TGFβ3 Regulates Periderm Removal Through ΔNp63 In The Developing Palate

Background and ObjectivesCleft lip/palate is the most common craniofacial deformity in human. It can be divided into several categories according to the genetic etiology:non-syndromic cleft lip with or without cleft palate (NSCL/P), non-syndromic cleft palate only (NSCPO), syndromic cleft lip with or without cleft palate, syndromic cleft palate only. NSCL/P has a multifactorial etiology that includes both genetic and environmental factors.Development of the palate comprises sequential stages of growth, elevation, adherence, and fusion of the palatal shelves. In all of these stages, palatal mesenchyme and epithelial components play major roles in the complete formation of the palate. Palatogenesis requires precise and chronological regulation of key physiological processes, such as growth, proliferation, development, differentiation, migration, signaling, epithelial-mesenchymal transition (EMT), and apoptosis. Failure in any of these steps results in cleft palate. There is compelling evidence for the vital role of transforming growth factor β (TGFβ) mediated regulation of palate development.Previous studies showed that the palatal mesenchyme expresses all three TGFβ isoforms (1,2 and 3) during palatogenesis. Around E11.0-E11.5, the palatal shelves outgrow from maxilla and grow vertically along both sides of the tongue. TGFβ3 was detected in the palatal epithelium at this stage. At E12.0-E12.5, palatal shelves continue growing vertically, TGFβ1 and TGFβ2 could be detected in both palatal mesenchymal and epithelial cells. At E13.0-E13.5, the two palatal shelves elevates to the horizontal position. A strong expression of TGFβ3 was detected in the palatal epithelial cells. TGFβ1 was also detected in the epithelial cells. Meanwhile, TGFβ1 and TGFβ2 were detected in the palatal mesenchymal cells. At E14.0-E14.5, three isoforms were detected before the adherence of the two palatal shelves. At E15.0-E15.5, the two palatal shelves start fusing with each other. The expression of TGFβ1 and 3 decreased while the expression of TGFβ2 was detected in the mesenchyme at a high level. At E16.0-E16.5, the two palatal shelves fused with each other to form a whole palate. TGFβ1 was detected in the ossification area while TGFβ2 and TGFβ3 were detected in the palatal mesenchyme.Interestingly, TGFβ3 (-/-) mice always have cleft palate, which can be reversed by addition of TGFβ3, implying an important role of TGFβ in palate development. However, the role of TGFβ in promoting palatal mesenchymal cell proliferation has never been explored. We hypothesized that TGFβ enhances palatal mesenchymal cell proliferation in palatogenesis. And the isoforms of TGFP regulate different cellular biofunctions of the palatal mesenchyme to various extents. During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth and fusion of the two palate shelves.The periderm is a flat layer of epithelium created during embryonic development. Chiefly, the periderm forms a protective layer against immature adhesion of oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate.The mechanism(s) of periderm generation from the differentiation of the basal MEE and its timely desquamation processes are not fully understood. Still, the transforming growth factor β3 (TGFβ3) gene has been attributed to the final phase of palatal confluence, palatal seam disintegration. Although the timing of TGFβB expression in the MEE coincides with periderm formation and degeneration, its roles in MEE differentiation and periderm desquamation have not been methodically studied.Recent studies indicate that regulation of truncated p63 (ΔNp63), which depends on the transcription factor interferon regulatory factor 6 (IRF6), facilitates epithelial differentiation and periderm vitality. Interestingly, murine knockout (-/-) models of three genes, ΔNp63, TGFβ3, and IRF6, are born with palatal clefts due to palatal shelves failure to adhere, suggesting that these genes coordinate effective signaling pathways to regulate palatal epithelial differentiation. Despite such observations of similar phenotypes in null mouse models of these three genes in independent studies, no studies have been undertaken to delineate the role of all three genes during palate development.We hypothesized that TGFβ3 regulates expression of IRF6 and p63 genes, thereby coordinating periderm desquamation. We performed biochemical analysis, gene activity