciliary body pigment epithelia only expressed cytokeratins 8 and 18, whereas the fetal retinal pigment epithelium and focally the adult epithelium, in the transition zone of retina and ciliary body, exhibited a reactivity for cytokeratin 19. In contrast, the non-pigmented ciliary epithelium was positive for vimentin only.In the rat conjunctiva distributed goblet cell clusters were selectively stained with cytokeratin 7, 8, 18 and 19 specific monoclonal antibodies. Among them a group of cytokeratin 8 and 18 specific monoclonal antibodies which stained the goblet cells as well as cytokeratin 8 and 18 positive internal controls did not react with either the cytokeratin 8 and 18 positive neuroectodermal cells of the rat eye nor the rat choroid plexus epithelium. This indicates differences in the phenotype e.g. conformational epitope changes, of neuroectodermal derived and other cytokeratins. The corneal and conjunctival epithelium showed a more complex distribution of squamous epithelium type cytokeratins. The limbal region as a transient zone connecting both epithelia exhibited a changing cytokeratin pattern. In general, the study emphasized the necessity to work with an enlarged antibody panel to avoid misleading results in the immunolocalization of cytokeratins.Dedicated to Prof. Dr. H.J. Scharf (Halle, FRG) on the occasion of his 70th birthday     

Origins and patterning of avian outflow tract endocardium

Outflow tract endocardium links the atrioventricular lining, which develops from cardiogenic plate mesoderm, with aortic arches, whose lining forms collectively from splanchnopleuric endothelial channels, local endothelial vesicles, and invasive angioblasts. At two discrete sites, outflow tract endocardial cells participate in morphogenetic events not within the repertoire of neighboring endocardium: they form mesenchymal precursors of endocardial cushions. The objectives of this research were to document the history of outflow tract endocardium in the avian embryo immediately prior to development of the heart, and to ascertain which, if any, aspects of this history are necessary to acquire cushion-forming potential. Paraxial and lateral mesodermal tissues from between somitomere 3 (midbrain level) and somite 5 were grafted from quail into chick embryos at 3-10 somite stages and, after 2-5 days incubation, survivors were fixed and sectioned. Tissues were stained with the Feulgen reaction to visualize the quail nuclear marker or with antibodies (monoclonal QH1 or polyclonals) that recognize quail but not chick cells. Many quail endothelial cells lose the characteristic nuclear heterochromatin marker, but they retain the species-specific epitope recognized by these antibodies. Precursors of outflow tract but not atrioventricular endocardium are present in cephalic paraxial and lateral mesoderm, with their greatest concentration at the level of the otic placode. Furthermore, the ventral movement of individual angiogenic cells is a normal antecedent to outflow tract formation. Cardiac myocytes were never derived from grafted head mesoderm. Thus, unlike the atrioventricular regions of the heart, outflow tract endocardial and myocardial precursors do not share a congruent embryonic history. The results of heterotopic transplantation, in which trunk paraxial or lateral mesoderm was grafted into the head, were identical, including the formation of cushion mesenchyme. This means that cushion positioning and inductive influences must operate locally within the developing heart tubes.     

Developmental expression of a neurofilament-M and two vimentin-like genes in Xenopus laevis

A hamster vimentin cDNA probe has been used to isolate and characterize three Xenopus laevis intermediate filament genes, named XIF1, XIF3 and XIF6. Of these, XIF6 shows 89% homology at the amino acid level to a portion of porcine neurofilament-M. XIF6 is transcribed solely in nervous tissue of embryos, commencing at the late neural tube stage. Expression is totally dependent on an interaction between mesoderm and ectoderm during gastrulation and can be used as a marker of neural induction. XIF1 shows 94% homology and XIF3 83% homology to hamster vimentin at the amino acid level over a region of the protein. Although XIF1 and XIF3 show more homology to vimentin than to any other intermediate filament gene, they have distinct temporal and spatial patterns of expression. XIF1 expression most resembles that of vimentin in higher vertebrates, being expressed in embryonic myotome and nerve cord, whilst XIF3 is unusual in that its expression is restricted predominantly to the head in tailbud embryos.     

