Zfp521 is required for OB maturation. (A) Cryosections of 3-wk-old Zfp521^−/− and control mice showing the distal femoral metaphysis were immunostained for Runx2 (green). Nuclear DAPI staining (dark blue) and colocalization with Runx2 (light blue) are shown. Higher magnification images of the marked areas show trabecular surfaces. Asterisks indicate the growth plate, and arrows indicate the trabecular bone. Bar graph shows the ratio of Runx2+/total cell number quantified in the primary spongiosa from confocal images (n = 3 mice/genotype). (B) An equal number of BM cells flushed from Zfp521^−/− and control mice was plated on 6-well plates and cultured in osteogenic medium (OM) for 10 and 21 d. The cells were stained for ALP activity on day 10 to count the number of CFU-Fs and with Alizarin red for osteoblastic colonies (CFU-OB) on day 21. (C) Calvarial cells from Zfp521^−/−, Zfp521_hOC^−/−, and respective control newborn mice were harvested and cultured on 6-well plates for 7 d in OM and stained for ALP activity. (D) Zfp521^−/−, Zfp521_hOC^−/−, and control calvarial cells were cultured for 21 d in OM and stained with Alizarin red to detect mineralized bone nodules. Alizarin red–positive nodules were quantified (bar graphs). (E) Time course of Zfp521 and hOC-Cre expression in Zfp521_hOC^−/− and control calvarial cells during OB differentiation was measured using quantitative RT-PCR (qRT-PCR) in RNA extracted from cultures in C and D. (F) Expression of late OB marker genes Bsp and Ocn was measured by qRT-PCR in Zfp521^−/− and control calvarial cells cultured for 21 d in OM. (G) Expression of late OB marker genes Bsp and Ocn was measured by qRT-PCR in Zfp521_hOC^−/− and control calvarial cells cultured for 21 d in OM. (H) Expression of early (Opn) and late (Ocn) marker genes by in situ hybridization in Zfp521^−/− and control bones. All data are means ± SD. Similar results were obtained in at least three separate experiments performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.