Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo.

Vital cell labeling techniques were used to trace the fate of the medial edge epithelial (MEE) cells during palatal fusion in vivo. Mouse palatal tissues were labeled in utero with DiI. The fetuses continued to develop in utero and tissues of the secondary palate were examined at several later stages of palatal ontogeny. The presence and distribution of DiI was correlated with the presence of cell phenotype-specific markers. During the initial stages of palatal fusion the DiI-labeled MEE were present in the midline position. These cells were attached to an intact laminin-containing basement membrane and contained keratin intermediate filaments. At later stages of palatogenesis the DiI-labeled MEE were not separated from the mesenchyme by an intact basement membrane and did not contain keratin. In late fetal development, DiI-labeled cells without an epithelial morphology were present in the mesenchyme. The transition of the DiI-labeled cells from an epithelial phenotype to a mesenchymal phenotype is consistent with a fate of epithelial-mesenchymal transformation rather than programmed cell death.     

Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion

Palatal fusion is a complex, multi-step developmental process; the consequence of failure in this process is cleft palate, one of the most common birth defects in humans. Previous studies have shown that regression of the medial edge epithelium (MEE) upon palatal fusion is required for this process, and TGF-beta signaling plays an important role in regulating palatal fusion. However, the fate of the MEE and the mechanisms underlying its disappearance are still unclear. By using the Cre/lox system, we are able to label the MEE genetically and to ablate Tgfbr2 specifically in the palatal epithelial cells. Our results indicate that epithelial-mesenchymal transformation does not occur in the regression of MEE cells. Ablation of Tgfbr2 in the palatal epithelial cells causes soft palate cleft, submucosal cleft and failure of the primary palate to fuse with the secondary palate. Whereas wild-type MEE cells disappear, the mutant MEE cells continue to proliferate and form cysts and epithelial bridges in the midline of the palate. Our study provides for the first time an animal model for soft palate cleft and submucous cleft. At the molecular level, Tgfb3 and Irf6 have similar expression patterns in the MEE. Mutations in IRF6 disrupt orofacial development and cause cleft palate in humans. We show here that Irf6 expression is downregulated in the MEE of the Tgfbr2 mutant. As a recent study shows that heterozygous mutations in TGFBR1 or TGFBR2 cause multiple human congenital malformations, including soft palate cleft, we propose that TGF-beta mediated Irf6 expression plays an important, cell-autonomous role in regulating the fate of MEE cells during palatogenesis in both mice and humans.     

The TGF-beta type III receptor is localized to the medial edge epithelium during palatal fusion

During palatal fusion, the medial edge epithelial cells (MEE) but not the oral/nasal palatal epithelium, selectively undergo epithelial-mesenchymal transformation. It is known that this process is regulated, at least in part, by endogenous TGF-beta3. One conceivable mechanism is that restricted expression of TGF-beta receptors (TbetaRs) in a subpopulation of cells may localize TGF-beta responsiveness (Brown et al., 1999). However, TGF-beta type II receptor (TbetaR-II) is expressed by all palatal epithelial cells during palatal fusion (Cui et al., 1998) and therefore cannot localize TGF-beta3 responsiveness. To investigate the role of TGF-beta type III receptor (TbetaR-III) in MEE transformation, we examined the expression pattern of TbetaR-III in the developing palate from E12 to E15 mice in vivo and in vitro by immunohistochemistry and compared the expression pattern to that of type I receptor (TbetaR-I). The expression of TbetaR-III was temporo-spatially restricted to the MEE during palatal fusion, while the expression of TbetaR-I was primarily localized in all palatal epithelia, consistent with the expression patterns of TbetaR-II and TGF-beta3 (Cui et al., 1998). These results support our hypothesis that TbetaR-III localizes and mediates the developmental role of TGF-beta3 on MEE transformation by specific expression in the MEE. TbetaR-III may modulate TGF-beta3 binding to TbetaR-II in the MEE cells to locally enhance TGF-beta3 autocrine signaling through the TbetaR-I/TbetaR-II receptor complex, which contributes to MEE selective epithelial-mesenchymal transformation.     

Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation

During fusion of the mammalian secondary palate, it has been suggested that palatal medial edge epithelial (MEE) cells disappear by means of apoptosis, epithelial-mesenchymal transformation (EMT) and epithelial cell migration. However, it is widely believed that MEE cells never differentiate unless palatal shelves make contact and the midline epithelial seam is formed. In order to clarify the potential of MEE cells to differentiate, we cultured single (unpaired) palatal shelves of ICR mouse fetuses by using suspension and static culture methods with two kinds of gas-mixtures. We thereby found that MEE cells can disappear throughout the medial edge even without contact and adhesion to the opposing MEE in suspension culture with 95% O2/5% CO2. Careful examination of MEE cell behavior in the culture revealed that apoptosis, EMT, and epithelial cell migration all occurred at various stages of MEE cell disappearance, including the transient formation and disappearance of epithelial triangles and islets. In contrast, MEE cells showed poor differentiation in static culture in a CO2 incubator. Furthermore, mouse and human amniotic fluids were found to prevent MEE cell differentiation in the cultured single palatal shelf, although paired palatal shelves fused successfully even in the presence of amniotic fluid. We therefore conclude that terminal differentiation of MEE cells is not necessarily dependent on palatal shelf contact and midline epithelial seam formation, but such MEE cell differentiation appears to be prevented in utero by amniotic fluid unless palatal shelves make close contact and the midline epithelial seam is formed.     

Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation

During palatogenesis, fusion of the palatine shelves is a crucial event, the failure of which results in the birth defect, cleft palate. The fate of the midline epithelial seam (MES), which develops transiently upon contact of the two palatine shelves, is still strongly debated. Three major mechanisms underlying the regression of the MES upon palatal fusion have been proposed: (1) apoptosis has been evidenced by morphological and molecular criteria; (2) epithelial-mesenchymal transformation has been suggested based on ultrastructural and lipophilic dye cell labeling observations; and (3) migration of MES cells toward the oral and nasal areas has been proposed following lipophilic dye cell labeling. To verify whether epithelial-mesenchymal transformation of MES cells takes place during murine palatal fusion, we used the Cre/lox system to genetically mark Sonic hedgehog- and Keratin-14-expressing palatal epithelial cells and to identify their fate in vivo. Our analyses provide conclusive evidence that rules out the occurrence of epithelial-mesenchymal transformation of MES cells.     

The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy.

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.     

Inhibition of embryonic palatal shelf horizontalization and medial edge epithelial breakdown by cortisol: role of H-2 in the mouse

We have investigated the effect of glucocorticoids on the development of the embryonic palate in vivo and of glucocorticoids and diphenylhydantoin (DPH) in culture in the cleft palate-sensitive B10.A strain and its resistant congenic partner strain, B10, as well as the effect of glucocorticoids in vivo in the sensitive A/J strain. The B10.A (H-2a) strain differs from its congenic partner strain, B10 (H-2b), only in the H-2 region of chromosome 17, and thus, differences between the two strains in the responses to the drugs can be ascribed to H-2-linked genes. The degree of corticoid-induced inhibition of shelf horizontalization in vivo is only slightly (if at all) greater in B10.A than in B10, but the degree of corticoid-induced inhibition of fusion following contact in vivo and the inhibition by cortisol and DPH of programmed cell death and breakdown of the medial edge epithelium (MEE) in vitro are much greater in B10.A than in B10. The corticoid-induced delay of shelf horizontalization produced in vivo in the A/J (H-2a) strain is considerably greater than that produced in either B10.A or B10. Thus, H-2-linked genes appear to influence slightly, if at all, the degree of corticoid-induced delay of shelf elevation, but they have a major effect on the corticoid-induced inhibition of fusion via the inhibition of breakdown of the medial edge epithelia. The delay of corticoid-induced shelf horizontalization appears to be a trait influenced primarily by non H-2-linked genes.     

