Defective organization and polarization of osteoblasts in type XII collagen–null bone. Osteoblast polarization was analyzed by the distribution of nucleus, Golgi apparatus, and F-actin staining with confocal fluorescence microscopy. (A) Wild-type osteoblasts (OB) in an epithelioid arrangement are well organized into a layer between the periosteum (PO) and cortical bone (CB), and F-actin fibers (green) are highly enriched in regions of the osteoblasts, which face the periosteum and cortical bone (white arrows). (B) In contrast, Col12a1^−/− osteoblasts are not well organized between the periosteum and cortical bone, and F-actin fibers appear to form aggregates distributed randomly within osteoblasts (arrows). (C) Wild-type osteoblasts exhibit a polarized distribution of the Golgi apparatus (red) and the nucleus (blue). Most Golgi reactivity is localized toward the bone matrix (arrows), whereas the nuclei were positioned to the opposite side. (D) Col12a1^−/− osteoblasts also fail to show a polarized distribution of Golgi and nucleus. It is noticeable that the F-actin fibers (yellow arrows) extend to the cortical bone matrix, establishing a network throughout the bone matrix in wild-type bone. This network is not as extensive in Col12a1^−/− bone. Bars: (A and B) 20 µm; (C and D) 10 µm.