Medial Edge Epithelium Research Articles

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.

Gsk3β is required in the epithelium for palatal elevation in mice

In Wnt/β-catenin signaling pathway, Gsk3β functions to facilitate β-catenin degradation. Inactivation of Gsk3β in mice causes a cleft palate formation, suggesting an involvement of Wnt/β-catenin signaling during palatogenesis. In this study, we have investigated the expression pattern, tissue-specific requirement and function of Gsk3β during mouse palatogenesis. We showed that Gsk3β is primarily expressed in the palatal epithelium, particularly in the medial edge epithelium overlapping with β-catenin. Tissue-specific gene inactivation studies demonstrated an essential role for Gsk3β in the epithelium for palate elevation, and disruption of which contributes to cleft palate phenotype in Gsk3β mutant. We observed that expression of Aixn2, a direct target gene of Wnt/β-catenin signaling, is ectopically activated in the mutant tongue, but not in the palate. Our results indicate that Gsk3β is an intrinsic regulator required in the epithelium for palate elevation, and could act through a pathway independent of Wnt/β-catenin signaling to regulate palate development.

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.

Analysis of the Presence of Cell Proliferation-Related Molecules in the Tgf-β3 Null Mutant Mouse Palate Reveals Misexpression of EGF and Msx-1

The Tgf-β₃ null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β₂, Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β₃, and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β₃ null mutant mouse palate plays a role in the cell proliferation defect observed.

Vitamin B12 Counteracts Dexamethasone-Induced Proliferation and Apoptosis During Key Periods of Palatogenesis in Mice

B vitamins rescue cleft palate induced by glucocorticoids in rodents; however, the mechanism of this effect remains largely unknown. The objective of our study was to assess the effect of dexamethasone and Vitamin B12 on cell proliferation and apoptosis during palatogenesis. In our study, mesenchymal cell proliferation in mouse embryonic palates decreased when the subjects were administered dexamethasone at embryo day 13.5 (E 13.5). However, mesenchymal cell proliferation was increased after dexamethasone exposure at E 14.0 and E 14.5 in comparison with the control group. After Vitamin B12 treatment, proliferation of mesenchymal cells was restored. No apoptosis was detected until bilaterial palatal shelves adhered and formed a medial epithelium seam in the control group and Vitamin B12-treated group. However, the apoptotic cells were detected under the medial edge epithelium before the palate contacted after dexamethasone treatment. The results suggested that Vitamin B12 restored proliferation, which had been reduced by dexamethasone via a delayed cellular cycle and apoptosis. This study implies that Vitamin B12 may be used to prevent or alleviate cleft palate induced by dexamethasone during embryonic palatogenesis.

Pyridoxine might not have a preventive effect on the retinyl palmitate-induced viscerocranial anomalies

Viscerocranial anomalies are induced in the presence of various teratogens. Vitamin A-induced cleft palate formation is one of the most frequently used experimental models in these studies. However, the underlying mechanisms are not yet fully understood. Several studies have shown that exogenous vitamin A disrupts the fusion of the palatal shelves by increasing the expression of epidermal growth factor receptor (EGFR). More recently, pyridoxine (vitamin B6) has been reported to have a potentially protective effect in regard to viscerocranial malformations. Therefore, in this study, we aimed to investigate whether pyridoxine has a preventive effect on retinyl palmitate-induced viscerocranial anomalies. The frequency of gross malformations induced by retinyl palmitate, the natural form of vitamin A, has been studied in a dose dependent manner. Low doses of retinyl palmitate (100 mg/kg) exposure on embryonic day (ED) 10 caused no gross anomalies in the rat fetuses. Teratogenic effects were observed only after exposure to higher dosages (1000 mg/kg) and primarily targeted the developing eyes and palates. On the other hand, co-administration of 10 mg/kg pyridoxine, at ED 9 and 10, significantly increased the frequencies of anomalies, even in the moderate dosage (500 mg/kg) group. In all cleft palates, sustained expression of EGFR in the medial edge epithelium was detected by immunohistochemistry. These results show that co-administration of pyridoxine has an inductive rather than protective effect on the formation of viscerocranial malformations after exposure to hypervitaminosis-A.

