Figure 1.
Western blot analysis of RUNX family proteins and CBFB. Proteins were extracted from calvaria (A–C), the trabecular bone in femurs and tibiae (D,E), ribs (F,G) and vertebrae (H,I) at 4 weeks of age in Cbfb2+/+, Cbfb2−/−, Runx2+/+ and Runx2+/− mice. Representative data are shown in (A), (D), (F), and (H). β-ACTIN was used as an internal control. The intensities of bands were normalized against β-ACTIN, and the normalized values in Cbfb2+/+ or Runx2+/+ mice were set as 1. The means ± SD of the relative levels of Cbfb2−/− mice (B,E,G,I) and Runx2+/− mice (C) are shown. The number of mice analyzed: B, n = 3–5; C, n = 3; E, n = 4–5; G, n = 4; I, n = 4.
Figure 1.
Western blot analysis of RUNX family proteins and CBFB. Proteins were extracted from calvaria (A–C), the trabecular bone in femurs and tibiae (D,E), ribs (F,G) and vertebrae (H,I) at 4 weeks of age in Cbfb2+/+, Cbfb2−/−, Runx2+/+ and Runx2+/− mice. Representative data are shown in (A), (D), (F), and (H). β-ACTIN was used as an internal control. The intensities of bands were normalized against β-ACTIN, and the normalized values in Cbfb2+/+ or Runx2+/+ mice were set as 1. The means ± SD of the relative levels of Cbfb2−/− mice (B,E,G,I) and Runx2+/− mice (C) are shown. The number of mice analyzed: B, n = 3–5; C, n = 3; E, n = 4–5; G, n = 4; I, n = 4.
Figure 2.
Skeletal development in Cbfb2+/+, Cbfb1−/− and Cbfb2−/− newborn mice. Cbfb1+/+ mice were similar to Cbfb2+/+ mice, and are not shown here. (A–C) Lateral view of skulls. (D–F) Neck and chest. (G–I) Lateral view of ribs and vertebrae. (J–L) Enlarged pictures of vertebrae. (M–O) Forelimbs. (P–R) Hind limbs. The arrow and arrowhead in (C) show delayed mineralization of interparietal bone and supraoccipital bone, respectively. The arrow and arrowheads in (F) show thin clavicle and delayed mineralization of sternum, respectively. The arrows in (O) and (R) show delayed mineralization of scapula and pelvic bones, respectively. Scale bars: 0.2 cm. (S) Whole area and mineralized area of scapulae and the ratios. The number of mice analyzed: Cbfb1+/+, n = 3; Cbfb1−/−, n =2; Cbfb2+/+, n = 5; Cbfb2−/−, n = 4. *** p < 0.001.
Figure 2.
Skeletal development in Cbfb2+/+, Cbfb1−/− and Cbfb2−/− newborn mice. Cbfb1+/+ mice were similar to Cbfb2+/+ mice, and are not shown here. (A–C) Lateral view of skulls. (D–F) Neck and chest. (G–I) Lateral view of ribs and vertebrae. (J–L) Enlarged pictures of vertebrae. (M–O) Forelimbs. (P–R) Hind limbs. The arrow and arrowhead in (C) show delayed mineralization of interparietal bone and supraoccipital bone, respectively. The arrow and arrowheads in (F) show thin clavicle and delayed mineralization of sternum, respectively. The arrows in (O) and (R) show delayed mineralization of scapula and pelvic bones, respectively. Scale bars: 0.2 cm. (S) Whole area and mineralized area of scapulae and the ratios. The number of mice analyzed: Cbfb1+/+, n = 3; Cbfb1−/−, n =2; Cbfb2+/+, n = 5; Cbfb2−/−, n = 4. *** p < 0.001.
Figure 3.
