Fig. 4. Reduced production of bone extracellular matrix and decreased osteoblast proliferation in Smad4 mutant mice. (A-D) In situ hybridization of proximal tibias from 4-week-old control (A,C) or mutant mice (B,D). The expression of (B) osteopontin (OPN) and (D) osteocalcin (OC) was decreased in Smad4 mutant mice compared with controls (A,C). (E-H) Real-time PCR of Akp1 (ALP), osteocalcin, Col1a2 and Runx2 from calvarial bone extracts of 7-week-old Smad4 mutant (grey bars) and control mice (white bars). Values are presented as relative expression. (I) Sections of calvaria from 3-week-old mice showing a reduced number of BrdU-positive cells (arrowhead) in Smad4 mutant mice compared with control littermates. (J) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in control (white bars) and Smad4 mutant mice (grey bars). ^*P<0.01. Bar, 400 µm (A-D); 26.5 µm (I).