Figure 2

Predominant expression of obif transcript in osteoblast-lineage cells. (A, B) Expressions of obif in mouse embryos examined by whole mount in situ hybridization. (C-P) In situ hybridization analysis of mouse obif and Sox9 in the developing mouse limb, rib, and mandible. Obif expression was complementary to that of Sox9 in the developing limb bud (C-J), rib (D, H) and mandible (K-P). FL, forelimb; HL, hindlimb; NT, neural tube; to, tongue; m.c., Meckel's cartilage. Scale bars = 200 μm. (Q-Y) Section in situ hybridization of mouse obif in endochondral ossification. At E14.5 obif transcripts were detected in the perichondrium but were absent from more centrally located chondrocytic cells (Q, R). In E15.5 humerus and E17.5 tibia, obif expression was found in cells associated with bone trabeculae and in cells associated with the formation of bone collars (S-Y). Scale bars = 200 μm. (Z) Northern blot analysis of obif and Runx2 expression in 4 week-old mouse tissues. The arrow corresponds to a 2.1 kb obif transcript. (AA) Northern blot analysis of obif and Runx2 expression in the calvaria and humeri harvested from newborn, 3 week-old, and adult mice. (BB, CC) Northern blot analysis of obif and Runx2 transcripts in cell differentiation models of osteoblasts and chondrocytes. Total RNAs, extracted from MC3T3-E1 cells (BB: lane 1, undifferentiated cells; lane 2, cells cultured in differentiation medium for 24 days), ATDC5 cells (BB: lane 3, undifferentiated cells; lane 4, cells cultured in differentiation medium for 21 days), NIH3T3 cells (BB, lane 5), C2C12 cells (BB: lane 6, undifferentiated cells; lane 7, cells cultured in myogenic differentiation medium for 6 days; lane 8, cells cultured in osteoblastic differentiation medium for 6 days), and ST2 cells (BB: lane 9, cells cultured in growth medium; lane 10, cells cultured in medium supplemented with Wnt3a and ascorbic acid for 4 days; and CC: lane 1, cells cultured in growth medium for 4 days; lane 2, cells cultured in medium with Wnt3a and ascorbic acid for 4 days; lane 3, cells cultured in medium with Wnt3a and ascorbic acid for 14 days; lane 4, cells cultured in medium with ascorbic acid for 14 days) were used for Northern blot analysis.