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.     

Bulging medial edge epithelial cells and palatal fusion

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

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.     

Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion

Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGFbetaRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGFbetaRII.     

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 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.     

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

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

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

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

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.     

Expression cloning and characterization of the TGF-beta type III receptor.

The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.     

In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type I receptor to bind TGF-beta2

Transforming growth factor beta (TGF-beta) ligands exert their biological effects through type II (TbetaRII) and type I receptors (TbetaRI). Unlike TGF-beta1 and -beta3, TGF-beta2 appears to require the co-receptor betaglycan (type III receptor, TbetaRIII) for high affinity binding and signaling. Recently, the TbetaRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-beta2 null mouse, implying the existence of TbetaRIII independent mechanisms for TGF-beta2 signaling. Because a variant of the type II receptor, the type II-B receptor (TbetaRII-B), has been suggested to mediate TGF-beta2 signaling in the absence of TbetaRIII, we directly tested the ability of TbetaRII-B to bind TGF-beta2. Here we show that the soluble extracellular domain of the type II-B receptor (sTbetaRII-B.Fc) bound TGF-beta1 and TGF-beta3 with high affinity (K(d) values = 31.7 +/- 22.8 and 74.6 +/- 15.8 pm, respectively), but TGF-beta2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTbetaRII.Fc). However, sTbetaRII.Fc or sTbetaRII-B.Fc in combination with soluble type I receptor (sTbetaRI.Fc) formed a high affinity complex that bound TGF-beta2, and this complex inhibited TGF-beta2 in a biological inhibition assay. These results show that TGF-beta2 has the potential to signal in the absence of TbetaRIII when sufficient TGF-beta2, TbetaRI, and TbetaRII or TbetaRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-beta receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures.     

Midline fusion in the formation of the secondary palate anticipated by upregulation of keratin K5/6 and localized expression of vimentin mRNA in medial edge epithelium.

Secondary palatal fusion is dependent on targeted removal of the epithelium between the palatal shelves. Aseptically delivered rat embryos 15 through 18 days post coitum (dpc) were probed with DIG-labeled antisense and sense ssDNA probes for spliced exon sequences flanking intron E of cytokeratins K5/6 and spliced exon sequences flanking intron F of vimentin. Cytokeratin K5/6 expression was upregulated in the medial edge epithelium (MEE) prior to rotation of the palatal shelves and in the vomerine epithelium in the region of fusion with the palate. K5/6 expression continued in the medial epithelial seam (MES) and in epithelial islands during breakdown of the MES. Vimentin expression was not detected in the MEE prior to rotation but was specifically upregulated in the MEE following rotation and prior to midline contact and continued in the MES and in epithelial cells identifiable during the breakdown of the MES. Initiation of vimentin upregulation in the MEE prior to contact of the palatal shelves was tested by serum-free organ culture of palates from embryos at 15.5 dpc with the shelves separated by a biocompatible membrane. Vimentin upregulation occurred in the epithelium specifically in the region of anticipated contact. These results are interpreted as indicating that i) cytokeratin K5/6 expression may play a critical role in the integration of the epithelial layers of the MES to ensure subsequent merging of the mesenchyme and ii) epithelial cells in the MEE are specifically 'primed' to upregulate expression of mesenchymal genes prior to integration into and breakdown of the MES.     

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.     

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.     

Endocytosis of the type III transforming growth factor-beta (TGF-beta) receptor through the clathrin-independent/lipid raft pathway regulates TGF-beta signaling and receptor down-regulation

Transforming growth factor-beta (TGF-beta) signals through three highly conserved cell surface receptors, the type III TGF-beta receptor (T beta RIII), the type II TGF-beta receptor (T beta RII), and the type I TGF-beta receptor (T beta RI) to regulate diverse cellular processes including cell proliferation, differentiation, migration, and apoptosis. Although T beta RI and T beta RII undergo ligand-independent endocytosis by both clathrin-mediated endocytosis, resulting in enhanced signaling, and clathrin-independent endocytosis, resulting in receptor degradation, the mechanism and function of T beta RIII endocytosis is poorly understood. T beta RIII is a heparan sulfate proteoglycan with a short cytoplasmic tail that functions as a TGF-beta superfamily co-receptor, contributing to TGF-beta signaling through mechanisms yet to be fully defined. We have reported previously that T beta RIII endocytosis, mediated by a novel interaction with beta arrestin-2, results in decreased TGF-beta signaling. Here we demonstrate that T beta RIII undergoes endocytosis in a ligand and glycosaminoglycan modification-independent and cytoplasmic domain-dependent manner, with the interaction of Thr-841 in the cytoplasmic domain of T beta RIII with beta-arrestin2 enhancing T beta RIII endocytosis. T beta RIII undergoes both clathrin-mediated and clathrin-independent endocytosis. Importantly, inhibition of the clathrin-independent, lipid raft pathway, but not of the clathrin-dependent pathway, results in decreased TGF-beta1 induced Smad2 and p38 phosphorylation, supporting a specific role for clathrin-independent endocytosis of T beta RIII in regulating both Smad-dependent and Smad-independent TGF-beta signaling.     

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.     

Ontogeny and localization of TGF-beta type I receptor expression during lung development

Transforming growth factor (TGF)-beta is a family of multifunctional cytokines controlling cell growth, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-beta are mediated by type I (TbetaR-I) and II (TbetaR-II) receptors. Our previous studies show that the expression of TbetaR-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of TbetaR-I during lung development. The expression level of TbetaR-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. TbetaR-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of TbetaR-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. TbetaR-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense TbetaR-I signal was observed in the epithelial lining of the developing bronchi. In gestational day 16 lungs, the expression of TbetaR-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of TbetaR-I expression. In postnatal lungs, TbetaR-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of TbetaR-I in cultured rat lung cells. TbetaR-I was expressed by cultured rat lung fibroblasts, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of TbetaR-I that is different from that of TbetaR-II during lung development. TbetaR-I, TbetaR-II, and TGF-beta isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for TbetaR-I in mediating TGF-beta signal transduction during lung development.