Figure 6.

Runx2 and Fgfr2 expression and BrdU labeling in the calvaria and mandible of wild-type, Sp7^−/−, and Runx2^−/− mice (A–U) Frontal sections of wild-type (A,D,G,J,M,P,S) and Sp7^−/− (B,E,H,K,N,Q,T), and Runx2^−/− (C,F,I,L,O,R,U) mice at E18.5 were reacted with the anti-Runx2 antibody (A–F,M–O) and anti-Fgfr2 antibody (G–I,P–R) or subjected to BrdU labeling (J–L,S–U). The boxed regions in A are magnified in D and M, the boxed regions in B are magnified in E and N, and the boxed regions in C are magnified in F and O. The boxed regions in (D–F) and (M–O) are magnified in (D’–F’) and (M’–O’), respectively. The similar regions of (D’–F’) are shown in (G–I) and (J–L), and those of (M’–O’) are shown in (P–R) and (S–U). Brackets in (D’–L) indicate the layers of osteoblastic cells or osteoblast progenitors in the calvarial region, short arrows in (G–I) indicate muscle fibers, arrowheads in (G–I) and (P–R) indicate chondrocytes, and long arrows in R indicate neurons. Bars: 500 μm (A–C), 100 μm (D–F,M–O), 20 μm (D’-L, M’–U). (V) Real-time RT-PCR analysis. RNA was extracted from the calvariae of wild-type, Sp7^−/−, and Runx2^−/− mice at E18.5. The values in wild-type mice were set as 1, and relative levels are shown. Data are the mean ± SE of 4–5 mice. *vs. wild-type mice. *p < 0.05, **^,††p < 0.01. (W) Western blot analysis. Protein was extracted from the calvariae of wild-type, Sp7^+/−, and Sp7^−/− mice at E18.5. β-actin was used as an internal control. (X) Quantification of Western blot bands. The normalized values of Runx2 protein bands in wild-type mice were set as 1, and the relative levels in Sp7^−/− embryos are shown. Data are the mean ± SE of 3 bands. (Y and Z) BrdU-positive osteoblastic cells and osteoblast progenitors in calvariae (Y) and mandibles (Z) were counted and shown as a percentage of the number of osteoblastic cells and osteoblast progenitors. n = 6. **^,††p < 0.01.