Immunohistochemical localization of prostaglandins E and F2 alpha in the developing murine palate

Prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) have been shown to cause changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels in a wide variety of tissues. In particular, murine palatal mesenchyme responds to PGE2 stimulation with dose-dependent increases in intracellular cAMP levels. These same mesenchymal cells also synthesize PGE2 and PGF2 alpha. The purpose of this study is to localize PGE and PGF2 alpha in the developing murine palate by using immunohistochemical techniques. Fresh frozen cryostat sections of murine C57BL/6J embryo palates (days 12-14 of gestation) were incubated with anti-PGE or PGF2 alpha monoclonal antibodies. On day 12 of gestation, PGE and PGF2 alpha, identified as 3',3-diaminobenzidine (DAB) reaction products, were localized throughout palatal mesenchyme and epithelium; on day 13 of gestation, reaction product indicative of both PGE and PGF2 alpha was detectable primarily in mesenchyme subjacent to palatal epithelium. Extracellular spaces of the adjacent mesenchyme in the central region of the day 13 palate exhibited less reaction product. Palatal epithelium, particularly the medial edge epithelium, exhibited a diminished amount of reaction product for both prostaglandins on day 13 as compared to the underlying mesenchyme. After formation of a midline epithelial seam between homologous palatal processes on day 14 of gestation, medial edge, oral, and nasal epithelium exhibited light staining for PGE or PGF2 alpha. Palate mesenchymal cells subjacent to the midline seam exhibited a diminished amount of reaction product for both PGE and PGF2 alpha as compared to day 13 of gestation. Overall, the results show local and temporal changes in the distribution of prostaglandins in the developing murine palate.     

Immunodetection of the transforming growth factors beta 1 and beta 2 in the developing murine palate.

The expression of some members of the TGF beta family of genes in embryonic craniofacial tissue suggests a functional role for these molecules in orofacial development. In other systems, TGF beta 1 and TGF beta 2 have been shown to regulate cell proliferation and extracellular matrix metabolism, processes critical to normal development of the secondary palate. We have thus determined the amount and tissue distribution of both TGF beta 1 and TGF beta 2 in embryonic palatal tissue. Cellular extracts of murine embryonic palatal tissue from days 12, 13 and 14 of gestation were assayed for the presence of TGF beta 1 and TGF beta 2 by immunoprecipitation. Physiological levels, ranging from 0.05-20 ng/micrograms protein, of both growth factors were detected in all tissues examined. Immunostaining with antibodies directed against either TGF beta 1 or TGF beta 2 demonstrated the presence of these growth factors in palatal epithelium and mesenchyme early during palatal development (gestational day [GD] 12) a period characterized by tissue growth. On GDs 13 and 14, characterized by palate epithelial differentiation, immunostaining for both growth factors predominated in epithelial tissue. Immunostaining for TGF beta 1 and TGF beta 2 was also intense in mesenchyme surrounding tooth germs and in perichondrium. Chondrocytes and cartilage extracellular matrix did not stain for either TGF beta 1 or beta 2. Combined with existing evidence for the presence of functional receptors for the transforming growth factor-beta s in the developing palate, these results provide rationale for studies designed to clarify a specific role for these signalling molecules in palate epithelial differentiation and/or epithelial-mesenchymal interactions.     

Ultrastructural changes in rat palatal epithelium after beta-aminopropionitrile.

Beta-Aminopropionitrile (BAPN) retarded or suppressed epithelial changes in the medial edge of the palatal process in later stages of gestation in rats. Programmed cell death did not follow the usual pattern, and only a few lysosomes were observed on day 18 of gestation. The sensitivity of the medial epithelium to BAPN appeared to be different in various areas of the palatal epithelium; the epithelium on the anterior region of the palatal process was hypertrophied and keratinized, while posteriorly the medial or neighboring epithelium was very thin and, in neonatal rats, the covering was absent. A basal lamina was distinct in the anterior region and indistinct or fragmented posteriorly. Collagen fibers did not develop adjacent to the basal lamina, and an amorphous material was scattered throughout the mesenchymal tissue. These findings suggest that BAPN decreases the "connecting capacity" between mesenchyme and epithelium, and results in a modification of epithelial changes.     

