Medial edge epithelial cell fate during palatal fusion

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.     

Disintegration of the medial epithelial seam: Is cell death important in palatogenesis?

During palatogenesis, the palatal medial edge epithelium (MEE) forms the medial epithelial seam (MES) on adhesion of the opposing palatal shelves. The MES eventually disappears, leading to mesenchymal confluence of the palate and completion of palatogenesis. Failure of these processes results in cleft palate, one of the most common congenital anomalies in human affecting around one case in 500-2500 live births. The cell fate of MEE has been controversial for more than 20 years. Recent studies suggest that the disappearance of MES is a complex process involving cell death, epithelial-mesenchymal transition (EMT) and epithelial migration. Interestingly, transforming growth factor-β3 (Tgf β3) expression in MEE and the tip epithelium of the nasal septum begins just before palatal shelf reorientation and lasts until MES disruption, and several works including targeted disruption of the gene have indicated that the process appears to be regulated mainly by the TGFβ3-TGFβR signaling. However, how MEE cells choose their fate and how the cell fate is altered in response to cellular environment remains to be elucidated.     

Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion

Malformations in secondary palate fusion will lead to cleft palate, a common human birth defect. Palate fusion involves the formation and subsequent degeneration of the medial edge epithelial seam. The cellular mechanisms underlying seam degeneration have been a major focus in the study of palatogenesis. Three mechanisms have been proposed for seam degeneration: lateral migration of medial edge epithelial cells; epithelial-mesenchymal trans-differentiation; and apoptosis of medial edge epithelial cells. However, there is still a great deal of controversy over these proposed mechanisms. In this study, we established a [Rosa26<-->C57BL/6] chimeric culture system, in which a Rosa26-originated ;blue' palatal shelf was paired with a C57BL/6-derived ;white' palatal shelf. Using this organ culture system, we observed the migration of medial edge epithelial cells to the nasal side, but not to the oral side. We also observed an anteroposterior migration of medial edge epithelial cells, which may play an important role in posterior palate fusion. To examine epithelial-mesenchymal transdifferentiation during palate fusion, we bred a cytokeratin 14-Cre transgenic line into the R26R background. In situ hybridization showed that the Cre transgene is expressed exclusively in the epithelium. However, beta-galactosidase staining gave extensive signals in the palatal mesenchymal region during and after palate fusion, demonstrating the occurrence of an epithelial-mesenchymal transdifferentiation mechanism during palate fusion. Finally, we showed that Apaf1 mutant mouse embryos are able to complete palate fusion without DNA fragmentation-mediated programmed cell death, indicating that this is not essential for palate fusion in vivo.     

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.     

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.     

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.     

Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis

During mammalian development, a pair of shelves fuses to form the secondary palate, a process that requires the adhesion of the medial edge epithelial tissue (MEE) of each shelf and the degeneration of the resulting medial epithelial seam (MES). It has been reported that epithelial-mesenchymal transformation (EMT) occurs during shelf fusion and is considered a fundamental process for MES degeneration. We recently found that cell death is a necessary process for shelf fusion. These findings uncovered the relevance of cell death in MES degeneration; however, they do not discard the participation of other processes. In the present work, we focus on the evaluation of the processes that could contribute to palate shelf fusion. We tested EMT by traditional labeling of MEE cells with a dye, by infection of MEE with an adenovirus carrying the lacZ gene, and by fusing wild-type shelves with the ones from EGFP-expressing mouse embryos. Fate of MEE labeled cells was followed by culturing whole palates, or by a novel slice culture system that allows individual cells to be followed during the fusion process. Very few labeled cells were found in the mesenchyme compartment, and almost all were undergoing cell death. Inhibition of metalloproteinases prevented basal lamina degradation without affecting MES degeneration and MEE cell death. Remarkably, independently of shelf fusion, activation of cell death promoted the degradation of the basal lamina underlying the MEE ('cataptosis'). Finally, by specific labeling of periderm cells (i.e. the superficial cells that cover the basal epithelium), we observed that epithelial triangles at oral and nasal ends of the epithelial seam do not appear to result from MEE cell migration but rather from periderm cell migration. Inhibition of migration or removal of these periderm cells suggests that they have a transient function controlling MEE cell adhesion and survival, and ultimately die within the epithelial triangles. We conclude that MES degeneration occurs almost uniquely by cell death, and for the first time we show that this process can activate basal lamina degradation during a developmental process.     

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.     

