TGF-β3-induced Palatogenesis Requires Matrix Metalloproteinases

Abstract

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membraneassociated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-␤3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-␤3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-␤3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion. INTRODUCTIONThe formation of the palate is of critical importance to separate the oropharynx from the nasopharynx. A dysfunction in one of the regulators of this developmental process can lead to a cleft palate, one of the most common birth defects in humans (Chenevix-Trench et al., 1992). In the mouse embryo, the entire process of palatal formation takes place between day 12 and 15 (E12 and E15) of development (Ferguson, 1988). The fusion itself occurs over a relatively short period of time during which the medial edge epithelia (MEE) of the shelves form a midline seam, which is then disrupted to allow mesenchymal continuity (Pourtois, 1966;Smiley and Koch, 1971). Complete fusion of the secondary palate requires disappearance of the MEE from the midline, as well as the breakdown of their basement membrane.The molecular mechanisms controlling palatal fusion are complex and not fully understood. However, studies in the mouse have pointed to primary and secondary causes of defective palatogenesis. In mice deficient for the epidermal growth factor receptor or the platelet-derived growth factor receptor, a cleft palate is often associated with a primary defect in the development of the first branchial arch (Shiota et al., 1990;Brunet et al., 1993;Robbins et al., 1999). In these cases, delayed development of the lower jaw interferes with forward displacement of the tongue and prevents the elevation and subsequent fusion of the shelves (Robbins et al., 1999). In transforming growth factor (TGF)-␤3-deficient mice a cleft palate develops in all mice due to ...

TGFB3 displays parent-of-origin effects among central Europeans with nonsyndromic cleft lip and palate

“…One of the key components in palatal fusion is transforming growth factor b3 (Tgf-b3) (Nogai et al 2008). Tgf-b3 expression, which is coordinated temporally during palatogenesis (Britto et al 2002), determines ECM macromolecule overaccumulation by stimulating ECM neosynthesis and by inhibiting many enzymes implicated in ECM degradation (Blavier et al 2001). Consequently, reduced activity of Tgf-b3 may increase the risk for oral clefting.…”

Jag2‐Notch1 signaling regulates oral epithelial differentiation and palate development

“…10A,B). We also examined the expression pattern of Mmp13, a known downstream target of Tgf␤3 signaling (Blavier et al, 2001), in wild-type and Jag2 ⌬DSL/⌬DSL mutant embryos. Mmp13 mRNA expression is restricted to the palatal MEE in the oral epithelium of wild-type embryos at E14.5 (Fig.…”

Orofacial clefting: recent insights into a complex trait

“…The chick, therefore, has the advantage of mirroring the pathology seen in cleft palate, whereas the mouse provides an excellent model to study palatal shelf fusion. Indeed, studies in these animal models have helped to identify a battery of genes essential for palatal formation: Tgfb3 [44]; Bmps [45]; Tbx22 [46]; Fgfrs [47]; Pdgfc [48]; RhoA [49]; the gene encoding PtdIns-3 kinase [43]; Gabrb3 [50]; Gad1 [51,52]; Cspg [53]; and Mmps and Timp2 [54].…”