Figure 3.

Samd4^PB/PB mice displayed osteopenic phenotype. (a) Alizarin red and alcian blue staining of newborn wild-type (WT) and Samd4^PB/PB mice. a1 and a2, whole mount mice; a3 and a4, skulls; a5 and a6, forelimbs; a7 and a8, hindlimbs. Red arrows indicated the lack of alizarin red staining in Samd4^PB/PB mice. (b) Von Kossa staining of calvarium and tibia from E16.5 WT and Samd4^PB/PB embryos, counterstained with Nuclear Fast Red. Mineral is stained black; nuclei are stained red. P, parietal bone; F, frontal bone. (c) Hematoxylin–eosin staining of tibia of 3-week-old WT and Samd4^PB/PBmale mice, showing the secondary ossification center (red), the proliferation zone (green), the hypertrophic zone (orange) and the trabecular region (black). (d–h) Micro-computed tomography analysis of distal femurs from 3-week-old Samd4^PB/PB male mice and WT control mice. (d) Representative three-dimensional reconstructions of distal femur trabecular bone. (e) The quantitative parameters bone volume/tissue volume (BV/TV), (f) trabecular bone number (Tb.N), (g) trabecular bone thickness (Tb.Th) and (h) cortical bone thickness (C.Th). (i) Immunohistochemistry staining of the indicated genes on the sections of tibia from 1-week-old Samd4^PB/PB mice and control WT mice. (j–l) Analysis of the transcript levels of the indicated genes by quantitative PCR using RNA isolated from 7-day-old WT and Samd4^PB/PB femurs. Values represent mean±s.d. (n=6 (e–h) and 3 (j–l) for each genotype). P-values were obtained from t-tests with paired or unpaired samples, **P<0.01. Bars=1 mm in panels (a and d); 200 μm in panels (b and i); 500 μm in (c).