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

Yasuo Sakai1, M.D., Ph.D.Junko Okano2 , M.D., Ph.D. Kohei Shiota3 , M.D., Ph.D.

Institutes1Department of Plastic Surgery, Osaka University School of Medicine, Japan2Developmental Skin Biology Section, NIAMS, National Institutes of Health, USA3Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Japan

Disclosure/Financial SupportSupported in part by Grant-in-Aid Scientific Research (C) (19590181) from the Ministry of Education, Culture, Sports, Science and Technology (to Y.S.).

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.

T

Palate Development

E14.5: Elevation

E15.0: Fusion

E11.5-13.5: Elongation

T, tongue; P, palatal shelf.

P

Bend Region

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

Results-1Both 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

Results-2Expression 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 TP P E11.5

E13.5 T P PT

Results-3Expression 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

Raldh2E11.0

RARE-lacZ

E13.5

T

P

T P

V2V2 V2

T P

T P T P

T P

Results-4Fgf10 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

Results-5Real-time PCR after RA treatment shows Fgf10

in the palatal shelves is significantly down-regulatedat E11.5 (*P<0.01) and at E12.5 (*P<0.05).

ControlRA-treated

Results-6Expression patterns of cleft palate related genes,

Bmp2 and Shh at E13.5.

T, tongue; P, palatal shelf.

Wild type Cyp26b1-/- RA-treated

T T

PP Bmp2

Shh

P

P P P

T T T

T

Conclusions

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.