Skeletal development in Runx2+/+ and Runx2+/− newborn mice. (A,B) Lateral view of skulls. (C,D) Neck and chest. (E,F) Lateral view of ribs and vertebrae. (G,H) Enlarged pictures of vertebrae. (I,J) Forelimbs. (K,L) Hind limbs. The arrow in (D) shows hypoplastic clavicle, and arrowheads in (D,F) show delayed mineralization of sternum. The arrows in (J,L) show delayed mineralization of scapula and pelvic bones, respectively. Scale bars: 0.2 cm. (M) Whole area and mineralized area of scapulae and the ratios. Six Runx2+/+ mice and nine Runx2+/− mice were analyzed. *** p < 0.001.
Figure 3.
Skeletal development in Runx2+/+ and Runx2+/− newborn mice. (A,B) Lateral view of skulls. (C,D) Neck and chest. (E,F) Lateral view of ribs and vertebrae. (G,H) Enlarged pictures of vertebrae. (I,J) Forelimbs. (K,L) Hind limbs. The arrow in (D) shows hypoplastic clavicle, and arrowheads in (D,F) show delayed mineralization of sternum. The arrows in (J,L) show delayed mineralization of scapula and pelvic bones, respectively. Scale bars: 0.2 cm. (M) Whole area and mineralized area of scapulae and the ratios. Six Runx2+/+ mice and nine Runx2+/− mice were analyzed. *** p < 0.001.
Figure 4.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P7. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B) and (D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–T) In-situ hybridization using Col1a1 (M–P) and Col2a1 (Q–T) probes. The boxed regions in the left columns were magnified in the right columns. Scale bars: 0.5 cm (A–D) and 100 μm (E–T). Three Cbfb2+/+ mice and two Cbfb2−/− mice were analyzed.
Figure 4.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P7. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B) and (D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–T) In-situ hybridization using Col1a1 (M–P) and Col2a1 (Q–T) probes. The boxed regions in the left columns were magnified in the right columns. Scale bars: 0.5 cm (A–D) and 100 μm (E–T). Three Cbfb2+/+ mice and two Cbfb2−/− mice were analyzed.
Figure 5.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P10. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B) and (D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–X) In-situ hybridization using a Col1a1 (M–P), Col2a1 (Q–T) and Col10a1 (U–X) probes. The boxed regions in the left columns were magnified in the right columns. In-situ hybridization using the sense probes showed no significant signals. Scale bars: 0.5 cm (A–D) and 100 μm (E–X). Three Cbfb2+/+ mice and two Cbfb2−/− mice were analyzed.
Figure 5.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P10. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B) and (D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–X) In-situ hybridization using a Col1a1 (M–P), Col2a1 (Q–T) and Col10a1 (U–X) probes. The boxed regions in the left columns were magnified in the right columns. In-situ hybridization using the sense probes showed no significant signals. Scale bars: 0.5 cm (A–D) and 100 μm (E–X). Three Cbfb2+/+ mice and two Cbfb2−/− mice were analyzed.
Figure 6.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P14. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B,D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–X) In-situ hybridization using a Col1a1 (M–P), Col2a1 (Q–T) and Col10a1 (U–X) probes. The boxed regions in the left columns were magnified in the right columns. Scale bars: 0.5 cm (A–D) and 100 μm (E–X). Three Cbfb2+/+ mice and two Cbfb2−/− mice were analyzed.
Figure 6.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P14. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B,D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–X) In-situ hybridization using a Col1a1 (M–P), Col2a1 (Q–T) and Col10a1 (U–X) probes. The boxed regions in the left columns were magnified in the right columns. Scale bars: 0.5 cm (A–D) and 100 μm (E–X). Three Cbfb2+/+ mice and two Cbfb2−/− mice were analyzed.
Figure 7.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P28. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B,D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–T) In-situ hybridization using Col1a1 (M–P) and Col2a1 (Q–T) probes. The boxed regions in the left columns were magnified in the right columns. Scale bars: 0.5 cm (A–D) and 100 μm (E–T). Three Cbfb2+/+ and Cbfb2−/− mice were analyzed.
Figure 7.