Adhesion molecules during somitogenesis in the avian embryo

In avian embryos, somites constitute the morphological unit of the metameric pattern. Somites are epithelia formed from a mesenchyme, the segmental plate, and are subsequently reorganized into dermatome, myotome, and sclerotome. In this study, we used somitogenesis as a basis to examine tissue remodeling during early vertebrate morphogenesis. Particular emphasis was put on the distribution and possible complementary roles of adhesion-promoting molecules, neural cell adhesion molecule (N-CAM), N-cadherin, fibronectin, and laminin. Both segmental plate and somitic cells exhibited in vitro calcium-dependent and calcium-independent systems of cell aggregation that could be inhibited respectively by anti-N-cadherin and anti-N-CAM antibodies. In vivo, the spatio-temporal expression of N-cadherin was closely associated with both the formation and local disruption of the somites. In contrast, changes in the prevalence of N-CAM did not strictly accompany the remodeling of the somitic epithelium into dermamyotome and sclerotome. It was also observed that fibronectin and laminin were reorganized secondarily in the extracellular spaces after CAM-mediated contacts were modulated. In an in vitro culture system of somites, N-cadherin was lost on individual cells released from somite explants and was reexpressed when these cells reached confluence and established intercellular contacts. In an assay of tissue dissociation in vitro, antibodies to N-cadherin or medium devoid of calcium strongly and reversibly dissociated explants of segmental plates and somites. Antibodies to N-CAM exhibited a smaller disrupting effect only on segmental plate explants. In contrast, antibodies to fibronectin and laminin did not perturb the cohesion of cells within the explants. These results emphasize the possible role of cell surface modulation of CAMs during the formation and remodeling of some transient embryonic epithelia. It is suggested that N-cadherin plays a major role in the control of tissue remodeling, a process in which N-CAM is also involved but to a lesser extent. The substratum adhesion molecules, fibronectin and laminin, do not appear to play a primary role in the regulation of these processes but may participate in cell positioning and in the stabilization of the epithelial structures.     

Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin

We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible "neoexpression" during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.     

Intermediate filament proteins and epithelial differentiation in the embryonic ovary of the rat

Abstract. The development and sexual differentiation of gonads in female rat embryos and fetuses between the ages of 11 and 17 days was studied by immunocytochemical analysis of intermediate filament proteins and laminin by light and electron microscopy. In the 11-day-old pregonadal embryo, the surface epithelial cells in the ventral cortex of the mesonephros contained desmin but not cytokeratin or vimentin. The development of the gonad began on the following day by proliferative growth of the mesonephric surface cells, which like the subepithelial cells soon expressed vimentin in addition to desmin. The differentiation continued by formation of separate epithelial cell clusters, which joined into cords, irregular in shape and size. Desmin disappeared from the cord cells and cytokeratins appeared while vimentin remained in all somatic cell types. Desmin was especially abundant in some stromal cells adjacent to the epithelial tissues. After the segration of the basic ovarian tissues, vimentin and desmin decreased and cytokeratins appeared in the surface epithelial cells. New changes in cytokeratin expression appeared with the differentiation of the embryonic cords in a sex-specific manner with gradual decrease of reactivity for cytokeratin 18. No immunoreaction to the neurofilament proteins was found at the present ages, and the germ cells were negative for intermediate filaments. The results show that desmin is expressed in several primitive ovarian and mesonephric cells even though they are not myogenic. The sexual differences emerge after the incipient formation of the genetically female gonad, as different organization of the internal epithelial tissue with different timing of changes in intermediate filament proteins when compared with the male gonad.     

Differentiation of mouse embryonic palatal epithelium in culture: selective cytokeratin expression distinguishes between oral, medial edge and nasal epithelial cells

During normal murine palatogenesis, regional specific differentiation of the epithelium results in three cell phenotypes: nasal (ciliated pseudostratified columnar cells), oral (stratified squamous cells) and medial edge (migratory, epithelio-mesenchymally transformed cells). We have developed a defined, serum-free, culture system which supports the growth and differentiation of isolated murine embryonic palatal epithelia in vitro. Using immunofluorescence microscopy, an established panel of antibodies was used to characterise the cytokeratin intermediate filament profile of palatal epithelial sheets at a precise developmental stage, following culture in serum-free medium with and without either transforming growth factor alpha (TGF alpha) or 10% donor calf serum (DCS). The morphologically discernable oral, medial edge and nasal phenotypes exhibited distinctive cytokeratin profiles, which remained consistent for all culture conditions, and which correlated with the known differentiation states of the epithelial types. The oral epithelia stained positively for cytokeratin 19 and cytokeratins characteristic of multilayered epithelia (1, 5, 14). Nasal epithelia stained similarly but in addition expressed the simple-epithelial cytokeratin pair, 8 and 18. Medial edge epithelia also expressed cytokeratins 1, 5 and 14 but with the exception of a few isolated cells there was no staining for cytokeratins 8 and 18. Cytokeratin 19 was absent specifically from the medial edge epithelial cells: this result may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformations. By exhibiting a complexity of expression linked to differentiation state and independent of culture conditions, cytokeratins constitute useful markers of palatal epithelial differentiation in vitro as well as in vivo.