Medial Edge Epithelium Research Articles

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.

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.

TGFbeta3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development.

Epithelial mesenchymal transformation (EMT) of the medial edge epithelial (MEE) seam creates palatal confluence. This work aims to elucidate the molecular mechanisms by which TGFβ3 brings about palatal seam EMT. We collected mRNA for PCR analysis from individual transforming MEE cells by laser microdissection techniques and demonstrated that TGFβ3 stimulates lymphoid-enhancing factor 1 (LEF1) mRNA synthesis in MEE cells. We show with antisense β-catenin oligonucleotides that up-regulated LEF1 is not activated by β-catenin in palate EMT. We ruled out other TGFβ3 targets, such as RhoA and MEK1/2 pathways, and we present evidence using dominant-negative Smad4 and dominant-negative LEF1 showing that TGFβ3 uses Smads both to up-regulate synthesis of LEF1 and to activate LEF1 transcription during induction of palatal EMT. When phospho-Smad2 and Smad4 are present in the nucleus, LEF1 is activated without β-catenin. Our paper is the first to show that the Smad2,4/LEF1 complex replaces β-catenin/LEF1 during activation of EMT in vivo by TGFβ3.

Snail family members and cell survival in physiological and pathological cleft palates

Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor β gene (TGF-β) superfamily members, and TGF-β 3 null mutant mice ( TGF-β 3 −/−) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-β 3 −/−mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-β 1 , in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-β 3 mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-β 3, TGF-β 1 is upregulated in the mesenchyme, and that in both physiological (avian) and pathological ( TGF-β 3 −/−mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium.

Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects.

Cleft palate and skull malformations represent some of the most frequent congenital birth defects in the human population. Previous studies have shown that TGFbeta signaling regulates the fate of the medial edge epithelium during palatal fusion and postnatal cranial suture closure during skull development. It is not understood, however, what the functional significance of TGFbeta signaling is in regulating the fate of cranial neural crest (CNC) cells during craniofacial development. We show that mice with Tgfbr2 conditional gene ablation in the CNC have complete cleft secondary palate, calvaria agenesis, and other skull defects with complete phenotype penetrance. Significantly, disruption of the TGFbeta signaling does not adversely affect CNC migration. Cleft palate in Tgfbr2 mutant mice results from a cell proliferation defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Our data suggests that TGFbeta IIR plays a crucial, cell-autonomous role in regulating the fate of CNC cells during palatogenesis. During skull development, disruption of TGFbeta signaling in the CNC severely impairs cell proliferation in the dura mater, consequently resulting in calvaria agenesis. We provide in vivo evidence that TGFbeta signaling within the CNC-derived dura mater provides essential inductive instruction for both the CNC- and mesoderm-derived calvarial bone development. This study demonstrates that TGFbeta IIR plays an essential role in the development of the CNC and provides a model for the study of abnormal CNC development.

Nicotine inhibits palatal fusion and modulates nicotinic receptors and the PI-3 kinase pathway in medial edge epithelia.

To analyze the effects of nicotine on palatal fusion inhibition in vitro and determine if nicotine modulated transforming growth factor beta3 or phosphatidylinositol-3 kinase signaling. A second objective was to determine the localization and regulation of nicotinic receptors in the medial edge epithelia (MEE) during palatal fusion. Palatal shelves from embryonic day (E) 13.5 mice were cultured in serum free media and treated with 0, 0.06, 0.6, or 6 mM nicotine, nicotinic receptor antagonist alpha-bungarotoxin, or the combination of nicotine and alpha-bungarotoxin. Tissues harvested at 72 h were analyzed for epithelial-mesenchymal transformation (EMT) and fusion. MEE samples collected at 20 h were analyzed for phosphorylated Akt-Ser473, phosphorylated Smad2, and nicotinic receptors. Nicotine inhibited palatal fusion in vitro in a dose dependent manner. Activated Akt-Ser473 was greater in control MEE than in nicotine treated tissues; while there was no difference in activated Smad2 between groups. The alpha7 subunit of nicotinic receptor was expressed in MEE during palate fusion and increased in nicotine treated tissues. Alpha-bungarotoxin did not rescue the nicotine treated palates. Nicotine treatment had no effect on Smad2, but caused a down regulation of the PI-3 kinase pathway that may have contributed to inhibiting palatal fusion in vitro.