Micro-CT and histological analyses of posterior frontal sutures in Cbfb2+/+ and Cbfb2−/− mice at P28. (A–D) Lateral (A,C) and dorsal (B,D) views of micro-CT images of skulls. The two lines in (B,D) show the anterior and posterior boundaries of the posterior frontal suture, and arrow heads indicate the location of coronal sections in the histological analyses. (E–H) H-E staining. (I–L) Safranin O staining. (M–T) In-situ hybridization using Col1a1 (M–P) and Col2a1 (Q–T) probes. The boxed regions in the left columns were magnified in the right columns. Scale bars: 0.5 cm (A–D) and 100 μm (E–T). Three Cbfb2+/+ and Cbfb2−/− mice were analyzed.
Figure 8.
The development of limb bones, ribs and vertebrae in Cbfb2−/− and Runx2+/− mice at 4 weeks of age. (A,B) Body weights of male mice. n = 21–29. (C–J) The lengths of the humerus (C,D), ulna (E,F), femur (G,H) and tibia (I,J) in female mice. The number of mice analyzed: Cbfb2+/+, n = 6; Cbfb2−/−, n = 4; Runx2+/+, n = 8; Runx2+/−, n = 7. (K–U) Three-dimensional images of frontal (K–N) and lateral views (O–R) of thoracic cage and vertebrae in micro-CT, the area of thoracic cage (S), real-time RT-PCR analysis using bony segments of rib RNA (T) and the length of the 1st vertebrae (U) in male mice. Cbfb2−/− mice with mild or severe rib deformity were analyzed separately in (T). Scale bars: 1 mm (C,E,G,I) and 1 cm (K–R). Data are shown as the mean ± SD. * p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 4; Cbfb2−/−, n = 8 (4 mild and 4 severe); Runx2+/+, n = 8; Runx2+/−, n = 7.
Figure 8.
The development of limb bones, ribs and vertebrae in Cbfb2−/− and Runx2+/− mice at 4 weeks of age. (A,B) Body weights of male mice. n = 21–29. (C–J) The lengths of the humerus (C,D), ulna (E,F), femur (G,H) and tibia (I,J) in female mice. The number of mice analyzed: Cbfb2+/+, n = 6; Cbfb2−/−, n = 4; Runx2+/+, n = 8; Runx2+/−, n = 7. (K–U) Three-dimensional images of frontal (K–N) and lateral views (O–R) of thoracic cage and vertebrae in micro-CT, the area of thoracic cage (S), real-time RT-PCR analysis using bony segments of rib RNA (T) and the length of the 1st vertebrae (U) in male mice. Cbfb2−/− mice with mild or severe rib deformity were analyzed separately in (T). Scale bars: 1 mm (C,E,G,I) and 1 cm (K–R). Data are shown as the mean ± SD. * p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 4; Cbfb2−/−, n = 8 (4 mild and 4 severe); Runx2+/+, n = 8; Runx2+/−, n = 7.
Figure 9.
Micro-CT analysis of calvaria, clavicles and thoracic cage in Cbfb2−/− and Runx2+/− mice. (A,B) Body weights of male mice at 10 weeks of age. n =14–19. (C–M) Calvaria (C–F), clavicles and thoracic cage (H–K), unmineralized area in calvaria (G), the length of clavicles (L) and the area of thoracic cage (M) in male Cbfb2+/+, Cbfb2−/−, Runx2+/+ and Runx2+/− mice at 14 weeks of age. Scale bars: 1 cm. Data are shown as the mean ± SD. *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 7; Cbfb2−/−, n = 4; Runx2+/+, n = 5; Runx2+/−, n = 6.
Figure 9.
Micro-CT analysis of calvaria, clavicles and thoracic cage in Cbfb2−/− and Runx2+/− mice. (A,B) Body weights of male mice at 10 weeks of age. n =14–19. (C–M) Calvaria (C–F), clavicles and thoracic cage (H–K), unmineralized area in calvaria (G), the length of clavicles (L) and the area of thoracic cage (M) in male Cbfb2+/+, Cbfb2−/−, Runx2+/+ and Runx2+/− mice at 14 weeks of age. Scale bars: 1 cm. Data are shown as the mean ± SD. *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 7; Cbfb2−/−, n = 4; Runx2+/+, n = 5; Runx2+/−, n = 6.