Quantification and localization of ornithine decarboxylase in the embryonic palate.

Ornithine decarboxylase (ODC; EC4.1.1.17), the key enzyme in polyamine biosynthesis, and intracellular polyamines increase rapidly and markedly in tissues and cells that are actively proliferating as well as differentiating and decrease as these processes cease. ODC activity has also been implicated as playing a role in the proliferation and differentiation of cells derived from the developing palate. Ornithine decarboxylase activity was thus quantified and ODC localized in the developing murine palate in vivo. Levels of ODC activity showed little variation during the ontogeny of the palate, averaging 126 pmol CO2/mg protein/hr. When difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity, was administered to pregnant mice throughout the period of palate development (days 11-14), palatal tissue ODC activity was reduced by 85%. No craniofacial malformations were observed, however. The lack of a teratogenic effect by DFMO treatment could be due to sufficient remaining ODC activity in craniofacial tissue and/or maintenance of intracellular polyamine levels by the activity of a polyamine transport system. The activity of this system was demonstrated by the ability of palatal tissue in vivo to take up radiolabeled putrescine. The presence of a polyamine transport system was previously suggested by the demonstration of such a system in palate mesenchymal cells in vitro. Dramatic temporal and spatial shifts in tissue patterns of immunolocalization for ODC in developing palatal tissue were also seen. Immunostaining for ODC was evenly distributed in oral, nasal, and medial edge palate epithelial cells on day 12 of gestation. The basal aspects of epithelial cells were, however, more intensely stained. Mesenchymal cells exhibited a peri-nuclear immunostaining pattern. On days 12 and 13 of gestation, the staining patterns for ODC in palate epithelial and mesenchymal cells were comparable. On day 14 of gestation, all regions of the palate epithelium, particularly the medial edge epithelia, were immunostained for ODC, whereas the intensity of staining in the mesenchymal cells was significantly reduced. This study represents essential initial observations toward understanding the role that ODC may play in normal craniofacial development.     

Expression of the epidermal growth factor receptor in developing fetal mouse palates: an immunohistochemical study

Epidermal growth factor (EGF) stimulates the growth of various tissues and, therefore, EGF receptor expression in fetal tissues may play a key role in organogenesis. We have examined immunohistochemically the ontogeny and localization of the EGF receptor in the fetal mouse palate during in vivo and in vitro palatogenesis using the anti-human EGF receptor rabbit antibody. Immunoreactive products against the EGF receptor were observed in the palatal tissue examined on days 12, 13, and 14 of gestation. On days 12 and 13, the immunoreactive products were predominantly positive on the oral and medial edge epithelia but were minimal on the epithelium of the vertical shelf. The EGF receptor immunoreactivity was less intense in the posterior palate as compared with the midpalatal region. In the fusing palate of day 14 fetuses, the cells forming the midline epithelial seam were continuously positive for EGF-R immunoreactivity. The mesenchyme of palatal shelves also showed regional heterogeneity and temporal sequence in EGF receptor expression. The localization of the EGF receptor in fetal mouse palates cultured in a serumless medium generally simulated that observed in vivo.     

Regulation of murine embryonic epithelial cell differentiation by transforming growth factors β

The expression of some members of the transforming growth factor beta (TGF beta) family of genes in embryonic craniofacial tissue suggests a functional role for these molecules in orofacial development. In an attempt to ascertain a role for the TGF beta s during palatal ontogeny, murine palatal shelves were excised on gestation day (GD) 12, prior to overt epithelial differentiation, grown in organ culture under serum-free conditions and exposed to TGF beta 1 and TGF beta 2 for 18 or 42 h. Shelves were labeled with [3H]-thymidine (20 microCi/ml) during the last 4 h in culture, fixed, dehydrated, embedded in paraffin and sections stained and examined by autoradiography. Treatment of GD12 palates with TGF beta 1 and TGF beta 2 resulted in precocious cessation of medial edge epithelium (MEE) DNA synthesis followed by elimination of the MEE. In addition, this response appeared to be dose-related with higher concentrations of growth factor eliciting a more marked biological response. TGF beta treatment of homologous shelves grown in apposition also resulted in precocious fusion of apposing MEE. Thus, members of the TGF beta family, known to be synthesized by palatal MEE, appear to act in an autocrine/paracrine fashion in this tissue and are capable of regulating differentiation of embryonic palatal medial edge epithelium.     