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.

TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion.

Transforming growth factor (TGF) -beta3 is known to selectively regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Previous studies suggested that the selective function of TGF-beta3 in MEE was conducted by TGF-beta receptors. Further studies were needed to demonstrate that the TGF-beta signaling mediators were indeed expressed and phosphorylated in the MEE cells. SMAD2 and SMAD3 were both present in the MEE, whereas SMAD2 was the only one phosphorylated during palatal fusion. SMAD2 phosphorylation was temporospatially restricted to the MEE and correlated with the disappearance of the MEE. No phosphorylated SMAD2 was found in MEE in TGF-beta3(-/-) mice, although nonphosphorylated SMAD2 was present. The results suggest that TGF-beta3 is required for initiating and maintaining SMAD2 phosphorylation in MEE. Phospho-SMAD3 was not detectable in palate during normal palatal fusion. Previous results suggested TGF-beta-induced cessation of DNA synthesis in MEE cells during palatal fusion in vitro. The present results provide evidence that inhibition of MEE proliferation in vivo was controlled by endogenous TGF-beta3. The number of 5-bromo-2'-deoxyuridine (BrdU) -labeled MEE cells was significantly reduced in TGF-beta3(+/+) compared with TGF-beta3(-/-) mice when the MEE seam formed (t-test, P < 0.05). This finding suggests that TGF-beta3 is required for inhibiting MEE proliferation during palatal fusion. The inhibition of MEE proliferation may be mediated by TGF-beta3-dependent phosphorylation of SMAD2.

Differential expression of decorin and biglycan genes during palatogenesis in normal and retinoic acid–treated mice

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine-rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor-beta. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid-induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid-gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid-treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid-treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up-regulation of decorin gene expression in the retinoic acid-treated mice might influence the pathogenesis of cleft palate.

Nonneuronal expression of the GABA A β3 subunit gene is required for normal palate development in mice

Cleft palate is one of the most common birth defects in humans, in which both genetic and environmental factors are involved. In mice, loss of the GABA A receptor β3 subunit gene ( Gabrb3) or the targeted mutagenesis of the GABA synthetic enzyme ( Gad1) leads to cleft palate. These observations indicate that a GABAergic system is important in normal palate development. To determine what cell types, neuronal or nonneuronal, are critical for GABA signaling in palate development, we used the neuron-specific enolase promoter to express the β3 subunit in Gabrb3 mutant mice. Expression of this construct was able to rescue the neurological phenotype, but not the cleft palate phenotype. Combined with the previous observation demonstrating that ubiquitous expression of the β3 subunit rescued the cleft palate phenotype, a nonneuronal GABAergic system is implicated in palate development. Using immunohistochemistry, we detected GABA in the developing palate, initially in the nasal aspect of palatal epithelium of the vertical shelves; later in the medial edge epithelium of the horizontally oriented palatal shelves and in the epithelial seam during fusion. Based on these observations, we propose that GABA, synthesized by the palatal epithelium, acts as a signaling molecule during orientation and fusion of the palate shelves.

Matrix metalloproteinases have a role in palatogenesis.

Mammalian palatogenesis depends on palatal shelf elevation, medial edge epithelium (MEE) breakdown, and mesenchyme flow. These all require matrix remodeling, which is controlled in part by the family of matrix metalloproteinases (MMPs). We used an organ culture system to examine the effect of a general MMP inhibitor (BB3103) on mouse palatogenesis. Palates cultured in 20 micro M BB3103 contained no active MMP-2, and only one palate fused from a sample size of 15. In this single palate, MMP-3 was present at higher levels than in palates that failed to fuse. MMP-3 is known to be involved in epithelial mesenchymal transformation (EMT), and its persistence may explain why this palate fused. This implies a role for MMPs in normal palatogenesis, and disruption of their activity may result in cleft palate.