Figure 10.
Micro-CT analyses of femurs and lumbar vertebrae in male mice at 4 weeks of age. (A–D) Three-dimensional trabecular bone architecture of distal femoral metaphysis (A,C) and quantification of the trabecular bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular bone mineral density (Tb.BMD) in Cbfb2+/+ and Cbfb2−/− mice (B) and Runx2+/+ and Runx2+/− mice (D). (E–H) Micro-CT images of cortical bone at mid-diaphysis in femurs (E,G) and quantification of the cortical area (CtAr/TtAr), cortical thickness (Ct.Th), periosteal perimeter (Ps.Pm), endosteal perimeter (Es.Pm) and cortical bone mineral density (Ct.BMD) in Cbfb2+/+ and Cbfb2−/− mice (F) and Runx2+/+ and Runx2+/− mice (H). The number of mice analyzed: Cbfb2+/+, n = 22; Cbfb2−/−, n = 14; Runx2+/+, n = 11; Runx2+/−, n = 8. (I–L) Three-dimensional trabecular bone architecture of 1st lumbar vertebrae (I,K) and trabecular bone parameters in Cbfb2+/+ and Cbfb2−/− mice (J) and Runx2+/+ and Runx2+/− mice (L). Scale bars: 1 mm. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 9; Cbfb2−/−, n = 7; Runx2+/+, n = 11; Runx2+/−, n = 8.
Figure 10.
Micro-CT analyses of femurs and lumbar vertebrae in male mice at 4 weeks of age. (A–D) Three-dimensional trabecular bone architecture of distal femoral metaphysis (A,C) and quantification of the trabecular bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular bone mineral density (Tb.BMD) in Cbfb2+/+ and Cbfb2−/− mice (B) and Runx2+/+ and Runx2+/− mice (D). (E–H) Micro-CT images of cortical bone at mid-diaphysis in femurs (E,G) and quantification of the cortical area (CtAr/TtAr), cortical thickness (Ct.Th), periosteal perimeter (Ps.Pm), endosteal perimeter (Es.Pm) and cortical bone mineral density (Ct.BMD) in Cbfb2+/+ and Cbfb2−/− mice (F) and Runx2+/+ and Runx2+/− mice (H). The number of mice analyzed: Cbfb2+/+, n = 22; Cbfb2−/−, n = 14; Runx2+/+, n = 11; Runx2+/−, n = 8. (I–L) Three-dimensional trabecular bone architecture of 1st lumbar vertebrae (I,K) and trabecular bone parameters in Cbfb2+/+ and Cbfb2−/− mice (J) and Runx2+/+ and Runx2+/− mice (L). Scale bars: 1 mm. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 9; Cbfb2−/−, n = 7; Runx2+/+, n = 11; Runx2+/−, n = 8.
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Figure 11.
Micro-CT analyses of femurs and lumbar vertebrae in male mice at 10 weeks of age. (A–D) Three-dimensional trabecular bone architecture of distal femoral metaphysis (A,C) and quantification of the trabecular bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular bone mineral density (Tb.BMD) in Cbfb2+/+ and Cbfb2−/− mice (B) and Runx2+/+ and Runx2+/− mice (D). (E–H) Micro-CT images of the cortical bone at mid-diaphysis in femurs (E,G) and quantification of the cortical area (CtAr/TtAr), cortical thickness (Ct.Th), periosteal perimeter (Ps.Pm), endosteal perimeter (Es.Pm) and cortical bone mineral density (Ct.BMD) in Cbfb2+/+ and Cbfb2−/− mice (F) and Runx2+/+ and Runx2+/− mice (H). The number of mice analyzed: Cbfb2+/+, n = 19; Cbfb2−/−, n = 14; Runx2+/+, n = 17; Runx2+/−, n = 15. (I–L) Three-dimensional trabecular bone architecture of 6th lumbar vertebrae (I,K) and trabecular bone parameters in Cbfb2+/+ and Cbfb2−/− mice (J) and 1st lumbar vertebrae of those parameters in Runx2+/+ and Runx2+/− mice (L). Scale bars: 1 mm (A,C,E,G) and 0.5 mm (I,K). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 9; Cbfb2−/−, n = 14; Runx2+/+, n = 14; Runx2+/−, n =13.
