Fig. 1

Treatment with RA or the Cyp26 inhibitor R115866 leads to impaired horizontal and vertical growth of calvaria. (A-C) Alizarin Red (AR) staining of calvarial pates of untreated juvenile wild-type zebrafish at the indicated standard length (SL); dorsal view of head; anterior to the right. For details, see text. (D-F) Magnified dorsal view of central head region of SL8-9 fish treated with DMSO (D), RA (E) or R115866 (rambazole; F) for 7days, after consecutive in vivo AR staining (red) before and calcein staining (green) after the treatment. The width of the green-only region is indicated by double-headed arrows. Arrowheads in D,E mark the position of the virtual transverse projections shown in Fig. S1A,B; asterisks in E,F mark diffuse and weak green staining in patent regions of the RA- and R115866-treated fish, but not in the control, most likely representing ectopic ossification of col1a2-encoded collagen in the overlying dermis (Le Guellec et al., 2004; see Fig. S1 and Discussion). (G,H) Transverse sections through frontal plate of SL8-9 fish treated with DMSO (G) or RA (H), in vivo stained with AR before and with DAPI after the treatment. Positions of osteogenic fronts before the treatment are indicated by regular arrows (revealed by the border of the AR-positive stripes); thicknesses of the frontal plates are indicated by double-headed arrows. Note the preferential vertical growth at the inner surface of the calvaria (larger distance between AR-positive stripe of ‘old’ bone and ventral surface of calvaria). (I,J) Quantification of reduced horizontal and vertical growth, expressed as percentage of added area (I) or width (J) of DMSO-treated controls. DMSO: n=7; RA: n=6; rambazol: n=4). Mean values±s.d. are shown. ****P<0.0001. cs, coronal suture; fp, frontal plate; ifs, interfrontal suture; of, osteogenic front; pp, parietal plate; ss, sagittal suture.