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.

This study suggests that an adequate folic acid/folate status plays a key role in the formation of ocular tissues and structures, whereas a vitamin deficiency is negatively associated with a normal eye development even after a short-term exposure.

The Tgf-β₃ null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β₂, Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β₃, and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β₃ null mutant mouse palate plays a role in the cell proliferation defect observed.