Figure 11.
Micro-CT analyses of femurs and lumbar vertebrae in male mice at 10 weeks of age. (A–D) Three-dimensional trabecular bone architecture of distal femoral metaphysis (A,C) and quantification of the trabecular bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular bone mineral density (Tb.BMD) in Cbfb2+/+ and Cbfb2−/− mice (B) and Runx2+/+ and Runx2+/− mice (D). (E–H) Micro-CT images of the cortical bone at mid-diaphysis in femurs (E,G) and quantification of the cortical area (CtAr/TtAr), cortical thickness (Ct.Th), periosteal perimeter (Ps.Pm), endosteal perimeter (Es.Pm) and cortical bone mineral density (Ct.BMD) in Cbfb2+/+ and Cbfb2−/− mice (F) and Runx2+/+ and Runx2+/− mice (H). The number of mice analyzed: Cbfb2+/+, n = 19; Cbfb2−/−, n = 14; Runx2+/+, n = 17; Runx2+/−, n = 15. (I–L) Three-dimensional trabecular bone architecture of 6th lumbar vertebrae (I,K) and trabecular bone parameters in Cbfb2+/+ and Cbfb2−/− mice (J) and 1st lumbar vertebrae of those parameters in Runx2+/+ and Runx2+/− mice (L). Scale bars: 1 mm (A,C,E,G) and 0.5 mm (I,K). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 9; Cbfb2−/−, n = 14; Runx2+/+, n = 14; Runx2+/−, n =13.
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Figure 12.
Bone histomorphometric analyses of trabecular bone in femurs and vertebrae in male mice at 10 weeks of age. (A–D) Bone histomorphometric analysis of femurs in Cbfb2+/+ and Cbfb2−/− mice (A,B) and Runx2+/+ and Runx2+/− mice (C,D). The osteoid surface (OS/BS), osteoid thickness (O.Th), osteoblast surface (Ob.S/BS), number of osteoblasts (N.Ob/BS), osteoclast surface (Oc.S/BS), number of osteoclasts (N.Oc/BS), eroded surface (ES/BS), mineral apposition rate (MAR), mineralizing surface (MS/BS) and bone formation rate (BFR/BS) are shown. BS, bone surface. The number of mice analyzed: Cbfb2+/+, n = 18; Cbfb2−/−, n = 14; Runx2+/+, n = 10; Runx2+/−, n = 14. (E–H) Bone histomorphometric analyses of lumbar vertebrae in Cbfb2+/+ and Cbfb2−/− mice (E,F) and Runx2+/+ and Runx2+/− mice (G,H). Scale bars: 0.2 mm. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 19; Cbfb2−/−, n = 14; Runx2+/+, n = 10; Runx2+/−, n = 11.
Figure 12.