In vitro development of the hamster and chick secondary palate

A series of experiments were undertaken to compare the in vitro behaviour of the medial edge epithelium (MEE) of hamster, in which palatal shelves normally fuse, and chick, in which they do not fuse. Homotypic pairs of hamster and chick embryo palatal processes, single palatal processes, and heterotypic palatal shelves of both animals were grown in vitro. The results indicated that contact between palatal shelves may not be crucial for MEE differentiation in mammals. The ability to acquire pre-fusion characteristics may be present in mammalian palatal tissue from their early development and may be expressed by cessation of DNA synthesis in the MEE, elevation of cAMP, and MEE cell death. Isolated chick palatal shelf cultured under identical conditions did not express these mammalian pre-fusion characteristics. When MEE of hamster and chick palatal shelves were placed in contact with one another, the intervening epithelia underwent cytolysis. This could be due to either the destruction of chick MEE by lysosomal enzymes liberated from adjacent degenerating hamster MEE cells, or by induction of cell death in chick MEE by hamster mesenchyme. Heterotypic palatal tissue combinations also suggest that release of lysosomal enzymes in the hamster MEE, which leads to its dissolution, may be the terminal event in epithelial differentiation prior to the establishment of mesenchymal continuity. It is suggested that an inverse relationship exists between DNA synthesis and cAMP levels during palatogenesis: when palate closes (as in mammals) the MEE is eliminated by increasing cAMP levels, whereas when palate remains open (as in birds) low level of cAMP preserve the integrity of MEE by supporting DNA synthesis.     

Retinoic acid-induced alterations in the expression of growth factors in embryonic mouse palatal shelves

Retinoic acid (RA) is teratogenic in many species, producing multiple malformations, including cleft palate. The effects of RA which lead to cleft palate vary depending on the stage of development exposed. After exposure of embryonic mice to RA on gestation day (GD) 10, abnormally small palatal shelves form. After exposure on GD 12 shelves of normal size form, but fail to fuse, as the medial cells proliferate and differentiate into a nasal-like epithelium. Growth factors and their receptors play an important role in regulating development, and the expression of EGF receptors, EGF, TGF-alpha, TFG-beta 1, and TGF-beta 2 has been reported in the mouse embryo. In a variety of cell types in culture, these growth factors are capable of regulating proliferation, differentiation, expression of matrix proteins, and other cellular events including epithelial-mesenchymal transformations. The present study examines immunohistochemically the expression of EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 in the control embryonic palatal shelves from GD 12 to 15 and the effects of RA treatment on GD 10 or 12 on their expression on GD 14 and 16. These growth factors were shown to have specific temporal and spatial expression in the palatal shelf. With advancing development the levels of TGF-alpha decreased while the expression of EGF increased. TGF-beta 2 localization became regional by GD 14-15, with higher levels found in epithelial cells and chondrogenic mesenchyme. TGF-beta 1 occurred in epithelial and mesenchymal cells and distribution did not change substantially with advancing development. RA exposure altered the expression of TFG-alpha, TGF-beta 1, and TGF-beta 2, but significant effects on EGF were not found. The effects on TGF-alpha and TGF-beta 1 expression were dependent on the gestational age exposed. Levels of TGF-alpha on GD 14 decreased after RA exposure on GD 10, but increased after GD 12 exposure. TGF-beta 1 expression in the mesenchyme was increased after exposure on GD 12, but was unaffected by RA on GD 10. After exposure on either day, the levels of TGF-beta 2 increased in GD 14 nasal epithelial cells. Acting in concert, growth factors could regulate events critical to formation of the secondary palate, including cessation of medial epithelial cell proliferation, synthesis of extracellular matrix proteins in the mesenchyme, programmed cell death of medial epithelial peridermal cells, and transformation of basal epithelial medial cells to mesenchymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tooth induction and temporal patterning in palatal epithelium of fetal mice