assays, and protein expression assays using palatal sections of TGFβ3 (-/-), ΔNp63 (-/-) and wild-type (WT) embryos, as well as primary MEE cells from WT palates.During elongation of palatal shelves, we observed restricted expression of ΔNp63 at the palatal periderm of TGFβ3 (-/-), but not in WT embryos, suggesting TGFβ3 regulates ΔNp63 during removal of the periderm. We also found that TGFβ3 activates transcription factor IRF6, which acts as a repressor for ΔNp63. In the absence of TGFβ3, ΔNp63 remains activated, which prevents removal of periderm and causes a palatalΔ cleft. Our results also show that cleft occurs in ΔNp63 (-/-) palates due to premature, rather than inadequate, fusion as observed in TGFβ3 (-/-). Overall, our data suggest that periderm degeneration in palatogenesis depends on functional TGFβ3 and IRF6 to repress ΔNp63. Thus, our study shows that the absence of TGFβ3 results in improper periderm removal that impedes palatal seam formation leading to the formation of cleft palate.Materials and Methods1. Immunohistochemistry (IHC) and immunofluorescence staining were performed on palatal sections of TGFβ3 (-/-), ΔNp63 (-/-) and wild-type (WT) embryos, in order to detect the expression pattern and the cellular localization of related proteins in tissues of different genotypes and different stages.2. Primary MEE cells from WT palates were treated with different doses of exogenous recombinant human TGFβ3 (rTGFβ3) for 12,24,36, and 48 hours. Cells were then harvested and protein was extracted for western blot analysis to analyze the expression change of ΔNp63 and IRF6. Also mRNA from the treated and untreated cells was collected and RA quantative real-time polymerase chain reactions (qRT-PCR) were performed to detect the change of the mRNA level of ΔNp63 and IRF6.3. The characteristics of palatal shelves from 14.0 to 15.0 dpc were evaluated using Scanning Electron Microscopy (SEM).4. The palate shelves from 14.25dpc were characterized using Atomic Force Scanning Microscopy tip-enhanced Raman scattering (TERS).5. Plasmid Constructs and Luciferase Activity Assay.Results1、Using structural and ultrastructural analysis (H&E staining, SEM scanning and AFM scanning), we demonstrate that during palatogenesis, the expression of ΔNp63 protein in the MEE at 14.25 dpc as well as the newly formed palatal seam at 14.5 dpc of the WT palates was significantly less than TGFβ(-/-) palates. These results imply a correlation between the absence of TGFβ3 in knockout palates and persistence of the periderm layer in relationship to increased expression of the ΔNp63 protein.2、TGFβ3 signaling can repress the expression of ΔNp63, which is essential for periderm desquamation. The TGFβ (-/-) palates showed increased expression of ANp63 in the MEE at 14.25 dpc as well as the newly formed seam at 14.5 dpc. In the absence of TGFβ3, ΔNp63 remains activated, which prevents removal of periderm and causes a palatal cleft.3、While TGFβ3 (-/-) palatal cleft occurs because the periderm fails to degenerate and its persistence blocks palatal fusion, the palatal cleft occurs in ΔNp63 (-/-) is caused by the absence of the periderm or the protective layer, promoting premature fusion of the palatal processes with developing nasal side epithelium of the nasal septum, following undisturbed palatal shelf lift from vertical progress to a horizontal position at 14.5 dpc.4、Periderm degeneration in palatogenesis depends on functional TGFP3 to repress ΔNp63. The absence of TGFβ3 results in improper periderm removal that impedes palatal seam formation, leading to the formation of cleft palate.Conclusions1、When two opposite palatal shelves are in proximity to adhere, subsequent elimination of the periderm is crucial to ensure precise fusion. However, in TGFβ3 (-/-) mice, as the presence of periderm, proper development of the palate does not happen;2、The periderm degeneration in palatogenesis depends on functional TGFβ3 to repress ANp63;3、Both TGFβ33 knock out and ΔNp63 knock out can result in cleft palate, but due to entirely contrasting mechanisms;4、At the beginning of palatal development, the activation of IRF6 transcription can upregulate ANp63 expression, which can result in the generation and maintainence of periderm. However, before palate shelves contacting each other, the increased TGFβ3 expression can reduce ANp63 expression, resulting in periderm degeneration, and then the palate shelves can fuse with each other, forming the main body of the hard palate.