Bone histomorphometric analyses of trabecular bone in femurs and vertebrae in male mice at 10 weeks of age. (A–D) Bone histomorphometric analysis of femurs in Cbfb2+/+ and Cbfb2−/− mice (A,B) and Runx2+/+ and Runx2+/− mice (C,D). The osteoid surface (OS/BS), osteoid thickness (O.Th), osteoblast surface (Ob.S/BS), number of osteoblasts (N.Ob/BS), osteoclast surface (Oc.S/BS), number of osteoclasts (N.Oc/BS), eroded surface (ES/BS), mineral apposition rate (MAR), mineralizing surface (MS/BS) and bone formation rate (BFR/BS) are shown. BS, bone surface. The number of mice analyzed: Cbfb2+/+, n = 18; Cbfb2−/−, n = 14; Runx2+/+, n = 10; Runx2+/−, n = 14. (E–H) Bone histomorphometric analyses of lumbar vertebrae in Cbfb2+/+ and Cbfb2−/− mice (E,F) and Runx2+/+ and Runx2+/− mice (G,H). Scale bars: 0.2 mm. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 19; Cbfb2−/−, n = 14; Runx2+/+, n = 10; Runx2+/−, n = 11.
Figure 13.
Dynamic bone histomorphometric analysis of cortical bone at the mid-diaphyses of femurs and serum markers for bone formation and resorption in male mice at 10 weeks of age. (A–L) Cross-sections of Cbfb2+/+ (A,E,I), Cbfb2−/− (B,F,J), Runx2+/+ (C,G,K) and Runx2+/− (D,H,L) mice, into which calcein was injected twice. The boxed regions in (A–D) are magnified in (E,I,F,J,G,K,H,L), respectively. Scale bars: 200 µm (A–D) and 100 µm (E–L). (M–P) Mineral apposition rate (MAR), mineralizing surface (MS/BS) and bone formation rate (BFR/BS) in the periosteum (M,N) and endosteum (O,P) of Cbfb2+/+ and Cbfb2−/− mice (M,O) and Runx2+/+ and Runx2+/− mice (N,P). Cbfb2+/+, n = 8; Cbfb2−/−, n = 8; Runx2+/+, n = 22; Runx2+/−, n = 21. (Q) Serum markers for bone formation (P1NP) and resorption (TRAP5b). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 14; Cbfb2−/−, n = 16; Runx2+/+, n = 20; Runx2+/−, n = 18.
Figure 13.
Dynamic bone histomorphometric analysis of cortical bone at the mid-diaphyses of femurs and serum markers for bone formation and resorption in male mice at 10 weeks of age. (A–L) Cross-sections of Cbfb2+/+ (A,E,I), Cbfb2−/− (B,F,J), Runx2+/+ (C,G,K) and Runx2+/− (D,H,L) mice, into which calcein was injected twice. The boxed regions in (A–D) are magnified in (E,I,F,J,G,K,H,L), respectively. Scale bars: 200 µm (A–D) and 100 µm (E–L). (M–P) Mineral apposition rate (MAR), mineralizing surface (MS/BS) and bone formation rate (BFR/BS) in the periosteum (M,N) and endosteum (O,P) of Cbfb2+/+ and Cbfb2−/− mice (M,O) and Runx2+/+ and Runx2+/− mice (N,P). Cbfb2+/+, n = 8; Cbfb2−/−, n = 8; Runx2+/+, n = 22; Runx2+/−, n = 21. (Q) Serum markers for bone formation (P1NP) and resorption (TRAP5b). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The number of mice analyzed: Cbfb2+/+, n = 14; Cbfb2−/−, n = 16; Runx2+/+, n = 20; Runx2+/−, n = 18.
Figure 14.
TRAP-positive cells in the endosteum of femurs at 4 weeks of age. (A–N) TRAP staining of femoral sections in Cbfb2+/+ (A–D), Cbfb2−/− (E–H), Runx2+/+ (I–L) and Runx2+/− (M–P) mice. The boxed regions in (A,E,I,M) are magnified in (B–D), (F–H), (J–L) and (N–P), respectively, and the boxed regions in (B–D), (F–H), (J–L) and (N–P) are magnified in (B’–D’), (F’–H’), (J’–L’) and (N’–P’), respectively. Black arrows indicate the TRAP-positive cells. Red lines show the area for counting in the endosteum. Scale bars: 500 µm (A,E,I,M) and 100 µm (B–D,F–H,J–L,N–P,B’–D’,F’–H’,J’–L’,N’–P’). (Q,R) The number of TRAP-positive cells in Cbfb2+/+ and Cbfb2−/− mice (Q) and Runx2+/+ and Runx2+/− mice (R). Data are shown as the mean ± SD. * p < 0.05. The number of mice analyzed: Cbfb2+/+ and Cbfb2−/−, n = 6; Runx2+/+ and Runx2+/−, n = 7.