Matrix metalloproteinase-25 has a functional role in mouse secondary palate development and is a downstream target of TGF-β3

BackgroundDevelopment of the secondary palate (SP) is a complex event and abnormalities during SP development can lead to cleft palate, one of the most common birth disorders. Matrix metalloproteinases (MMPs) are required for proper SP development, although a functional role for any one MMP in SP development remains unknown. MMP-25 may have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the MMP-25 gene. We report on the gene expression profile of MMP-25 in the developing mouse SP and identify its functional role in mouse SP development.ResultsMMP-25 mRNA and protein are found at all SP developmental stages in mice, with the highest expression at embryonic day (E) 13.5. Immunohistochemistry and in situ hybridization localize MMP-25 protein and mRNA, respectively, to the apical palate shelf epithelial cells and apical mesenchyme. MMP-25 knockdown with siRNA in palatal cultures results in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium. MMP-25 mRNA and protein levels significantly decrease when cultured palate shelves are incubated in growth medium with 5 μg/mL of a TGF-β3-neutralizing antibody.ConclusionsOur findings indicate: (i) MMP-25 gene expression is highest at E12.5 and E13.5, which corresponds with increasing palate shelf growth downward alongside the tongue; (ii) MMP-25 protein and mRNA expression predominantly localize in the apical epithelium of the palate shelves, but are also found in apical areas of the mesenchyme; (iii) knockdown of MMP-25 mRNA expression impairs palate shelf fusion and results in significant medial edge epithelium remaining in contacted areas; and (iv) bio-neutralization of TGF-β3 significantly decreases MMP-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development and are the first to identify a role for a single MMP in mouse SP development.

Integration of IRF6 and Jagged2 signalling is essential for controlling palatal adhesion and fusion competence

In mammals, adhesion and fusion of the palatal shelves are essential mechanisms during the development of the secondary palate; failure of these processes leads to the congenital anomaly, cleft palate. The mechanisms that prevent pathological adhesion between the oral and palatal epithelia while permitting adhesion and subsequent fusion of the palatal shelves via their medial edge epithelia remain obscure. In humans, mutations in the transcription factor interferon regulatory factor 6 (IRF6) underlie Van der Woude syndrome and popliteal pterygium syndrome. Recently, we have demonstrated that mice homozygous for a mutation in Irf6 exhibit abnormalities of epithelial differentiation that results in cleft palate as a consequence of adhesion between the palatal shelves and the tongue. In the current paper, we demonstrate that Irf6 is essential for oral epithelial differentiation and that IRF6 and the Notch ligand Jagged2 function in convergent molecular pathways during this process. We further demonstrate that IRF6 plays a key role in the formation and maintenance of the oral periderm, spatio-temporal regulation of which is essential for ensuring appropriate palatal adhesion.

A TGF‐β‐induced gene, βig‐h3, is crucial for the apoptotic disappearance of the medial edge epithelium in palate fusion

TGF-beta3, TbetaR-I, and TGF-beta-activated Smad2 has been suggested to be a series of signaling molecules for secondary palate fusion. In this article, we show that a gene induced by TGF-beta, betaig-h3, is coincidentally expressed with TGF-beta3 in medial edge epithelial (MEE) cells undergoing apoptosis during normal palatal fusion. betaig-h3 was also highly expressed in the areas of post-weaning mammary gland cells and developing phalangeal joints in which TGF-beta3 or BMP-4-induced apoptosis occurs, respectively. Blocking of betaig-h3 expression in E12.5 embryos with antisense oligodeoxynucleotides (ODN) resulted in cleft of the secondary palate in 84% of the treated mice that were born. Moreover, the antisense ODN treatment resulted in a failure of apoptosis in the MEE between palatal shelves in physical contact in organ culture. We conclude that betaig-h3 expression in the MEE is stimulated by TGF-beta3, causes cell death, and consequently results in complete fusion of the apposed palatal shelves.

Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant or spontaneous disorder characterized by multiple cutaneous basal cell carcinomas, odontogenic keratocysts, skeletal anomalies and facial dysmorphology, including cleft lip and palate. Causative mutations for NBCCS occur in the PTCH1 gene on chromosome 9q22.3–q31, which encodes the principle receptor for the Hedgehog signalling pathway. We have investigated the molecular basis of craniofacial defects seen in NBCCS using a transgenic mouse model expressing Shh in basal epithelium under a Keratin-14 promoter. These mice have an absence of flat bones within the skull vault, hypertelorism, open-bite malocclusion, cleft palate and arrested tooth development. Significantly, increased Hedgehog signal transduction in these mice can influence cell fate within the craniofacial region. In medial edge epithelium of the palate, Shh activity prevents apoptosis and subsequent palatal shelf fusion. In contrast, high levels of Shh in odontogenic epithelium arrests tooth development at the bud stage, secondary to a lack of cell proliferation in this region. These findings illustrate the importance of appropriately regulated Hedgehog signalling during early craniofacial development and demonstrate that oro-facial clefting and hypodontia seen in NBCCS can occur as a direct consequence of increased Shh signal activity within embryonic epithelial tissues.

Hand2 is required in the epithelium for palatogenesis in mice

The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in the development of multiple organs, including craniofacial organs. Mice carrying Hand2 hypomorphic alleles (Hand2LoxP/−) display a cleft palate phenotype. A specific deletion of the Hand2 branchial arch-specific enhancer also leads to a hypoplastic mandible and cleft palate formation in mice. However, the underlying mechanism of Hand2 regulation of palate development remains unknown. Here we show that Hand2 is expressed in both the epithelium and mesenchyme of the developing palate. While mesenchymal specific inactivation of Hand2 has no impact on palate development, epithelial specific deletion of Hand2 creates a cleft palate phenotype. Hand2 appears to exert distinct roles in the anterior and posterior palate. In the anterior palate of Hand2LoxP/− mice, premature death of periderm cells and a down-regulation of Shh are observed in the medial edge epithelium (MEE), accompanied by a decreased level of cell proliferation in the palatal mesenchyme. In the posterior palate, a lower dose of Hand2 causes aberrant periderm cell death on the surface of the epithelium, triggering abnormal fusion between the palatal shelf and mandible and preventing palatal shelf elevation. We further demonstrate that BMP activities are essential for the expression of Hand2 in the palate. We conclude that Hand2 is an intrinsic regulator in the epithelium and is required for palate development.

Cleft Lip and/or Palate with Monogenic Autosomal Recessive Transmission in Pyrenees Shepherd Dogs

To document the genetic background of Pyrenees shepherd dogs as it relates to the incidence of cleft lip and/or cleft palate, to describe the phenotype, and to determine possible candidate genes. Pedigree analysis was performed and blood samples were taken from five affected pups, their siblings, and parents. Seven candidate genes were selected and linkage analysis was performed. Further methods used included sequencing and histology. In 37 litters consisting of 163 pups, we found 47 affected pups in a total population of 2104. The male:female ratio was 1:0.96. Affected pups showed isolated cleft lip and/or cleft palate; no attendant disorders have been reported. Despite a high degree of relationship, two affected pups displayed a cleft palate (- H S H -) and a cleft lip with or without cleft palate (L A -) cleft formation. Histology of affected pups showed that the medial edge epithelium remained intact and did not undergo an epithelial-mesenchymal transformation. There was no evidence for linkage between the trait and TGFb3 or Msx1. Subsequent sequencing excluded the coding sequence of Fst as well. Pedigree analysis showed that cleft palate is not genetically distinct from cleft lip with or without cleft palate but is inherited in this breed as a monogenic autosomal recessive trait. Linkage analysis and sequencing excluded TGFb3, Msx1, and Fst as candidate genes. Histology of affected pups showed that the medial edge epithelium is still intact.

Interactions between TGF- β1 and TGF- β3 and their role in medial edge epithelium cell death and palatal fusion in vitro

In recent decades, studies have shown that both TGF- β 1 and TGF- β 3 play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF- β 1 and TGF- β 3 are present. Few studies have addressed the existence of interactions between TGF- β 1 and TGF- β 3, which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF- β 1 and TGF- β 3 actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF- β 1 or anti-TGF- β 3 antibodies and TUNEL, added rhTGF- β 1 or rhTGF- β 3 or blocked the TGF- β 1 and TGF- β 3 action at different concentrations to WT or Tgf-β 3 null mutant palate cultures, performed in situ hybridizations with Tgf-β 1 or Tgf-β 3 riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF- β 1 and TGF- β 3 in the developing palate and confirm that TGF- β 3 has a more active role in MES cell death than TGF- β 1, although both are major inductors of MES disappearance. Finally, the co-localization of TGF- β 1, but not TGF- β 3, with TUNEL in the MES allows us to suggest a possible role for TGF- β 1 in MES apoptotic clearance.

