Fig. 4. Impaired calvarial osteogenesis in En1 mutant mice. (A) Whole-mount in situ hybridization analysis of calvarial Opn expression, showing that En1^-/- embryos exhibit a delay in the commencement of osteogenesis (E14.5), followed by deficient osteoblast differentiation (arrows, E16.5-E18.5). (B) Section ISH analysis demonstrating the absence of Osx expression in the mesenchyme of En1^-/- calvariae at E13.5. At E16.5, Osx is expressed by wild-type osteoblasts of the frontal bone, but is reduced in En1^-/- osteoblasts (arrowheads). (C) Analysis of osteopontin (Opn) and osteocalcin (Ocn) on sagittal sections of wild-type and En1^-/- calvariae at P1. Although Opn normalizes to wild-type levels in En1^-/- calvariae postnatally, it is aberrantly expressed by terminally differentiated osteocytes of the mutant frontal bones (inset, arrowheads). By comparison, normal Ocn expression in ectoperiosteal osteoblasts is nearly abolished in En1^-/- calvariae. (D) Northern blot analysis of Opn, Bsp, Ocn and Osx expression by calvarial osteoblasts over a 21-day culture period. p, parietal bone; f, frontal bone. Scale bars: 1.5 mm in A; 0.1 mm in B; 0.2 mm in C.