Figure 14.
TRAP-positive cells in the endosteum of femurs at 4 weeks of age. (A–N) TRAP staining of femoral sections in Cbfb2+/+ (A–D), Cbfb2−/− (E–H), Runx2+/+ (I–L) and Runx2+/− (M–P) mice. The boxed regions in (A,E,I,M) are magnified in (B–D), (F–H), (J–L) and (N–P), respectively, and the boxed regions in (B–D), (F–H), (J–L) and (N–P) are magnified in (B’–D’), (F’–H’), (J’–L’) and (N’–P’), respectively. Black arrows indicate the TRAP-positive cells. Red lines show the area for counting in the endosteum. Scale bars: 500 µm (A,E,I,M) and 100 µm (B–D,F–H,J–L,N–P,B’–D’,F’–H’,J’–L’,N’–P’). (Q,R) The number of TRAP-positive cells in Cbfb2+/+ and Cbfb2−/− mice (Q) and Runx2+/+ and Runx2+/− mice (R). Data are shown as the mean ± SD. * p < 0.05. The number of mice analyzed: Cbfb2+/+ and Cbfb2−/−, n = 6; Runx2+/+ and Runx2+/−, n = 7.
Figure 15.
Real-time RT-PCR analyses of the expression of the genes related to osteoblast differentiation and osteoclastogenesis, RUNX family genes and Cbfb isoforms using RNA from tibiae and vertebrae at 4 weeks of age. Real-time RT-PCR analyses were performed using RNA from the tibiae (A,B) and vertebrae (C,D) in Cbfb2+/+ and Cbfb2−/− mice (A,C) and Runx2+/+ and Runx2+/− mice (B,D). The primer pair for Bglap&Bglap2 amplifies both Bglap and Bglap2, and that of Cbfb1+2 amplified both Cbfb1 and Cbfb2. The ratios of Tnfsf11 and Tnfrsf11b are also shown. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The values of Cbfb2+/+ or Runx2+/+ mice were defined as 1 and relative levels are shown. The number of mice analyzed: Cbfb2+/+, n = 16; Cbfb2−/−, n = 13; Runx2+/+, n = 19; Runx2+/−, n = 15 in the tibiae and Cbfb2+/+, n = 9; Cbfb2−/−, n = 6; Runx2+/+, n = 7; Runx2+/−, n = 7 in the vertebrae.
Figure 15.
Real-time RT-PCR analyses of the expression of the genes related to osteoblast differentiation and osteoclastogenesis, RUNX family genes and Cbfb isoforms using RNA from tibiae and vertebrae at 4 weeks of age. Real-time RT-PCR analyses were performed using RNA from the tibiae (A,B) and vertebrae (C,D) in Cbfb2+/+ and Cbfb2−/− mice (A,C) and Runx2+/+ and Runx2+/− mice (B,D). The primer pair for Bglap&Bglap2 amplifies both Bglap and Bglap2, and that of Cbfb1+2 amplified both Cbfb1 and Cbfb2. The ratios of Tnfsf11 and Tnfrsf11b are also shown. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. The values of Cbfb2+/+ or Runx2+/+ mice were defined as 1 and relative levels are shown. The number of mice analyzed: Cbfb2+/+, n = 16; Cbfb2−/−, n = 13; Runx2+/+, n = 19; Runx2+/−, n = 15 in the tibiae and Cbfb2+/+, n = 9; Cbfb2−/−, n = 6; Runx2+/+, n = 7; Runx2+/−, n = 7 in the vertebrae.
Figure 16.
