FGF9 Promotes Expression of HAS2 in Palatal Elevation via the Wnt/beta-Catenin/TCF7L2 Pathway

Background: Fgf9 mutation was found in cleft palate patients. Our previous study indicated that Fgf9 promotes timely elevation of palate by regulating hyaluronic acid (HA) accumulation at embryonic day 13.5 (E13.5). HA is synthesized by hyaluronic acid synthases (HAS) isoforms 1, 2, or 3. However, how FGF9 regulates HA in palatogenesis is still unclear. Methods: Using Ddx4-Cre mice, we generated the Fgf9(-/-) mouse model (with exon 2 deletion). Immunohistochemistry was used to detect the location and expression of HAS2 in WT and the Fgf9(-/-) palate at E13.5. We also predicted the association between Fgf9 and Has2 within the developing palate by performing a bioinformatics analysis. The expression of beta-catenin, HAS2, and TCF7L2 were verified by Western blotting after knockout of Fgf9. Rescue experiments were performed by ELISA in vitro. Results: Fgf9(-/-) mice exhibited 100% penetrance of the cleft palate. A knockout of Fgf9 confirmed that HAS2 and TCF7L2 expression was positively correlated with FGF9. TCF7L2 binds to the Has2 promoter, exhibiting the high specificity predicted by JASPAR. Additionally, increased HA expression by BML-284, TCF-dependent agonist, was blocked in Fgf9(-/-) palate because of the significant decline in TCF7L2 expression. Conclusions: FGF9 promotes HAS2 expression via Wnt/beta-catenin/TCF7L2 pathway with TCF7L2 activating transcription of Has2 in the palate.