The present study examined the effect of aging on epithelium and on its ability to respond to an inductive stimulus provided by murine dental papillae. In fetal CD-1 mice, 15- to 17-day molar mesenchyme was combined with 15- to 19-day epithelium from the secondary palates. Enamel organs were separated from the dental papillae, and palatal epithelium was peeled away from its underlying mesenchyme after treatment with 1% trypsin. Recombinants of epithelium and papillae were initially cultured on a solidified complex medium for 24 hr followed by an additional 10-14 days of intraocular explanation. Control specimens consisted of isolated molar papillae. Nineteen of 88 isochronal, heterotypic recombinations formed teeth. None of the 46 heterochronal, heterotypic grafts of 18- and 19-day palatal epithelium combined with 15- to 17-day molar papillae-produced teeth. Instead, keratin-filled epithelial cysts and bone spicules were formed. Isolated control molar papillae often formed bone in the intraocular sites but did not form teeth or contain epithelium. These results show that palatal epithelium is first restricted to its developmental pathway at 18 days of gestation. Younger epithelium can convert to functional ameloblasts that secrete enamel protein. In addition to the change in gene expression, normal tooth morphology is attained. The loss of competence of the palatal epithelium at 18 days gestation coincided with the acquisition of stratum corneum and the attainment of the fully differentiated state. The oral surface of palatal epithelium appears to be determined histogenically and morphogenically at 18 days of gestation in mice.     

Retinoic acid alters EGF receptor expression during palatogenesis

Various growth factors are necessary for normal embryonic development and EGF receptors are present in developing palatal shelves of embryonic/fetal mice at least from day 12 of gestation. The medial epithelium of the palatal shelf undergoes a series of developmental events which do not occur in the oral and nasal epithelia. In utero and in organ culture, the control palatal medial epithelium shows a developmental decline in EGF receptors, demonstrated both by a decrease in the binding of antibody to EGF receptors and a decrease in the binding of 125I-EGF; decreases which are not observed in cells of the adjacent oral or nasal epithelium. During this period, medial cells cease DNA synthesis and undergo programmed cell death. Medial epithelial cells exposed to all-trans-retinoic acid continue to express EGF receptors, bind EGF, proliferate, fail to undergo programmed cell death and exhibit a morphology typical of nasal cells. The data suggest that this disturbance by retinoic acid of EGF receptor localization and subsequent alterations in differentiation of the epithelial cells plays a role in the retinoic-acid-mediated induction of cleft palate.     

Bulging medial edge epithelial cells and palatal fusion

The surface of the medial edge epithelium of embryonic day 12, 13 and 14 mouse palatal shelves was observed utilising Environmental Scanning Electron Microscopy (ESEM). This technique offers the advantage of visualisation of biological samples after short fixation times in their natural hydrated state. Bulging epithelial cells were observed consistently on the medial edge epithelium prior to palatal shelf fusion. Additionally, we have used ESEM to compare the morphology and surface features of palatal shelves from embryonic day 13 to 16 mouse embryos that are homozygous null (TGF-beta3 -/-), heterozygous (TGF-beta3 +/-) or homozygous normal (TGF-beta3 +/+) for transforming growth factor beta-3 (TGF-beta3). At embryonic day 15 and 16 most TGF-beta3 +/- and +/+ embryos showed total palatal fusion, whilst all TGF-beta3 null mutants had cleft palate: the middle third of the palatal shelves had adhered, leaving an anterior and posterior cleft. From embryonic day 14 to 16 abundant cells were observed bulging on the medial edge epithelial surface of palates from the TGF-beta3 +/- and +/+ embryos. However, they were never seen in the TGF-beta3 null embryos, suggesting that these surface bulges might be important in palatal fusion and that their normal differentiation is induced by TGF-beta3. The expression pattern of E-Cadherin, beta-catenin, chondroitin sulphate proteoglycan, beta-Actin and vinculin as assayed by immunocytochemistry in these cells shows specific variations that suggest their importance in palatal shelf adhesion.     

