Hdac3 Regulates Bone Density Figure 3. Growth plates are abnormal in Osx-Cre: Hdac3 CKO mice. A. Femurs are shorter in male Hdac3 CKO and CKOOsx mice as compared to wildtype and heterozygous littermates at 5.5 weeks of age. (n = 11 WT, 3 Het, 5 CHet, 7 CKO, and 9 CKOOsx), **p#0.01. B–C. The hypertrophic zone within growth plates of male mice was measured from Goldner’s trichrome stained sections of the distal femurs. (n = 10 WT, 4 Het, 4 CHet, 6 CKO, and 6 CKOOsx), *p,0.05. D Von Kossa staining of representative distal femurs. doi:10.1371/journal.pone.0011492.g003 performed with eight paired wildtype and CKO or CKOOsx littermates. In these experiments, 768 and 786 genes were significantly changed (fold change .1.1, p,0.05 and 95% confidence) in the Hdac3 CKOOsx mice, but only 176 genes were present in both data sets (Table S1). Expression changes of 1.4 fold or greater were verified by qPCRs. With this more stringent cutoff (fold change .1.4), 64 genes were identified (Table 1). Of these, 43 genes were upregulated in Hdac3 CKO mice and 21 genes were downregulated. As expected, Hdac3 was one of these most suppressed genes. The most upregulated gene in both experiments was cyclin- dependent kinase inhibitor 1a (Cdkn1a, also known as p21Cip1), a known Hdac regulated gene [35,36,37]. In reporter assays we found that Hdac3 repressed the p21(WAF1/CIP1) promoter in a concentration dependent manner (Figure S4). Gene ontogeny analysis of genes differentially regulated more than 1.4-fold identified 11 pathways as being affected by Hdac3 deletion (Figure 9). These pathways include signal transduction cascades (e.g. Wnt, Akt, G- proteins), cell cycle and DNA damage pathways, and cytoskeletal/cell adhesion. Notably, osterix mRNA levels were unchanged (data not shown). Thus, Hdac3 depletion modulates several pathways that are crucial for osteoblast development and function. Discussion Hdac3 is essential for embryonic development [11,12]. It associates with numerous transcription factors, co-repressors and class II Hdacs [18,19,20,21,22]. We previously showed that Hdac3 binds and represses Runx2-dependent activation of osteoblast genes [24]. In this study, we demonstrate that conditional deletion of Hdac3 in cells where Cre recombinase is expressed from the endogenous osterix promoter causes severe deficits in calvarial, trabecular and cortical bone volume. Some Osx-Cre expressing mice (Hdac3 wildtype) also had mild skull defects (Figure S5); however, all Hdac3 CKO mice had severe craniofacial abnormal- ities. Importantly, none of the most differentially regulated genes (Table 1) were significantly changed in Osx-Cre mice with wildtype Hdac3 alleles. Interestingly, incomplete skull bone ossification was also observed in fetal rats exposed to the Hdac inhibitor vorinostat in utero [38]. Distal femur trabecular bone volumes were not affected by the expression of Cre or loss of a single Hdac3 allele (e.g. CHet mice), but were severely reduced when both copies of Hdac3 were inactivated. Trabecular and cortical bone formation rates were markedly depressed in both groups of CKO mice (Hdac3fl/fl or fl/2) with concomitant increases in bone marrow fat. Together these data demonstrate that Hdac3 is essential for proper osteoblast function and bone formation. In this study we focused on the postnatal consequences of Hdac3 depletion on bone structural and cellular properties. Because the Hdac3 CKO mice were smaller than wildtype and heterozyogous littermates, we also examined growth plates in skeletally immature mice. The hypertrophic zone was thicker in Hdac3-haploinsuffi- cient and conditional knockout animals; suggesting that Hdac3 Figure 4. Hdac3-depletion in Osx-expressing cells decreases trabecular bone volume in distal femora. A. Representative microCT reconstructions of trabecular bone in distal femurs. B–F. Structural parameters of distal femurs from microCT reconstructions of 5.5 week-old male mice. n = 5 per group, **p,0.01. doi:10.1371/journal.pone.0011492.g004 PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11492