Runx1 is involved in the fusion of the primary and the secondary palatal shelves

Runx1 is expressed in medial edge epithelial (MEE) cells of the palatal shelf. Conditionally rescued Runx1−/− mice showed limited clefting in the anterior junction between the primary and the secondary palatal shelves, but not in the junction between the secondary palates. In wild type mice, the fusing epithelial surface exhibited a rounded cobblestone-like appearance, while such cellular prominence was less evident in the Runx1 mutants. We also found that Fgf18 was expressed in the mesenchyme underlying the MEE and that locally applied FGF18 induced ectopic Runx1 expression in the epithelium of the palatal explants, indicating that Runx1 was induced by mesenchymal Fgf18 signaling. On the other hand, unpaired palatal explant cultures revealed the presence of anterior–posterior (A–P) differences in the MEE fates and fusion mechanism. Interestingly, the location of anterior clefting in Runx1 mutants corresponded to the region with different MEE behavior. These data showed a novel function of Runx1 in morphological changes in the MEE cells in palatal fusion, which is, at least in part, regulated by the mesenchymal Fgf signaling via an epithelial–mesenchymal interaction.

P.019 Maxillary growth according to the severity of cleft

Reported expression patterns for TGF-β receptors (TβR-I, -II, and -III) during palatogenesis suggest that they play essential roles in the mechanisms leading to palatal fusion. The purpose of this study was to compare the functions of the three TβRs during palatal fusion.Using organ culture of mouse palatal shelves, expression levels of TβR-I, -II, and -III were suppressed by transfecting the siRNAs siTβR-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signalling via each TβR. Linkage between TGF-β signalling and critical events in palatal fusion led to the use of, MMP-13 expression as an outcome measure for the function of the TGF-β receptors.The siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siTβR-I or -II, palatal shelves at E13 + 72 h were not fused, with complete clefting in the anterior and posterior regions. The middle palatal region following treatment with either siTβR-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siTβR-III resulted in a persistent midline seam of medial edge epithelium (MEE) in the anterior region with islands of persistent MEE in the middle and posterior regions of the midline. Treatment with all three siTβRs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siTβR-I or -II, but not -III, showed altered MMP-13 expression levels.The ability to identify and recover MEE and palatal mesenchymal cells during palatal fusion will aid in the evaluation of the different mechanistic events regulated by each TβR during palatogenesis.

The role of twist during palate development

In palatogenesis, the MEE (Medial Edge Epithelium) cells disappear when palates fuse. We hypothesize that the MEE cells undergo EMT (Epithelial-Mesenchymal Transition) to achieve mesenchyme confluence. Twist has an important role in EMT for tumor metastasis. The purpose of this study was to analyze Twist function during palatal fusion. Twist protein was expressed in palatal shelves and MEE both in vivo and in vitro just prior to fusion. Twist mRNA increased in chicken palates 3 and 6 hr after TGFbeta3 treatment. Palatal fusion was decreased when cultured palatal shelves were treated with 200 nM Twist siRNA and the subcellular localization of beta-catenin was altered. Twist mRNA decreased in palatal shelves treated with TGFbeta3 neutralizing antibody or LY294002, a specific phosphatidylinositol-3 kinase (PI-3K) inhibitor. In summary, Twist is downstream of TGFbeta3 and PI-3K pathways during palatal fusion. However, decreasing Twist with siRNA did not completely block palate fusion, indicating that the function of Twist may be duplicated by other transcription factors.

