Fig. 7. Msx2 and Twist cooperatively regulate the differentiation and proliferation of skeletogenic mesenchyme. Cross-sections through the frontal bone rudiments of E12.5 (A-F) embryos of the indicated genotypes were tested for alkaline phosphatase activity (A,B), Runx2 expression (C,D) or Wnt1-Cre-driven-R26R-lacZ expression (E,F). Note reduced expression of ALP and Runx2, but unchanged expression of lacZ in Msx2^+/–; Twist^+/– embryos compared with Msx2^+/– embryos. (G-J) Cross-sections of E14.5 frontal bones stained for ALP activity (G,I) and adjacent sections stained for 10-phosphorylated histone H3 (H,J) to mark proliferating cells. P-H3-positive cells (arrowheads) were counted in the regions of developing frontal bones shown in H and J. The resultant counts were normalized to the area of ALP-staining shown in G and I, and are plotted in K. Error bars represent standard deviations derived from three independent experiments. Note the statistically significant (Student's t-test) reduction of labeling index in Msx2^+/–; Twist^+/– mutant embryos compared with Msx2^+/– embryos. ch, cerebral hemisphere; ep, epithelium; fb, frontal bone. Scale bars: 100 µm.