Apoptotic epithelial cell death: a prerequisite for palatal fusion. An in vivo study in rabbits

The aim of the present study was to investigate stage-specific changes in medial edge epithelium during in vivo fusion of embryonic palatal shelves in 25 Russian rabbits. Material and Methods: The embryos were dissected following Caesarian section at day 18. Palatal shelves of specific developmental steps (approximation, contact, fusion) were examined by light microscopy, immunohistochemistry and transmission electron microscopy. Results: Light microscopy revealed that the superfical peridermal cells underwent apoptosis prior to contact of the basal epithelial cells. Following contact, an epithelial monolayer was left on each shelf with an intact basement membrane. Apoptosis of the epithelial cells was followed by discontinuity of the basement membrane. Islands of epithelial cells remained. Conclusion: This paper presents new data on palatal development in vivo. The results support our theory that apoptotic medial edge epithelial cell death is a precondition for palatal fusion. There were no indications of epithelial mesenchymal transformation or migration. Copyright 2002 European Association for Cranio-Maxillofacial Surgery. Published by Elsevier Science Ltd. All rights reserved.

TGF-β3-Induced Chondroitin Sulphate Proteoglycan Mediates Palatal Shelf Adhesion

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by β- d -Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-β 3 ) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-β 3 is added to TGF-β 3 null mutant palatal shelves in culture. When chick palatal shelves (that do not adhere in vivo nor express TGF-β 3 , nor CSPG in the MEE) are cultured in vitro , they do not express CSPG and partially adhere, but when TGF-β 3 is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-β 3 .

PI-3 kinase activity is required for epithelial-mesenchymal transformation during palate fusion.

Epithelial-mesenchymal transformation (EMT) is the primary mechanism for the disappearance of medial edge epithelia (MEE) during palate fusion. This phenotype transition is highly regulated by growth factors, extracellular matrix, cell surface receptors, and a variety of intracellular signaling. Phosphatidylinositol-3 (PI-3) kinase regulates cytoskeleton reorganization, cell migration, and transforming growth factor (TGF) beta-regulated EMT. Therefore, we investigated the role of PI-3 kinase in EMT during palatal fusion in vitro. Palatal shelves from embryonic (E) 13.5 day mouse embryos were collected and cultured for up to 72 hr. A specific PI-3 kinase inhibitor, LY294002, was added to the medium at concentrations of 100 etaM, 1 microM, and 10 microM. The fate of midline epithelia was traced by carboxyfluorescence labeling and analyzed by confocal microscopy. Harvested tissues were also processed for immunohistochemical analysis of a specific marker for basal lamina (laminin). Palatal fusion stages were scored on a scale of 1 to 5, with 1 equal to complete nonfusion and 5 equal to complete fusion. The mean fusion score (MFS) was calculated for each treatment group. Palatal shelves fused after 72 hr of culture in control and 100 etaM LY294002 inhibitor-treated groups, with MFS of 4.67 and 4.5, respectively. Laminin was absent in the midline and epithelia transformed into mesenchyme. However, when cultured palates were treated with 1 and 10 microM LY294002, MEE persisted in the midline and the basal lamina remained intact after 72 hr. The MFS was significantly less in the 1 and 10 microM LY294002-treated tissues at 2.08 and 1.33, respectively. Our results demonstrate that EMT during palatal fusion in vitro is dependent on PI-3 kinase activity.

TGF-β 3-Induced Chondroitin Sulphate Proteoglycan Mediates Palatal Shelf Adhesion

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by β- d-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-β 3) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-β 3 is added to TGF-β 3 null mutant palatal shelves in culture. When chick palatal shelves (that do not adhere in vivo nor express TGF-β 3, nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-β 3 is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-β 3.

Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis.

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1(-/-) palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.

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.

TGF-beta3 is required for the adhesion and intercalation of medial edge epithelial cells during palate fusion.

