Hdac3 Regulates Bone Density Figure 2. Calvarial bone formation is defective in Osx-Cre: Hdac3 CKO mice. A–C. Staining of one day-old wildtype, het (Hdac3+/2:Osx-Cre2), and Hdac3 CKO mice with alizarin red and alcian blue. D. MicroCT reconstructions of skulls from 5.5 week-old wildtype, Het (Hdac3+/2: Osx-Cre+), and Hdac3 CKO mice. doi:10.1371/journal.pone.0011492.g002 Periosteal and endocortical areas at the mid-diaphysis of the femur were reduced more than 60% in the Hdac3 CKOOsx mice (Figures 7G and 7H). Cortical area and section modulus, which are estimates of axial strength and bending strength [33], were reduced 50 and 70%, respectively, in the Hdac3 CKOOsx mice (Figures 7I and 7J). Together the data presented thus far demonstrate that Hdac3-deficiency prevents proper calvarial, trabecular, and cortical bone formation. Adipogenesis is Increased in Bone Marrow of Hdac3 Conditional Knockout Mice Bone marrow osteoblasts and adipocytes are derived from common multipotent mesenchymal progenitor cells and an inverse correlation between bone and white fat volumes are well characterized [34]. Goldner’s trichrome-stained sections from 5.5 week-old Hdac3 CKO femurs revealed the presence of a large number of adipocyte ghosts (Figure 8A). The adipocytes were present throughout the femur but seemed to be more prevalent towards the midshaft and along cortical surfaces. Adipocyte density, perimeter and number were elevated 11- to 25-fold in both CKO and CKOOsx groups, but not in Het or CHet mice (Figure 8B–D). Similar increases in adipocyte density were observed in female animals and in 2 and 4 week-old mice (data not shown). Adipocytes were not seen in 4 day-old mice (data not shown), indicating that bone marrow adipogenesis resulting from Hdac3-deficiency occurred postnatally. Microarray analysis of Osteoblasts from Hdac3 CKO mice To understand the molecular changes that result from Hdac3 depletion in osteoblast progenitor cells, the gene expression patterns in calvarial extracts were determined by microarray analysis and confirmed by qPCR. Two independent microarray analyses were PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11492