Folic acid and the cleft palate of Tgf-β₃ null mice

Tgf-ß₃ null mutant mice (Tgf-ß₃ -/-) have cleft palate (CP). This CP is mainly due to lack of bilateral medial edge epithelia (MEE) adhesion and disappearance, although a decrease of palatal mesenchymal cell proliferation may also cooperate. A low intake of dietary Folic Acid (FA) has been correlated with CP appearance, whilst supplements of FA have been seen to reduce the risk of this congenital malformation. Objective: Our aim has been to determine whether a supplement of FA added either to Tgf-ß₃ +/- dams or Tgf-ß₃ -/- palatal shelf cultures benefits the CP presented by the Tgf-ß₃ -/- mice. Methods: The diet of Tgf-ß₃ +/- females was 2 mg FA/kg diet (control) or supplemented with 40 mg FA/kg diet (20 fold the standard levels) for 2 to 16 weeks. Pregnant females were sacrificed at embryonic day (E) 17. The palate of Tgf-ß₃ -/- embryos was observed macro and microscopically. In addition, E13.5 Tgf-ß₃ -/- palatal shelves were apposed and cultured for 18 or 36 hours in 1% ascorbic acid DMEM/F12, in the absence (control) or presence of 4 or 20 fold supplement of FA. Analysis of the presence of dead cells (TUNEL assay) and palatal shelf adhesion and fusion was then performed. Results: The Tgf-ß₃ -/- progeny of Tgf-ß₃ +/- dams FA supplemented had 17.24% presence of a milder form of CP, whilst this was only observed in 3.4% of the controls. In the milder cases, palatal shelves appeared either partially adhered or in closer contact. No differences between groups were observed in the culture study. Conclusion: Dietary FA supplement to Tgf-ß₃ +/- females softens the severity of the cleft palate presented by Tgf-ß₃ -/- embryos, through mechanisms other than MEE cell death, adhesion and fusion.

Supported by grants from FIS (PI06/0184) and UCM-CM Research Group 920202, 2006.