Analysis of Zfhx1a mutant mice reveals palatal shelf contact-independent medial edge epithelial differentiation during palate fusion

Cleft palate is a common birth defect that involves disruptions in multiple developmental steps such as growth, differentiation, elevation, and fusion. Medial edge epithelial (MEE) differentiation is essential for palate fusion. An important question is whether the MEE differentiation that occurs during fusion is induced by palate shelf contact or is programmed intrinsically by the palate shelf itself. Here, we report that the loss of Zfhx1a function in mice leads to a cleft palate phenotype that is mainly attributable to a delay in palate elevation. Zfhx1a encodes a transcription regulatory protein that modulates several signaling pathways including those activated by members of the transforming growth factor-beta (TGF-beta) superfamily. Loss of Zfhx1a function in mice leads to a complete cleft palate with 100% penetrance. Zfhx1a mutant palatal shelves display normal cell differentiation and proliferation and are able to fuse in an in vitro culture system. The only defect detected was a delay of 24-48 h in palatal shelf elevation. Using the Zfhx1a mutant as a model, we studied the relationship between MEE differentiation and palate contact/adhesion. We found that down-regulation of Jag2 expression in the MEE cells, a key differentiation event establishing palate fusion competence, was independent of palate contact/adhesion. Moreover, the expression of several key factors essential for fusion, such as TGF-beta3 and MMP13, was also down-regulated at embryonic stage 16.5 in a contact-independent manner, suggesting that differentiation of the medial edge epithelium was largely programmed through an intrinsic mechanism within the palate shelf.

Morphological and immunohistochemical studies on cleft palates induced by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin in mice

Morphological and immunohistological examinations were performed to reveal the mechanisms of cleft palate induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). ICR strain mice 8-10 weeks of age were used in the study. TCDD was administered in olive oil on gestation day (GD) 12.5 with gastric tubes at 40 microg/kg. From GD 13.5 to 16.5, palates were examined by scanning electron microscopy (SEM), hematoxyline-eosin (HE) staining, and immunohistochemical staining of FGFR1/2, TGF-beta3, MSX1 and LHX8. In the control group, both of the palatal shelves began elevating on GD 14.0 and finished within 6 h. After the elevation, all of the shelves had completely fused with each other on GD 14.5. In the TCDD-treated group, palatal shelves elevated 1 day later than in the control group. However, all palates had elevated by GD 15.0. After the elevation, the shelves contacted each other and fused; however, they were separated on GD16.0. HE staining showed that medial edge epithelium (MEE) was thinner in the TCDD group than in the control group. MEE observed under a high magnification (x2500) exhibited filopodia-like filaments and the cells were bulged in the control group. In contrast, in the TCDD group, no filaments were observed and the cells were flat with unclear boundaries. Immunohistologically, there were no characteristic findings except for FGFR1. FGFR1 was not expressed in the TCDD group after the fusion phase (GD 14.5). TCDD induces many morphological and molecular changes to MEE cells and causes cleft palates.

Follistatin antagonizes transforming growth factor-β3-induced epithelial–mesenchymal transition in vitro: implications for murine palatal development supported by microarray analysis

Epithelial–mesenchymal transition (EMT) is involved in normal embryonic development as well as in tumor progression and invasiveness. This process is also known to be a crucial step in palatogenesis during fusion of the bi-lateral palatal processes. Disruption of this step results in a cleft palate, which is among the most frequent birth defects in humans. A number of genes and encoded proteins have been shown to play a role in this developmental stage. The central role is attributed to the cytokine transforming growth factor-β3 (TGF-β3), which is expressed in the medial edge epithelium (MEE) already before the fusion process. The MEE covers the tips of the growing palatal shelves and eventually undergoes EMT or programmed cell death (apoptosis). TGF-β3 is described to induce EMT in embryonic palates. With regard to the early expression of this molecule before the fusion process, it is not well understood which mechanisms prevent the TGF-β3 producing epithelial cells from undergoing differentiation precociously. We used the murine palatal fusion to study the regulation of EMT. Specifically, we analyzed the MEE for the expression of known antagonists of TGF-β molecules using in situ hybridization and detected the gene coding for Follistatin to be co-expressed with TGF-β3. Further, we could show that Follistatin directly binds to TGF-β3 and that it completely blocks TGF-β3-induced EMT of the normal murine mammary gland (NMuMG) epithelial cell line in vitro. In addition, we analyzed the gene expression profile of NMuMG cells during TGF-β3-induced EMT by microarray hybridization, detecting strong changes in the expression of apoptosis-regulating genes.

Alteration of medial-edge epithelium cell adhesion in two Tgf-β 3 null mouse strains

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β 3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β 3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α 5- and β 1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β 3 or neutralizing antibodies against fibronectin or the α 5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β 3 null mutants; the importance of TGF-β 3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α 5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β 3 null mutant mice.