Hdac3 Regulates Bone Density Figure 1. Hdac3-deficient mice are smaller and have shorter lifespans. A. PCR amplicons of Hdac3 alleles. B. Western blot analysis of Hdac3 expression in calvaria. C. Representative 5.5 week-old male wildtype (WT) and Hdac3 CKO mice are shown in the left panel. Average weekly weights of male and female wildtype (closed squares) and Hdac3 CKO (open squares) mice at and after the time of weaning are presented in the graphs. D. Hdac3- CKO and -CKOOsx mice had shorter lifespans than wildtype and heterozygous littermates. Each symbol represents a mouse that died from natural causes. doi:10.1371/journal.pone.0011492.g001 and F). However, mineral apposition rate (MAR), which is defined as the distance between two labels formed over a defined period of time, was not altered by Hdac3 deficiency in distal femurs (Figure 6G). Thus, in the rare places where bone was present and two labels were detected on a bone surface, the activity of osteoblasts in the Hdac3 CKOOsx mice was indistinguishable from that of wildtype mice. Reductions in mineralized surface (30%) and bone formation rates (50%) were also observed in vertebral trabeculi (Figures 6H–L). The MAR in vertebrae was reduced by 30% (Figures 6J). Similar results were present in the CKO (Hdac3fl/2:Cre+) mice (data not shown). Together, these data indicate that there are widespread deficits in trabecular bone formation in Hdac3 CKO mice. Hdac3 Conditional Knockout Mice Have Low Cortical Bone Density Few naturally occurring fractures were observed in Hdac3 CKO and CKOOsx mice; however, the long bones broke frequently along the diaphysis during dissection and processing (Figure 7A), suggesting cortical bone defects. Fluorochrome-labeling experi- ments demonstrated dramatic defects in bone formation on the periosteal and endocortical bone surfaces in both groups of Hdac3 CKO mice (Figure 7B). The bone formation rate, mineral apposition rate, and mineralized surface percentage were all significantly decreased in both CKO and CKOOsx mice (Figure 7C, 7D, and 7E). Meanwhile, mineralization lag time was increased 5- to 6-fold in the CKO groups (Figure 7F). PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11492