Rescue of an in vitro palate nonfusion model using interposed embryonic mesenchyme

The authors previously established an in vitro palate nonfusion model on the basis of a spatial separation between prefusion embryonic day 13.5 mouse palates (term gestation, 19.5 days). They found that an interpalatal separation distance of 0.48 mm or greater would consistently result in nonfusion after 4 days in organ culture. In the present study, they interposed embryonic palatal mesenchymal tissue between embryonic day 13.5 mouse palatal shelves with interpalatal separation distances greater than 0.48 mm in an attempt to "rescue" this in vitro palate nonfusion phenotype. Because no medial epithelial bilayer (i.e., medial epithelial seam) could potentially form, palatal fusion in vitro was defined as intershelf mesenchymal continuity with resolution of the medial edge epithelia bilaterally. Forty-two (n = 42) palatal shelf pairs from embryonic day 13.5 CD-1 mouse embryos were isolated and placed on cell culture inserts at precisely graded distances (0, 0.67, and 0.95 mm). Positive controls consisted of shelves placed in contact (n = 6). Negative controls consisted of shelves placed at interpalatal separation distances of 0.67 mm (n = 6) and 0.95 mm (n = 7) with no interposed mesenchyme. Experimental groups consisted of embryonic day 13.5 palatal shelves separated by 0.67 mm (n = 11) and 0.95 mm (n = 12) with interposed lateral palatal mesenchyme isolated at the time of palatal shelf harvest. Specimens were cultured for 4 days (n = 19) or 10 days (n = 23), harvested, and evaluated histologically. All positive controls at 4 and 10 days in culture showed complete histologic palatal fusion. All negative controls at 4 days and 10 days in culture remained unfused. Five of six palatal shelves separated at 0.67 mm interpalatal separation distance with interposed mesenchyme were fused at 4 days, and all five were fused at 10 days. At an interpalatal separation distance of 0.95 mm with interposed mesenchyme (n = 12), no palates (zero of four) were fused at 4 days, but seven of eight were fused at 10 days. These data suggest that nonfused palatal shelves can be "rescued" with an interposed graft of endogenous embryonic mesenchyme to induce fusion in vitro.     

Retinoids and epidermal growth factor alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture

The mechanism by which retinoids (RA) induce cleft palate is not known. During normal palatogenesis, the medial epithelia of opposing palatal shelves cease DNA synthesis, come into contact, adhere, and undergo programmed cell death (PCD). In organ cultures of day 12 embryonic mouse palatal shelves, epidermal growth factor (EGF) blocks PCD, and DNA synthesis continues. In the present study, the effects of trans-RA, 13-cis-RA, EGF, and combinations of EGF and RA on surface morphology, DNA synthesis, and cellular ultrastructure are determined for CD-1 embryonic mouse palatal shelves cultured on day 12 of gestation. DNA synthesis in the medial cells was sustained and PCD was blocked by EGF, trans-RA, and 13-cis-RA. Exposure to trans-RA, but not to 1-cis-RA, induced the medial epithelia to undergo hyperplasia, and addition of EGF enhanced the effect. In the presence of RA, particularly trans-RA, medial epithelial cells acquired nasal cell characteristics, and EGF enhanced this effect. Expansion of the mesenchymal extracellular spaces was blocked by trans-RA and to a lesser degree by 13-cis-RA. The RA-induced alterations in normal epithelial and mesenchymal cell differentiation may be relevant to the etiology of RA-induced cleft palate in vivo.     

Developmental pattern of cAMP, adenyl cyclase, and cAMP phosphodiesterase in the palate, lung, and liver of the fetal mouse: alterations resulting from exposure to methylmercury at levels inhibiting palate closure