Molecular and morphologic changes during the epithelial-mesenchymal transformation of palatal shelf medial edge epithelium in vitro.

The fate of the medial edge epithelial (MEE) cells during palatal fusion has been proposed to be either programmed cell death or epithelial-mesenchymal transformation. Vital cell labeling techniques were used to mark the MEE and observe their fate during palatal fusion in vitro. Fetal mouse palatal shelves were labeled with Dil and allowed to proceed through fusion while maintained in an organ culture system. The tissues were examined at several stages of palatal fusion for the distribution of Dil, presence of specific antigens and ultrastructural appearance of the cells. The MEE labeled with Dil occupied a midline position at all stages of palatal fusion. Initially the cells had keratin intermediate filaments and were separated from the underlying mesenchyme by an intact basement membrane. During the process of fusion the basement membrane was degraded and the Dil-labeled MEE were in contact with the mesenchymal-derived extracellular matrix. In the late stages of fusion the Dil-labeled MEE altered their cellular morphology, had vimentin intermediate filaments, and were not associated with an identifiable basement membrane. Dil-labeled cells, without an epithelial phenotype, remained present in the midline of the completely fused palate. The data indicate that the MEE did not die but underwent a phenotypic transformation to viable mesenchymal cell types, which were retained in the palatal mesenchyme.     

Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse

The disappearance of palatal medial edge epithelium (MEE) after fusion of secondary palatal shelves is often cited as a classical example of embryonic remodeling by programmed cell death. We reinvestigated this phenomenon in 16-day rat embryos, using light and electron microscopy. We confirm reports that the periderm of the two-layered MEE begins to slough after shelves assume horizontal positions. In vitro, peridermal cells are not able to slough and are trapped during the adhesion process. In vivo, however, surface cells shed before the shelves in the anterior palate adhere, allowing junctions to form between opposing basal epithelial cells. Midline seams so formed consist of two layers of basal cells, all of which appear healthy. Even though its cells are dividing, growth of the seam fails to keep pace with palatal growth and it thins to one layer of cells, and then breaks up into small islands. The basal lamina disappears and elongating MEE cells extend filopodia into adjacent connective tissue. Electron micrographs reveal transitional steps in loss of epithelial characteristics and gain of fibroblast-like features by transforming MEE cells. One such feature, observed with the aid of immunofluorescence, is the turn of the mesenchymal cytoskeletal protein, vimentin. No cell death or macrophages are observed after adhesion and thinning over most of the palate. These data indicate that MEE is an ectoderm that retains the ability to transform into mesenchymal cells. Epithelial-mesenchymal transformation may be expressed in other embryonic remodelings (R.L. Trelstad, A. Hayashi, K. Hayashi, and P.K. Donahue, 1982, Dev. Biol. 92, 27), resulting in heretofore unsuspected conservation of embryonic cell populations.     

Localisation of acidic and basic fibroblast growth factors during mouse palate development and their effects on mouse palate mesenchyme cells in vitro

Summary The distribution of acidic and basic fibroblast growth factors (aFGF, bFGF) was mapped during mouse embryonic palate development. Generally, they localised most intensely in the basement membrane and epithelia rather than the mesenchyme. Localisation was predominantly restricted to the palatal nasal, and medial edge epithelia. Staining was particularly intense in the medial edge epithelia at the time of mid-line epithelial seam formation. Intense staining persisted in the epithelia of the degenerating seam and later in the oral and nasal epithelial triangles. Mouse embryonic palate mesenchyme (MEPM) cells cultured in vitro on a variety of substrata (on plastic, on the surface of a collagen gel and within a collagen gel) responded to treatment with aFGF or bFGF. These responses were modulated by the culture substratum. The FGFs stimulated MEPM cell proliferation on plastic and on collagen, but inhibited cell growth in collagen. The FGFs had little effect on protein production when cells were cultured on plastic, but caused a large reduction in on-collagen and incollagen cultures. This reduction was greater in collagenous than non-collagenous proteins. Generally, treatment with FGFs stimulated the production of glycosaminoglycans (GAGs), particularly hyaluronan (HA) and dermatan sulphate (DS). In addition, the size class of HA was shifted to a higher molecular weight form. These data indicate that aFGF and bFGF may play a role in modulating mesenchymal cell matrix biosynthesis, so facilitating palatal epithelial seam degeneration. Correspondence to: M.W.J. Ferguson     

Epithelial-mesenchymal transformation during palatal fusion: carboxyfluorescein traces cells at light and electron microscopic levels.

