Fig. 2. Restricted domains of alkaline phosphatase and Runx2 expression in Msx2 mutant embryos. We examined alkaline phosphatase (ALP) activity and Runx2 expression in the developing frontal bones of Msx2 mutant and control embryos. (A-L) Whole-mount ALP stains of heads at the indicated stages. (A-D) Dorsal views; (E-L) Lateral views. Boxed areas are shown at higher magnification in the panels below. Note the reduced area of ALP staining in mutant embryos (C,D arrowheads; H, arrowhead, broken line). The arrow in K indicates a halo of ALP-positive cells which is not present in the Msx2 mutant (L). (M,N) Cross-sections at the levels indicated by lines in K and L. The area of ALP staining in three mutant and three wild-type individuals was measured by quantitating pixels (O). Error bars indicate one standard deviation. The wild-type and Msx2^–/– data sets were compared using Student's t-test. (P-S) In situ hybridization analysis of Runx2 expression in wild type (P,R) and Msx2 mutant (Q,S) embryos. Tissue sections through the prospective frontal bone of E12.5 (P,Q) and E11.5 (R,S) embryos were incubated with a ³³P-labeled Runx2 antisense probe. The sections were subjected to autoradiography and photographed under dark field. Schematics depicting key anatomical features of the sections are shown on the right. Note that Runx2 expression is first detectable between E11.5 and E12.5. Note also that the hybridization signal is reduced in the frontal bone rudiment of the Msx2 mutant compared with the wild type. ch, cerebral hemisphere; cs, coronal suture; e, eye; fb, frontal bone rudiment; pb, parietal bone rudiment. Scale bars: 1 mm in B,F; 500 µm in D,H,J; 200 µm in L,N,Q.