TGFβ3 Regulates Periderm Removal Through ΔNp63 in the Developing Palate

Abstract

The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the 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. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFβ3, interferon regulatory factor 6 (IRF6) (-/-), and truncated p63 (ΔNp63) (-/-) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion.

p63 exerts spatio-temporal control of palatal epithelial cell fate to prevent cleft palate

“…As p63 expression is maintained in the MEE of Tgfb3 -/- mice which exhibit cleft palate and persistent periderm cells over the MEE [12,19,23,24], we manipulated the level of p63 in the MEE of Tgfb3 -/- mice genetically. Initially, we generated Tgfb3 +/- ; p63 +/- compound heterozygous mice which did not exhibit any gross abnormalities and in which fusion of the secondary palate proceeded normally.…”

“…During palatogenesis in wild-type embryos, confocal imaging of GFP expression over a 24-hour period revealed that periderm cells migrated out of the MEE towards the epithelial triangles and into the oral and nasal epithelia of the palatal shelves allowing MES degradation to be completed (S1 Video). As p63 is down-regulated in the MEE of wild-type but not Tgfb3 -/- mice which exhibit cleft palate and persistent periderm cells over the MEE [12,19], we crossed the mKrt17 -GFP reporter onto a Tgfb3 -/- background and noted that GFP-labelled periderm cells failed to migrate out of the MEE despite forced contact between the palatal shelves (Fig 2D–2F; S2 Video). In contrast, the migratory periderm phenotype observed in the MEE of wild-type mice was restored in Tgfb3 -/- ; p63 +/- embryos (Fig 2G–2I; S3 Video).…”

“…Moreover, the latter process does not occur in Tgfb3 -/- embryos in which the palatal shelves remain cleft. Intriguingly, while p63 is down-regulated in the MEE of wild-type embryos, p63 expression is maintained in the Tgfb3 -/- MEE and the periderm cells fail to migrate to the oral and nasal epithelial triangles [12, 19] (Figs 1 and 2). …”

“…Although the mechanisms by which MES degeneration is completed have been controversial, the prevailing evidence supports a major role for cell death [9–11]. Moreover, competence for palatal shelf fusion is precisely regulated: periderm acts as a barrier which prevents premature adhesion of the oral epithelia and removal of periderm from the MES is therefore a prerequisite for palatal fusion [8,12]. However, the molecular mechanisms underlying the precise spatio-temporal control of palatal adhesion/fusion remain incompletely characterized.…”

Activation of Notch1 inhibits medial edge epithelium apoptosis in all-trans retinoic acid-induced cleft palate in mice

“…TGF-b, Shh, BMP, and Wnt signaling pathways have all been considered to contribute to palatal morphogenesis [2]. TGF-b3, which plays an essential role in palatogenesis, has been extensively studied [6]. It has been proposed that the MES independently but sequentially undergoes cell cycle arrest, cell migration, and apoptosis following the treatment of isolated MES cells with TGF-b3 [7].…”