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.     

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37 degrees C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37 degrees C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.     

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.     

Palatal shelf epithelium: A morphologic and histochemical study in X-irradiated and normal mice

Synopsis The palatal shelf epithelium of normal and irradiated mice was examined morphologically and histochemically, utilizing the periodic acid-Schiff (PAS) technique for the demonstration of the basement membrane and the Nitro BT method for succinate dehydrogenase activity in order to demonstrate the metabolic competence of its cells. The programmed cell death theory was not supported by the present investigation, since the cells of the medial ridge epithelium retained their structural and metabolic integrity even subsequent to the formation of cell nests. Additionally, the medial ridge epithelium of mice with radiation-induced cleft palates demonstrated normal structural and metabolic integrity long past the prospective time of fusion.     

Alterations in the lipid content of rat palatal epithelium during carcinogenesis

Summary The hard palates of 150 female albino rats of the Sprague-Dawley strain were painted 3 times a week either with the fat-soluble carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) for 11 months with inhibited secretion of saliva or with the water-soluble carcinogen 4-nitrochinoline N-oxide (4NQO) for 8 months with intact salivary secretion. Specimens were taken regularly from the mucosa of the hard palate, and the content of lipids in the epithelium was studied histochemically and biochemically during the carcinogenesis.Changes in the lipid content could be observed histochemically as there was a focal loss of lipid stainability in the epithelium during the more advanced stages of carcinogenesis with severe epithelial dysplasia, carcinoma in situ and early invasive carcinoma.The biochemical method used (TLC) could not, however, verify the histologically observed changes in the lipid content of the epithelium, probably because the changes were very local.The investigations have been performed with financial support from Maggie Stephens Foundation, Colgate-Palmolive Limited and from the Swedish Dental Society.     

Comparison of glycosidase activities in epidermis, palatal epithelium and buccal epithelium.

1. beta-Glucosidase, alpha-glucosidase, beta-galactosidase and alpha-mannosidase were measured in epidermis, palatal and buccal epithelium of the pig (Sus scrofa). 2. All three epithelia contained similar alpha-mannosidase activity (1.7-3.2 nmol mg tissue-1 hr-1 at pH 4), and none contained significant alpha-glucosidase. 3. Specific activity of beta-glucosidase was high (9-13 nmol mg tissue-1 hr-1 at pH 4) in epidermis and palate, but activity was low (less than 2 nmol mg tissue-1 hr-1) in buccal epithelium. 4. Only epidermis contained a high level of beta-galactosidase (5.8 nmol mg tissue-1 hr-1). 5. Differences in glycosidase profiles may underlie differences in permeability barrier properties in these epithelia.     

Alterations in apoptosis and epithelial-mesenchymal transformation in an in vitro cleft palate model

The processes of apoptosis and epithelial-mesenchymal transformation have been identified as two major mechanisms by which secondary palatal shelves achieve fusion. The aim of this study was to investigate alterations in these mechanisms by changing the physical distance between paired palatal shelves in an in vitro model of palatogenesis. Wild-type palatal pairs were dissected from E13.5 CD1 mouse embryos and allowed to grow in tissue culture for 48 hours at various intershelf distances. During the fusion process, medial edge epithelial cell fate was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, to evaluate apoptosis, and carboxyfluorescence (carboxy-2,7'-dichlorofluorescein diacetate succinimidyl ester) labeling, to measure transformation to mesenchymal cells. Palatal pairs separated in culture greater than or equal to 0.4 mm failed to fuse. TUNEL staining showed that the number of apoptotic cells in the palatal shelves increased as the intershelf distance increased, becoming marked in shelves that did not achieve fusion. The amount of epithelial-mesenchymal transformation, however, decreased with increasing intershelf distance. These results suggest that the contribution of epithelial-mesenchymal transformation and apoptosis to palatal shelf development and fusion can be altered by physical proximity. Therefore, one mechanism behind clefting in utero may result from an imbalance in epithelial-mesenchymal transformation and apoptosis as observed in vitro where palatal shelves are challenged to fuse by physical separation. This effect could be significant in the understanding and treatment of developmental palatal abnormalities. Perhaps in utero manipulation of intershelf spacing or epithelial-mesenchymal transformation and/or apoptosis could reverse the clefting paradigm.     

Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro

A number of adult and embryonic epithelia, when suspended within native type I collagen gels, give rise to elongate bipolar cells that migrate freely within the three-dimensional matrix. The morphology of these newly formed mesenchyme-like cells is indistinguishable from "true" mesenchymal cells at the light and ultrastructural level. In this report, we extend previous observations on the transformation of embryonic avian lens epithelium to mesenchyme-like cells. Lens epithelia, dissected from 12-day chick embryos, were cultured either within a collagen matrix or on a two-dimensional surface. Cells derived from explants on the surface of type I collagen express the epithelial phenotype. The cells form new basal lamina, continue to express delta-crystallin protein and secrete both type IV collagen and laminin. In contrast, epithelia suspended within collagen gels lose epithelial morphology, phenotype, and cytodifferentiation. The newly formed mesenchyme-like cells lack the ability to synthesize lens-specific delta-crystallin protein, type IV collagen, and laminin. They do, however, express type I collagen de novo, a characteristic of mesenchymal cells. The changes in cytodifferentiation and tissue phenotype which occur during the transformation are stable under the conditions studied here. When mesenchyme-like cells are removed from the gel and replated onto two-dimensional surfaces, they remain bipolar, will invade collagen matrices, and are unable to synthesize delta-crystallin protein.     

The effect of extracellular matrix interactions on morphologic transformation in vitro

There is emerging evidence that the structure and function of a cell is dependent in part on the contacts that cells make with the extracellular matrix. We report here the effect of extracellular matrices secreted from both normal and tumor cells have on the structure of normal rat kidney epithelial cells. Normal rat kidney cells plated on the basement membrane secreted by tumor cells adopt a morphology and phenotype which closely resembles a Kirsten-ras transformed normal rat kidney cell. This morphologic transformation was not observed for cells plated on individual extracellular matrix components or on basement membrane secreted by normal placenta cells. This suggests that tumor derived basement membrane has unique characteristics which may cause morphologic transformation of normal rat kidney cells.     

Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium

Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event.     

Role of pleated septate junctions in the epithelium of miracidia of Schistosoma mansoni during transformation to sporocysts in vitro

The surfaces of miracidia of Schistosoma mansoni were examined ultrastructurally during in vitro transformation to sporocysts. Before transformation, the surface was composed of ciliated epithelial plates (EP) that were set into a reticulum of narrow syncytial ridges (SR). The EP were attached to SR by extensive pleated septate junctions that had 18-24 strands of intramembrane particles (IMP) on the protoplasmic faces and complementary pits on the ectoplasmic faces. These junctions also appeared to separate the EP plasma membrane into apical and basolateral domains with a larger number of IMPs on the latter. Transformation was induced by placing the miracidia in salt containing medium which also halted ciliary beating. In 2-5 hr, the SR expanded until they formed a syncytium covering the parasite surface, while the EP retracted and rounded up. During this time, the EP and SR were held in contact with one another by the septate junctions which became progressively convoluted. Subsequently, the EP detached from the parasite. When transforming miracidia were returned to fresh water, the cilia resumed beating and the EP detached from the parasite surface and exposed the underlying basement membrane. Those EP that remained attached were connected only by septate junctions to the expanded SR. These studies demonstrate that the formation of the syncytium occurs gradually with contact maintained between EP and SR via the septate junctions. Further, the septate junctions are similar to occluding junctions in mammalian epithelia since they segregate the plasma membrane of the EP and they have an adhesive function.