Involvement of type XII collagen in bone formation. (A–F) Type XII collagen is localized to bone-forming regions. Reactivity for type XII collagen is localized to the periosteum (PO), endosteum (EO), and trabecular bone (TB). Immunofluorescence analysis of the mid-diaphysis in cross (A and B) and longitudinal (D) sections of the femur and calvaria (E and F) from P30 wild type (Col12a1^+/+) and the mid-diaphysis in cross sections of the femur in type XII collagen–null (Col12a1^−/−) mice (C). Green, anti–type XII collagen; blue, nuclei reactive with DAPI; CB, cortical bone; BM, bone marrow; OB, osteoblast. (G) A schematic representation of the wild-type Col12a1 exons 1–8 and target locus to create type XII collagen–null mice. After homologous recombination, the exon 2–5 locus was replaced by the selection cassette that includes the phosphoglycerolkinase (PGK) neomycin-polyadenylation (NEO polyA) signal. (H) Analysis of type XII collagen expression in tissues from wild-type and Col12a1^−/− mice by immunoblotting. FDL, flexor digitorum longus. (I) X-ray and skeletal staining with alizarin red of 5-mo-old Col12a1^+/+ and Col12a1^−/− mice. Col12a1^−/− mice demonstrated kyphosis (asterisk) in the x-ray image. Abnormal double spinous processes (arrows) were present in the vertebral column of Col12a1^−/− mice visualized by skeletal staining. L3, third lumbar. (J–L) Body weight, femur length, and percentage of tissue area/length were measured in P30 wild-type (control; n = 9) and type XII collagen–null (n = 10) mice. (J) Body weight is decreased in Col12a1^−/− mice. (K) Col12a1^−/− mice have shorter femurs compared with wild-type mice when normalized to body weight. (L) The percentage of tissue area/length normalized to body weight revealed that Col12a^−/− mice have more slender femurs compared with wild-type controls. **, P < 0.01. Error bars are mean ± SD. Bars: (A, D, and E) 25 µm; (B, C, and F) 250 µm; (I, left) 2 cm; (I, right) 1 mm.