Proliferation of osteoblast-like and osteoprogenitor-like cells in the trabecular and cortical bone of femurs. (A–H) BrdU staining of trabecular bone (A–D) and cortical bone (E–H) in femurs from Cbfb2+/+ (A,E), Cbfb2−/− (B,F), Runx2+/+ (C,G) and Runx2+/− (D,H) mice at P7. The boxed regions in (A–H) are magnified in (A’–H’), respectively. Arrows in (A’–H’) show BrdU-positive osteoblast-like or osteoprogenitor-like cells. Scale bars: 100 µm (A–H) and 20 µm (A’–H’). (I,J) Frequencies of BrdU-positive osteoblast-like and osteoprogenitor-like cells in trabecular and cortical bone in Cbfb2+/+ and Cbfb2−/− mice (I) and Runx2+/+ and Runx2+/− mice (J). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. The number of mice analyzed: Cbfb2+/+, n = 7; Cbfb2−/−, n = 5; Runx2+/+, n = 5; Runx2+/−, n = 9.
Figure 16.
Proliferation of osteoblast-like and osteoprogenitor-like cells in the trabecular and cortical bone of femurs. (A–H) BrdU staining of trabecular bone (A–D) and cortical bone (E–H) in femurs from Cbfb2+/+ (A,E), Cbfb2−/− (B,F), Runx2+/+ (C,G) and Runx2+/− (D,H) mice at P7. The boxed regions in (A–H) are magnified in (A’–H’), respectively. Arrows in (A’–H’) show BrdU-positive osteoblast-like or osteoprogenitor-like cells. Scale bars: 100 µm (A–H) and 20 µm (A’–H’). (I,J) Frequencies of BrdU-positive osteoblast-like and osteoprogenitor-like cells in trabecular and cortical bone in Cbfb2+/+ and Cbfb2−/− mice (I) and Runx2+/+ and Runx2+/− mice (J). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. The number of mice analyzed: Cbfb2+/+, n = 7; Cbfb2−/−, n = 5; Runx2+/+, n = 5; Runx2+/−, n = 9.
Figure 17.
Proliferation of osteoblast-like and osteoprogenitor-like cells in vertebrae. (A–H) H-E staining (A,B,E,F) and BrdU staining (C,D,G,H) of the sections of 1st lumbar vertebrae in Cbfb2+/+ (A,C), Cbfb2−/− (B,D), Runx2+/+ (E,G) and Runx2+/− (F,H) mice at 4 weeks of age. The boxed regions in (A–H) are magnified in (A’–H’), respectively. Arrows in (C’,D’,G’,H’) show osteoblast-like or osteoprogenitor-like cells. Scale bars: 100 µm (A–H) and 20 µm (A’–H’). (I) The frequencies of BrdU-positive osteoblast-like and osteoprogenitor-like cells, which were counted in (C’,D’,G’,H’). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. The number of mice analyzed: Cbfb2+/+, n = 3; Cbfb2−/−, n = 3; Runx2+/+, n = 3; Runx2+/−, n = 4. Two regions were counted in each mouse.
Figure 17.
Proliferation of osteoblast-like and osteoprogenitor-like cells in vertebrae. (A–H) H-E staining (A,B,E,F) and BrdU staining (C,D,G,H) of the sections of 1st lumbar vertebrae in Cbfb2+/+ (A,C), Cbfb2−/− (B,D), Runx2+/+ (E,G) and Runx2+/− (F,H) mice at 4 weeks of age. The boxed regions in (A–H) are magnified in (A’–H’), respectively. Arrows in (C’,D’,G’,H’) show osteoblast-like or osteoprogenitor-like cells. Scale bars: 100 µm (A–H) and 20 µm (A’–H’). (I) The frequencies of BrdU-positive osteoblast-like and osteoprogenitor-like cells, which were counted in (C’,D’,G’,H’). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. The number of mice analyzed: Cbfb2+/+, n = 3; Cbfb2−/−, n = 3; Runx2+/+, n = 3; Runx2+/−, n = 4. Two regions were counted in each mouse.