Transforming growth factor-beta3 (TGF-beta3) plays a critical role during palate development, since mutations of the TGF-beta3 gene give rise to cleft palate in both humans and mice. Striking alterations have been reported in the behaviour and differentiation of medial edge epithelial (MEE) cells in TGF-beta3 knockout mouse palates. In the present paper, we provide evidence of alterations in MEE intercellular adhesion in TGF-beta3 -/- mouse palates using immunohistochemistry with monoclonal antibodies to a panel of cell adhesion and cytoskeletal molecules including E-cadherin, alpha and beta catenin, beta actin, vinculin and beta2 integrin. In vitro labeling of opposing MEE with two different lipophilic markers and subsequent analysis by confocal microscopy revealed that wild type MEE cells intercalate as soon as the midline epithelial seam forms. This finding indicates that the palate may elongate in a dorso-ventral direction by means of convergent extension, as occurs in other embryonic developmental processes. In contrast, this intercalation does not occur in the TGF-beta3 -/- MEE but it can be rescued by the exogenous addition of TGF-beta3. Thus, the substantial alteration of MEE intercellular adhesion observed in TGF-beta3 -/- palates may account for the defect in palatal shelf adhesion and the formation of cleft palate.

Programmed Cell Death Is Required for Palate Shelf Fusion and Is Regulated by Retinoic Acid

The actual role of programmed cell death (PCD) in embryonic processes and the extrinsic signals that define the death fate in developing cells are still poorly understood. Here, we show that during secondary palate shelf fusion in the mouse, PCD appeared in the medial edge epithelia (MEE) of the anterior region only after shelf contact. Contact was necessary for efficient cell death activation in the MEE. However, exogenous all-trans-retinoic acid (RA) increased cell death independently of contact. Competence to induce cell death by contact or by RA exposure was obtained when the MEE were close to touch. Endogenous RA is a relevant regulator of the secondary palate PCD since this was reduced by a retinol dehydrogenase inhibitor and an RAR specific antagonist. Bmp-7 expression was positively regulated by RA. However, BMP-7 was unable to activate cell death within the palate tissue and NOGGIN, a natural BMP antagonist, did not block PCD. Reduction of PCD at the MEE directly with a caspase inhibitor or by inhibiting retinol dehydrogenase resulted in unfused palate shelves, but adhesion was not affected. In contrast, exogenous RA also blocked fusion, but in this situation the increased cell death within the MEE appeared to affect adhesion, thereby causing cleft palate in vivo.

Toward pathogenesis of Apert cleft palate: FGF, FGFR, and TGF beta genes are differentially expressed in sequential stages of human palatal shelf fusion.

Critical cellular events at the palatal medial edge epithelium (MEE) occur in unperturbed mammalian palatogenesis, the molecular control of which involves a number of growth factors including transforming growth factor beta 3 (TGF beta 3). Apert syndrome is a monogenic human disorder in which cleft palate has been significantly correlated to the fibroblast growth factor receptor (FGFR) 2-Ser252Trp mutation. We report the relative expression of these genes in human palatogenesis. The expression of the IgIIIa/b and IgIIIa/c transcript isoforms of FGFR2 and the proteins FGFR1, FGFR2, and FGFR3 was studied in situ throughout the temporospatial sequence of human palatal shelf fusion and correlated with the expression of TGF beta 3. In addition, the immunolocalization of the ligand FGFs 2, 4, and 7 was undertaken together with the intracellular transcription factor STAT1, which is activated by FGFR signaling. FGFRs are differentially expressed in the mesenchyme and epithelia of fusing palatal shelves, in domains overlapping those of their ligands FGF4 and FGF2 but not FGF7. Coexpression is seen with TGF beta 3, which is implicated in MEE dynamics and FGF and FGFR upregulation, and STAT1, an intracellular transcription factor that mediates apoptosis. The coregulation of molecules of the FGFR signaling pathway with TGF beta 3 throughout the stages of human palatal fusion suggests their controlling influence on apoptosis and epitheliomesenchymal transdifferentiation at the MEE. Experimental evidence links FGFR2-IgIIIa/b loss of function with palatal clefting, and these correlated data suggest a unique pathological mechanism for Apert cleft palate.