During the fusion of rodent embryo palatal shelves, the cells of the outer epithelial layer slough off, allowing the cells of the medial edge basal layer to form a midline seam that undergoes epithelial-mesenchymal transformation, as judged by electron microscopy and immunohistochemistry. In this study, we analyze the fate of the transformed cells using a lipid soluble dye to label the medial edge epithelium in situ. Prefusion E14 mouse palates were exposed in vitro or in vivo to a fluoresceinated lipid soluble marker, carboxydichlorofluorescein diacetate succinimidyl ester (CCFSE), which localizes in epithelia as a lipid insoluble compound that does not pass into the connective tissue compartment. The midline seam that formed after 24 hours contained labelled epithelial cells that were replaced by individually labelled mesenchymal cells where the seam transformed. By light microscopy, the labelled cells were seen to contain intensely fluorescent bodies that do not react for acid phosphatase. We were able for the first time to identify these structures by electron microscopy as CCFSE isolation bodies. The cells with isolation bodies are clearly healthy and able to participate in subsequent development of the palate. At 4 days after labelling, individual CCFSE containing cells present in the palate mesenchyme occupy both midline and lateral areas and can clearly be classified as fibroblasts by electron microscopy. CCFSE is a far more useful marker than another lipid soluble marker, DiI, for following cells, because the cells can be fixed and identified both at the light and electron microscope levels. Interestingly, if labelled palatal shelves are not allowed to fuse in vitro, the basal epithelial cells do not form mesenchyme after sloughing, indicating that formation of the epithelial midline seam is necessary to trigger its epithelial-mesenchymal transformation.     

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.     

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.     

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.     

Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.     

The expression of TGF-β3 for epithelial-mesenchyme transdifferentiated MEE in palatogenesis

The fate of the palatal medial edge epithelial (MEE) cells undergoes programming cell death, migration, and epithelial-mesenchymal transdifferentiation (EMT) coincident with the process of palatal fusion and disappearance of MEE. Mesenchymal cells in the palate have both cranial neural crest (CNC) and non-CNC origins. The objectives of this study were to identify the populations of palatal mesenchymal cells using β-galactosidase (β-gal) and DiI cell lineage markers, and to determine whether MEE-derived cells continued to express transforming growth factor-β3 (TGF-β3) and transforming growth factor-β type III receptor (TβR-III), which were specific for MEE. A model has been developed using Wnt1 tissue specific expression of Cre-recombinase to activate β-gal solely in the CNC. The expressions of TGF-β3 and TβR-III in MEE were temporally correlated with critical events in palatogenesis. Three cell populations could be distinguished in the palatal mesenchymal CNC-derived, non-CNC derived and MEE-derived. After fusion, β-gal (−) and DiI (+) mesenchymal cells continued to express TGF-β3, however TβR-III was expressed only in the epithelial MEE, as well as keratin expression. In addition, we performed laser capture microdissection to identify mRNA expression of isolated DiI (+) MEE cells. Both epithelial and transdifferentiated MEE have expressed TGF-β3, however, TβR-III was only expressed in epithelium. Extracellular matrix, especially MMP13 has been expressed coincident with fused stage which can be strongly associated with TGF-β3. These results demonstrate that combining a heritable marker and a cell lineage dye can distinguish different populations of mesenchymal cells in the developing palate. Furthermore, TGF-β3 and MMP13 could be strongly associated with EMT in palatogenesis.     

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.     

Programmed cell death is not a necessary prerequisite for fusion of the fetal mouse palate

It has been widely accepted that programmed cell death (PCD) is an essential event in palatogenesis and that its failure can result in cleft palate, one of the most common birth defects in the human. However, some conflicting results have been reported concerning the timing of cell death occurring in the fusing palate and therefore the role of PCD in palatal fusion is controversial. In order to clarify whether cell death is indispensable for mammalian palatogenesis, we cultivated the palates of day-13 mouse fetuses in vitro and prevented cell death by treating them with the inhibitors of caspases-1 and -3 or with aurintricarboxylic acid which inhibits the activity of caspase-activated DNase. Even when cell death was almost completely inhibited, palatal fusion took place successfully. Histological examination revealed that in the absence of apoptotic cell death, the medial edge epithelia of opposing palatal shelves adhered to each other and subsequently, the midline epithelial seam was disrupted and disappeared to bring about mesenchymal confluence across the palate. It seems that cell death is not a necessary prerequisite for palatal fusion but it may help to efficiently eliminate unnecessary cells which failed to migrate or differentiate properly.