Retinoic Acid Induces Cleft Palate by Suppressing Fgf10 in the Bend Region of the Palatal Shelves

Retinoic acid (RA) is essential for normal embryonic development in vertebrates. Administration of excess RA during pregnancy can cause various craniofacial anomalies including cleft palate. At Plastic Surgery 2008, we reported that excess RA might prevent tongue withdrawal in mouse fetuses by down-regulating Tbx1 expression in the mesenchymal cells of the developing tongue, resulting in abnormal morphogenesis of tongue intrinsic muscles. In the present study, we observed that RA caused suppression of Fgf10 in the bend region of the palatal shelves, which was considered to be critical for elevation of the palatal shelves.

Both RA-degrading enzymes (Cyp26a1 and Cyp26b1) and one of the RA-synthesizing enzymes (retinaldehyde dehydrogenase2: RALDH2) are expressed at early stages of normal palatal development, and RA induces remarkable up-regulation of Cyp26a1 and Cyp26b1. Disruption of Cyp26 can cause a higher level of RA in a stage- and region-fashion in mouse development. Although Cyp26a1 knockout (KO) mice reveal no phenotype in the palate, Cyp26b1 KO mice possess cleft palate in a rate of 100%, mimicking the phenotype of Fgf10 KO mice. In developing mouse palate, Cyp26a1 was expressed in the epithelium on the lateral sides of the palatal shelf between embryonic day (E) 11.0 and E13.5. On the other hand, Cyp26b1 began to be expressed in the mesenchyme of the bend region of palatal primordia at E11.0 and E11.5 just before expression of Fgf10. By E12.5 and E13.5, its expression was displaced ventrally and detected in close to the oral epithelium. We also examined the RA distribution by b-galactosidase staining using RARE-hsplacZ reporter mice. RARE-hsplacZ positive cells was observed in palatal primordia in normal fetuses, but in Cyp26b1 KO fetuses, it was exhibited a diffuse distribution in the palatal mesenchyme.

Fgf10 is one of target genes of Tbx1, a candidate gene of DiGeorge/velocardiofacial syndrome (DGS/VCFS). Tbx1 can induce expressions of Cyp26s and is also down-regulated in response to excess RA. DGS/VCFS patients often show cleft palate, which was considered to be due to the change of a RA level in developing palate. Therefore, our findings provide a new insight into the important role of the bend region for the elevation of palatal shelves and the pathogenetic and molecular mechanisms of cleft palate caused by excess RA.