Cbfβ deletion in mice recapitulates cleidocranial dysplasia and reveals multiple functions of Cbfβ required for skeletal development

Abstract

The pathogenesis of cleidocranial dysplasia (CCD) as well as the specific role of core binding factor β (Cbfβ) and the Runt-related transcription factor (RUNX)/Cbfβ complex in postnatal skeletogenesis remain unclear. We demonstrate that Cbfβ ablation in osteoblast precursors, differentiating chondrocytes, osteoblasts, and odontoblasts via Osterix-Cre, results in severe craniofacial dysplasia, skeletal dysplasia, abnormal teeth, and a phenotype recapitulating the clinical features of CCD. Cbfβ(f/f)Osterix-Cre mice have fewer proliferative and hypertrophic chondrocytes, fewer osteoblasts, and almost absent trabecular bone, indicating that Cbfβ may maintain trabecular bone formation through its function in hypertrophic chondrocytes and osteoblasts. Cbfβ(f/f)Collagen, type 1, alpha 1 (Col1α1)-Cre mice show decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage, indicating that osteoblast lineage-specific ablation of Cbfβ results in milder bone defects and less resemblance to CCD. Activating transcription factor 4 (Atf4) and Osterix protein levels in both mutant mice are dramatically reduced. ChIP assays show that Cbfβ directly associates with the promoter regions of Atf4 and Osterix. Our data further demonstrate that Cbfβ highly up-regulates the expression of Atf4 at the transcriptional regulation level. Overall, our genetic dissection approach revealed that Cbfβ plays an indispensable role in postnatal skeletal development and homeostasis in various skeletal cell types, at least partially by up-regulating the expression of Atf4 and Osterix. It also revealed that CCD may result from functional defects of the Runx2/Cbfβ heterodimeric complex in various skeletal cells. These insights into the role of Cbfβ in postnatal skeletogenesis and CCD pathogenesis may assist in the development of new therapies for CCD and osteoporosis.

Keywords: chondrocyte differentiation; endochondral bone formation; growth plate formation; ossification; osteoblast differentiation.

Fig. 1.

Cbfβ^f/f Osx-Cre mice have decreased bone mineralization and skeletal deformities, resulting in a CCD-like phenotype. (A) Photographic analysis of 3-wk-old Cbfβ^f/f Osx-Cre (ff/Δ) mice and WT (ff) mice. (B–D) X-ray analysis of (B) femurs, (C) mandibles, and (D) clavicles. Yellow arrows in C and D indicate that Cbfβ^f/f Osx-Cre mice have a severe anterior open bite and mandibular retrognathism as well as hypoplastic/aplastic clavicles, respectively. (E and F) The µCT scans (E) and quantification (F) show that bones from Cbfβ^f/f Osx-Cre mice are smaller and less mineralized than those of WT. (G and H) Photographic analysis (G) and high magnification (H) of incisor tooth development. Yellow arrows indicate normal (left mouse), stunted (middle mouse), or abnormal (right mouse) tooth development. (I) High-magnification analysis of molar tooth development. (J) Photographic analysis shows defective, supernumerary-like teeth (red arrow) in Cbfβ^f/f Osx-Cre mice compared with WT. (K) All mice were genotyped by PCR from tail snip DNA.

Fig. 2.

Cbfβ^f/f Osx-Cre mice have shortened limbs, decreased bone ossification, and disproportionately defective skull, calveria, and mandible. (A) Whole-mount skeletal was stained by Alizarin red and Alcian blue staining of newborn Cbfβ^f/f Osx-Cre (ff/Δ) and WT (ff) mice. Yellow arrows show reduced bone ossification in the limbs of the mutant mice. (B–E) The skull (B), forelimbs (C), clavicles (D), and vertebrae (E) of the mutant mice display decreased ossification. Yellow arrows in C show that the mutant mice have less calcified trabecular bone adjacent to the hypertrophic zone.

Fig. 3.

