Delayed terminal differentiation in type XII collagen–null osteoblasts. (A–E) Primary osteoblasts were derived from neonatal calvaria of Col12a1^+/+ and Col12a1^−/− mice and cultured in osteogenic medium. The cells are harvested after 0, 7, 14, and 21 d in culture and used for quantitative real-time PCR analysis. Each group represents the mean of triplicate determinations. mRNA was normalized to Gapdh expression. (A) Type XII collagen mRNA expression is stable during osteoblast development. (B and C) Similar expression patterns are detected in mRNA expressions of Runx2 (B) and type I collagen (C) in Col12a1^+/+ and Col12a1^−/− osteoblasts. (D and E) The expression of osteocalcin (Ocn; D) and osteopontin (Opn; E), which are terminal osteoblast differentiation markers, is significantly decreased in Col12a1^−/− osteoblasts as compared with wild-type controls. **, P < 0.01. Error bars are mean ± SD. (F) Immunoblot analysis of P30 femurs from Col12a1^+/+ (n = 2) and Col12a1^−/− (n = 3) mice was performed to validate the in vitro data. Opn and Ocn expression is decreased, whereas type I expression is stable in Col12a1^−/− femurs compared with the mRNA data.