1. Retinoic Acid Induces Cleft Palate by Suppressing Fgf10 in the Bend Region of the Palatal Shelves. Yasuo Sakai 1, M.D., Ph.D. Junko Okano 2, M.D., Ph.D. Kohei Shiota 3, M.D., Ph.D.

2. Institutes 1 Department of Plastic Surgery, Osaka University School of Medicine, Japan 2 Developmental Skin Biology Section, NIAMS, National Institutes of Health, USA 3 Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Japan Disclosure/Financial Support Supported in part by Grant-in-Aid Scientific Research (C) (19590181) from the Ministry of Education, Culture, Sports, Science and Technology (to Y.S.).

3. Objective of the Study Retinoic acid (RA) is essential for normal embryonic development in vertebrates. We have examined the affect of RA on the palate formation. In the study, we focused on the bend region of the palatal shelves where is critical for their elevation.

4. T Palate Development E14.5: Elevation E15.0: Fusion E11.5-13.5: Elongation T, tongue; P, palatal shelf. P Bend Region

5. Materials and Methods Cyp26b1 knock out (KO) mice RA treatment –Gastric intubation to E11.5 pregnant ICR mice (RA100mg/kg) In situ hybridization RARE (RA response element)-hsp lacZ transgenic mice Real-time RT-PCR

6. Results-1 Both Cyp26b1 (RA-degrading enzyme) KO embryos and RA- treated ones revealed cleft palate without elevation of the palatal shelves. T, tongue; P, palatal shelf; gg, genioglossus muscle. Note abnormal protrusions on the tongue (arrows) and poorly differentiated muscles (arrow heads) in the mutant. Wild type Cyp26b1-/- T T gg P P E15.5

7. Results-2 Expression patterns ofCyp26b1 in the developing palate. T, tongue; P, palatal shelf. Note Cyp26b1 is temporally expressed in the future bend region (arrow) at E11.5. Wild type RA-treated T T P P E11.5 E13.5 T P P T

8. Results-3 Expression patterns ofRaldh2 (RA-metabolizing enzyme) and RARE-laZ in the developing palate. T, tongue; P, palatal shelf; V2, cranial nerve V2. Wild type Cyp26b1-/- RA-treated Raldh2 E11.0 RARE- lacZ E13.5 T P T P V2 T P T P T P T P

9. Results-4 Fgf10 in the bend region is down-regulated both in Cyp26b1-/- embryos and RA-treated ones. T, tongue; P, palatal shelf; sg, submandibular gland. Altered expression patterns of Fgf10in the bend region (arrows). Wild type Cyp26b1-/- RA-treated T T sg P P E11.5 E13.5 P P P P T T T T

10. Results-5 Real-time PCR after RA treatment shows Fgf10 in the palatal shelves is significantly down-regulated at E11.5 (*P<0.01) and at E12.5 (*P<0.05). Control RA-treated

11. Results-6 Expression patterns of cleft palate related genes, Bmp2 and Shh at E13.5. T, tongue; P, palatal shelf. Wild type Cyp26b1-/- RA-treated T T P P Bmp2 Shh P P P P T T T T

12. Conclusions

13. Significance of the Findings 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. 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.