Cbfβ^f/fOsx-Cre newborn tibiae have impaired endochondral bone ossification, and Goldner’s Trichrome staining revealed a decrease in osteoblast numbers. (A–C) H&E staining (A), Von Kossa and Alcian blue staining (B), and Safranin O staining (C) of tibiae from newborn Cbfβ^f/f Osx-Cre (ff/Δ) and WT (ff) mice. Yellow arrows in A and C indicate that Cbfβ^f/f Osx-Cre mice have a reduced bone collar and fewer hypertrophic chondrocytes in the growth plate, respectively. (D) H&E staining shows defective intramembranous bone formation in newborn mutant mice compared with WT. (E) Von Kossa staining of tibia from 10-wk-old Cbfβ^f/f Osx-Cre and WT bones. (F) Goldner’s Trichrome stain of hard-tissue sections of tibia from 3-wk-old Cbfβ^f/f Osx-Cre (ff/Δ) and WT (ff) mice. For histological detail of trabeculae, the bottom row shows a higher magnification of areas in yellow boxes. (G) Quantification of the data shown in F.

Fig. 4.

Cbfβ^f/fCol1α1-Cre mice have decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage. (A–D) Photographic analysis (A), X-ray analysis focusing on femoral bone density (B), µCT scans of femurs (C), and X-ray analysis of calvaria (D) of 3-wk-old Cbfβ^f/f Col1α1-Cre (ff/Δ) and WT (ff) mice. White arrows in D show that the calvaria are less calcified in the mutant mice. (E) H&E staining shows that intramembranous bone formation was not dramatically affected in Cbfβ^f/fCol1α1-Cre mice compared with WT. (F and G) Safranin O staining of tibia and comparison of growth plate development among 4-wk-old Cbfβ^f/f Osx-Cre mutant, Cbfβ^f/fCol1α1-Cre mutant, and WT mice.

Fig. 5.

Cbfβ deficiency decreases chondrocyte proliferation, reduces expression of Cbfβ and Osterix, and impairs chondrocyte hypertrophy in Cbfβ^f/f Osx-Cre mice. (A and B) PCNA staining for cellular proliferation (A) and immunohistochemistry (IHC) staining with anti-Cbfβ, anti-Runx2, and anti-osterix antibodies (B) of tibial paraffin sections from 4-wk-old Cbfβ^f/f Osx-Cre (ff/Δ), Cbfßf/f Col1α1-Cre (ff/Δ), and WT (ff) mice. (Insets) The magnified images of the red boxed areas, which show a decrease in chondrocyte proliferation in Cbfβ^f/f Osx-Cre mice and reduced expression of Cbfβ and Osterix in both mutant mice compared with WT. (C) Immunofluorescence staining of the tibia from newborn Cbfβ^f/f Osx-Cre (ff/Δ) and WT (ff) mice.

Fig. 6.

Cbfβ deficiency in primary calvarial cells cultured from Cbfβ^f/fOsx-Cre and Cbfβ^f/fCol1α1-Cre mice inhibits osteoblastogenesis. (A) Calvarial cells from Cbfβ^f/f Osx-Cre (ff/Δ) and WT (ff) newborn mice were applied to osteoblastogenesis assays. (B) GeneChip analysis of the expression of Atf4, Ocn, Spp1, Alpl, and Col1α1 in mouse calvarial cells. (C) qPCR analysis of the mRNA expression level of Atf4 in mouse calvarial cells after culturing in the osteoblast differentiation media. (D) Protein expression levels were analyzed by Western blot analysis. (E) qPCR analysis of mRNA expression levels of Col1α1, Spp1, Runx2, Sox9, OPG, RANKL, and OCN in calvaria-derived osteoblasts from Cbfβ^f/f Osx-Cre (ff/Δ) and WT (ff) mice. Results are expressed as mean ± SD, n ≧ 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig. 7.

Cbfβ regulates Osx, Runx2, and Atf4 expression by directly associating with their promoters. (A–C) ChIP analysis of Cbfβ binding to the (A) Osx promoter, (B) Runx2 promoter, and (C) Atf4 promoter in calvaria-derived osteoblasts using primers as indicated on the x axes. Results are presented as ChIP/Input. Results are presented as mean ± SD, n ≧ 6 in each group. *P < 0.05, **P < 0.01, ***P < 0.005. (D) Western blot analysis of the expression of Cbfβ, Osx, Runx1, Runx2, Runx3, Sox9, PCNA, ColX, and Atf4 in the calvaria of newborn Cbfβ^f/fOsx-Cre, Cbfβ^f/fCol1α1-Cre, and WT mice. β-tubulin is used as the loading control.