hypertrophy and endochondral ossification [14]. Hdac6-deficiency modestly enhanced trabecular bone formation [15]. Recently, Hdac8 was shown to be essential for neural crest progenitor cell differentiation and skull bone formation [13]. Hdac4 and Hdac6 bind and inhibit the activity of Runx2, a transcription factor essential for osteoblast development [14,24,25]. Hdac3 also interacts with Runx2 to repress the expression of osteoblast- specific genes, osteocalcin and bone siaoloprotein, in vitro [24,26,27,28,29]. Suppression of Hdac3 in immortalized osteo- blast cell lines by RNA interference promoted the expression of these genes and other markers of osteoblast maturation [24]. Germline Hdac3-deficiency caused embryonic lethality at approx- imately 9 days post coitum [11,12], several days before skeletons form in the mouse embryo. In this study, we determined the consequences of Hdac3 depletion on bone formation by crossing mice with loxP sites flanking exon 7 of Hdac3 [11] to mice wherein Cre recombinase is expressed from the endogenous osterix (Osx) promoter [30]. The data presented here show that Hdac3 depletion impaired both trabecular and cortical bone properties and promoted bone marrow adipogenesis. Results Hdac3 Conditional Knockout Mice Have Defects in Intramembranous Bone Formation The effects of Hdac3 depletion on skeletal maturation were determined by crossing Hdac3fl/2 mice [11,31] with mice expressing Cre recombinase from the osterix (Osx) promoter [30]. This breeding strategy eventually created five groups of progeny: wildtype (Hdac3fl/+:Cre2), heterozygotes (Hdac3+/2: Cre+ or 2 or Hdac3 fl/2:Cre2; Hets), conditional heterozygotes (Hdac3fl/+:Cre+; CHet), conditional knockouts with a single germline allele (Hdac3fl/2:Cre+; CKO), and osterix-driven CKO (Hdac3 fl/fl: Cre+; CKOOsx) (Figure 1A). Immunoblot, qPCR, and microarray analyses of parietal bone protein and mRNA extracts from 4 to 5 day- old mice demonstrated that Hdac3 levels were significantly reduced in both groups of CKO mice (Figure 1B and Table 1). The expression of other Hdacs was not altered (data not shown). Hdac3 CKO and CKOOsx mice were indistinguishable in size at birth from wildtype and heterozygous littermates, but were smaller at the time of weaning and remained smaller throughout their lifespan, with male and female CKO animals being approx- imately 40% and 20% smaller than wildtype littermates, respectively (Figure 1C). Heterozygous mice were not signif- icantly different in size and weight from wildtype animals (Figure S1). The Hdac3 CKO and CKOOsx mice had shorter lifespans with some mice dying as early as 3 weeks of age from undetermined causes (Figure 1D). The oldest CKO mouse survived 77 weeks, but was still outlived by heterozygous and wildtype mice. The consequences of Hdac3 depletion in osterix-expressing cells on skeletal development were examined by whole mount skeletal staining. One day-old Hdac3 CKO mice exhibited grossly normal skeletal patterning with both cartilaginous and calcified skeletal elements as detected by Alician blue and Alizarin red dyes, respectively (Figure 2A). However, early calvarial development was impaired (Figure 2B–2D). Parietal bone formation was notably delayed in Hdac3 CKO mice. Microcomputed tomogra- phy reconstruction of skulls from 5.5 week-old adult Hdac3 CKO mice showed that the calvarial bones remained extremely thin and porous through adulthood (Figure 2D and Figure S2). These data indicated that Hdac3 is necessary for proper intramembranous bone formation. Hdac3 Regulates Bone Density Hdac3 Conditional Knockout Mice Have Growth Plate Defects The smaller stature of the Hdac3-depleted mice and potential reduction of Hdac3 levels in cartilage due to osterix expression in chondrocytes [3,32] prompted the assessment of femoral growth plate structure. Femur length was decreased 18 to 25% in male Hdac3- CKO and CKOOsx mice (Figure 3A) and 17% in female mice (data not shown). The average growth plate depth of male 5.5 week-old Hdac3-CKOOsx mice was the same size as the average from wildtype mice and had both proliferating and hypertrophic zones; however, the hypertrophic zone constituted a larger percentage of the growth plate in Hdac3- heterozygous and CKO groups than in wildtype mice (Figure 3B and 3C). Von Kossa staining revealed fewer trabeculi below the growth plate in 5.5 week-old CKO animals (Figure 3D). These results suggest that Hdac3 deletion might delay or arrest terminal chondrocyte differentiation, which could adversely affect endochondral bone formation; however, the normal femur length of heterozygous animals despite an extended hypertrophic zone indicates a severe mineralization defect. Hdac3 Conditional Knockout Mice Have Low Trabecular Bone Density The extent of trabecular bone loss in long bones from Hdac3 CKO mice was measured by microcomputed tomography (Figure 4). Reconstructions of the trabecular bone in distal femurs illustrated the striking reduction in bone volume in both CKO and CKOOsx mice as compared to their wildtype and heterozygous littermates (Figure 4A). At 5.5 weeks of age, trabecular bone volume density (BV/TV) in the distal femurs of male CKO mice was reduced by 75% (Figure 4B). The reductions were due to a 43% decrease in trabecular number (Tb.N) (Figure 4C). Trabec- ular thickness (Tb.Th) was unchanged (Figure 4D). In accordance with decreased trabecular number in both groups of Hdac3 CKO mice, trabecular separation (Tb.Sp) was increased and connectiv- ity density (ConnD) was decreased in these animals (Figures 4E–F). Similar differences were also observed in 12 week-old male mice (Figure S3) and in females (data not shown). To understand how Hdac3-depletion in osterix-positive cells affected trabecular bone formation, Goldner’s Trichrome-stained sections of distal femurs were examined by static histomorphom- etry (Figure 5). There was no change in the number of osteoblasts per bone surface (Figure 5A); however, in accordance with the significant decrease in bone density, there were fewer osteoblasts per tissue area in the Hdac3 CKO mice as compared to wildtype and heterozygous littermates (Figure 5B). Osteoclast surface was slightly elevated in the Hdac3 CKO mice; however the changes did not reach statistical significance (Figure 5C). These data indicate that Hdac3 depletion in osterix-expressing cells is detrimental to bone formation, but that the bony structures present in these animals remained lined with osteoblasts and osteoclasts. To determine if osteoblasts in Hdac3 CKOOsx mice produce a mineralized matrix at the same rate and to the same extent as wildtype and heterozygous littermates, 5.5 week-old male mice were injected once with tetracycline and twice with calcein and newly formed mineralized surfaces were measured (Figure 6A). The amount of mineralized and double-labeled surface (MS and dL.S, respectively) per bone surface (BS) were reduced by 38% and 55%, respectively, in the distal femurs of Hdac3 CKOOsx mice (Figures 6B and C). Similar to what was observed in the microCT scans (Figure 4D), trabecular number was decreased in histological sections (Figure 6D). Trabecular bone formation rates (BFR) per bone surface and tissue volume were also significantly reduced by 44% and 87%, respectively, in Hdac3 CKOOsx mice (Figures 6E PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11492