Exposure to methylmercury (MeHg: 10 mg Hg/kg maternal body weight) on 12(6) (days hours) of gestation significantly delays palate closure in the Swiss Webster CFW mouse. The cAMP content and activity of adenyl cyclase and phosphodiesterase (PDE) were measured in the tissues of control and MeHg-induced cleft palates between 13(6) and 17(6) of gestation. Lung and liver were investigated similarly to determine if MeHg affected the adenyl cyclase system of the palate in a unique manner. In control palatal tissue, cAMP levels increased sharply from 13(22) (undetectable) to 14(6) (maximum). PDE activity increased similarly up to 14(2), but decreased 50% between 14(2) and 14(6). Since it has been reported that cAMP induces the synthesis of PDE, the difference in cAMP/PDE from 13(22) to 14(2) and from 14(2) to 14(6) suggests the localization of relatively high levels of cAMP in at least two separate compartments. Between 14(6) and 14(10), the adenyl cyclase activity of control palates decreased significantly. This rapid decrease suggests relatively high adenyl cyclase activity in the medial edge epithelial cells which undergo autolysis prior to shelf fusion (centered at 14(15). Maternal MeHg administration at 12(6) delayed the median time of palatal shelf rotation (14(13)) by 5 hours, and significantly altered the developmental pattern of the adenyl cyclase system. Thus, the increase in cAMP between 14(2) and 14(6) was abolished and the decrease in adenyl cyclase activity between 14(6) and 14(10) was delayed by almost 20 hours. These changes may be manifestions of a MeHg-induced delay in medial edge epithelial cell differentiation. In a previous study, we observed that the fetal liver exhibits the highest MeHg concentration of all tissues. Since MeHg only slightly altered the adenyl cyclase system of the fetal liver compared to the lung and palate (in which MeHg uptake is considerably less), it may be that the effects of MeHg on palatal tissue are not due to a direct effect of MeHg on components of the adenyl cyclase system.     

Mesenchymal fusion of the palatal processes and of heterotypic explants

Summary Two palatal sheleves were cultured for 3 to 6 h, and palatal shelves and half lips were associated and cultured for 24, 48 or 72 h on an agar culture medium. In all cases an epithelial wall was observed between the two mesenchymes. In homotypical palatal shelf cultures, the medial palatal wall disappears and mesenchymal fusion occurs. The epithelial barrier in palate-lip associations is constituted at first by palatal and labial cells, then purely labial, the medial palatal epithelium undergoing destruction. This labial wall pushed away by the actively growing mesenchyme finally disappears so that the palatal and labial mesenchymes are continuous. This phenomenon is completely different from the mesenchymal fusion and has not the same characteristics.     

Subcellular compartmentalization of cAMP-dependent protein kinase regulatory subunits during palate ontogeny

Mammalian palatal ontogeny involves epithelial-mesenchymal interactions, cell differentiation, and cell movements. These events occur on days 12, 13, and 14 of gestation in the C57BL/6J mouse embryo. During this period intracellular cAMP levels and cAMP-dependent protein kinase (cAMP-dPK) levels in the palate transiently elevate. Cyclic AMP activates cAMP-dPK by binding primarily to two types of regulatory subunits of this enzyme, designated as RI and RII. To assess whether differential compartmentalization of the regulatory subunits occurs during palatal ontogeny, cytosolic, nuclear, and particulate fractions were prepared from day 12, 13, and 14 embryonic maxillary and palatal tissue. After photo-affinity labeling of each fraction with 8-azido [32P] cAMP, SDS-PAGE, and autoradiography, autoradiograms were analyzed densitometrically. The RI isoform predominated in the nuclear and particulate fractions on all three developmental days; whereas RII predominated in the cytosolic fractions. Thus, differential compartmentalization of cAMP-dPK may be a means by which cAMP dependent responses are regulated during palatogenesis.     

Transient and recurrent expression of the Egr-1 gene in epithelial and mesenchymal cells during tooth morphogenesis suggests involvement in tissue interactions and in determination of cell fate.

We have analyzed the expression of early growth response gene (Egr-1) by mRNA in situ hybridization during mouse embryonic tooth development and in experimental recombinations of dental epithelium and mesenchyme. Egr-1 was transiently and recurrently expressed both in epithelial and mesenchymal cells starting from day 13 of gestation and up to 4 days after birth. The expression correlated with developmental transition points of dental mesenchymal and epithelial cells suggesting a role for Egr-1 in sequential determination and differentiation of cells. In recombination cultures of early dental epithelium and mesenchyme Egr-1 RNA was localized at the epithelial-mesenchymal interface in mesenchymal cells, and in two cases also in epithelial cells. These data indicate that Egr-1 expression may be regulated by epithelial-mesenchymal interactions when they are specific enough to initiate differentiation. We have also analyzed by in situ hybridization whether Wilms' tumour-1 gene (wt-1) is expressed in the developing tooth as it was proposed on the bases of in vitro studies that it may inhibit Egr-1 expression. No wt-1 expression was detected at any stage of tooth development showing that wt-1 is not obligatory for regulation of Egr-1 expression.     

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.