Fig. 7. Increased remodeling and osteoclast recruitment in En1^-/- calvariae. (A) Alizarin Red stained wild-type and En1^-/- calvariae at P1 showing multiple perforations in the mutants (arrows). (B) Whole-mount detection of osteoclast-specific TRAP activity in calvariae at P5, depicting increased staining in En1 mutants (compare with wild type, arrow). (C) Histological detection of TRAP⁺ osteoclasts located along the endocranial (arrows) and trabecular (arrowheads) bone surfaces of the frontal bones at P1. (D) Quantitative histomorphometric assessment of osteoclast number in sections of wild-type and En1^-/- frontal bones. (E,F) Northern blot and semi-quantitative analysis of OPG and Rankl expression in cultured calvarial osteoblasts. At day 7 of postconfluent growth, wild-type and En1^-/- cells express similar levels of OPG, whereas expression of Rankl is strongly upregulated in mutant osteoblasts. F, frontal bone; P, parietal bone. Scale bars: 2 mm in A,B; 0.1 mm in C.