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-b 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-b 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 a 5 -and b 1 -integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-b 3 or neutralizing antibodies against fibronectin or the a 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-b 3 null mutants; the importance of TGF-b 3 to restore their normal pattern of expression; and the crucial role of fibronectin and the a 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-b 3 null mutant mice.Key words cleft palate Á Tgf-b 3 Á mouse Á collagen Á laminin Á fibronectin Á a 5 b 1 -integrin Á ICAM-1 Á Nectin-1

Folic acid (FA) is essential for numerous bodily functions. Its decrease during pregnancy has been associated with an increased risk of congenital malformations in the progeny. The relationship between FA deficiency and the appearance of cleft palate (CP) is controversial, and little information exists on a possible effect of FA on palate development. We investigated the effect of a 2–8 weeks’ induced FA deficiency in female mice on the development of CP in their progeny as well as the mechanisms leading to palatal fusion, i.e. cell proliferation, cell death, and palatal-shelf adhesion and fusion. We showed that an 8 weeks’ maternal FA deficiency caused complete CP in the fetuses although a 2 weeks’ maternal FA deficiency was enough to alter all the mechanisms analyzed. Since transforming growth factor-β₃ (TGF-β₃) is crucial for palatal fusion and since most of the mechanisms impaired by FA deficiency were also observed in the palates of Tgf-β₃null mutant mice, we investigated the presence of TGF-β₃ mRNA, its protein and phospho-SMAD2 in FA-deficient (FAD) mouse palates. Our results evidenced a large reduction in Tgf-β₃ expression in palates of embryos of dams fed an FAD diet for 8 weeks; Tgf-β₃ expression was less reduced in palates of embryos of dams fed an FAD diet for 2 weeks. Addition of TGF-β₃ to palatal-shelf cultures of embryos of dams fed an FAD diet for 2 weeks normalized all the altered mechanisms. Thus, an insufficient folate status may be a risk factor for the development of CP in mice, and exogenous TGF-β₃ compensates this deficit in vitro.