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. 2001 Apr 2;153(1):87-100.
doi: 10.1083/jcb.153.1.87.

Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes

C Ueta  1 , M Iwamoto, N Kanatani, C Yoshida, Y Liu, M Enomoto-Iwamoto, T Ohmori, H Enomoto, K Nakata, K Takada, K Kurisu, T Komori
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Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes (VSports注册入口)

"VSports app下载" C Ueta et al. J Cell Biol. .

V体育ios版 - Abstract

During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes VSports手机版. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones. .

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Figures

Figure 2
Figure 2
Examination of skeletal system. Skeletal examination of wild-type mice (A, E, I, and M) and type I Cbfa1 (B, F, J, and N), type II Cbfa1 (C, G, K, and O), and DN-Cbfa1 (D, H, L, and P) transgenic mice at E18.5. Calcified tissues are stained red with Alizarin red and the cartilage is stained blue with Alcian blue. Representative skeletons are shown. (A–D) Whole skeletons. In wild-type mice, cartilaginous tissues are observed in occipital bone, joints, the ventral portion of ribs, and vertebral bodies (A). In both type I and II Cbfa1 transgenic mice, most of the skeleton, including occipital bone, most of the ribs, and all of the vertebrae, is calcified (B and C). In DN-Cbfa1 transgenic mice, calcification is limited in flat bones of the head, mandible, clavicle, and long bones (D). (E–H) Thoracic cages. In wild-type mice, the ventral portion of all ribs is cartilaginous and sternum is segmentally calcified (E). In both type I and II Cbfa1 transgenic mice, the thoracic cage is small and bell-shaped, and the major portion of the ribs and sternum are calcified (F and G). In DN-Cbfa1 transgenic mice, the major portion of the ribs and the entire region of the sternum are cartilaginous (H). (I–L) Vertebral skeletons. In wild-type mice, centers of vertebral bodies and most vertebral arches are calcified (I). In both type I and II Cbfa1 transgenic mice, all of the vertebral bodies and arches are united and calcified (J and K). In DN-Cbfa1 transgenic mice, all of the vertebral bodies and arches are cartilaginous (L). (M–P) Forelimb skeletons. In wild-type mice, calcification is limited in diaphyses of long bones and the center of the scapula (M). In both type I and II Cbfa1 transgenic mice, the humerus is short and the humerus, radius, and ulna are thick and united (N and O). Shoulder joints and some carpal bones are also fused (N and O). Most of the elbow joint region is calcified and the calcified region is wider than that in wild-type mice in all long bones and scapula (N and O). In DN-Cbfa1 transgenic mice, calcification is limited around diaphyses of the humerus, radius, and ulna (P). Bars: (A–D) 2 mm; (E–P) 1 mm.
Figure 1
Figure 1
Generation of transgenic mice. (A) Diagrams of the DNA constructs used to generate Col2a1-type I Cbfa1, Col2a1-type II Cbfa1, and Col2a1–DN-Cbfa1 transgenic mice. DNA fragments covering the entire coding region of the mouse type I or II Cbfa1 isoforms were inserted into a Col2a1-based expression vector, which contains promoter and enhancer of mouse Col2a1 gene. DNA fragments encoding the runt domain only or runt domain with NH2-terminal domain of type I Cbfa1 were also inserted into the Col2a1-based expression vector to generate DN forms of Cbfa1. Type I, type I Cbfa1; Type II, type II Cbfa1; DN, DN Cbfa1; Pr, Col2a1 promoter; En, Col2a1 enhancer; N, NotI; H, HindIII; E, EcoRI; B, BamHI. (B) Northern blot hybridized with Cbfa1 probe. RNA was extracted from the head or trunk of E12.5–18.5 embryos, and 20 μg of total RNA was loaded per lane. Representative data using RNA from heads of E12.5 embryos are shown. Lanes of wild-type (left), type I Cbfa1, and type II Cbfa1 transgenic mice are on one filter and lanes of wild-type (right, W) and DN-Cbfa1 transgenic mice (T) are on the other filter. Arrows indicate the bands of endogenous Cbfa1 and transgenes. Hybridization with the GAPDH probe was used as internal control for the loading of equal amounts of RNA. (C) Gross appearance of wild-type, type I Cbfa1, type II Cbfa1, and DN-Cbfa1 transgenic mice at E18.5. Both type I and II Cbfa1 transgenic mice exhibit dwarfism with domed skull, short snout and mandible, protruding tongue, and short limbs and tail. DN-Cbfa1 transgenic mice exhibit dwarfism with short limbs. In DN-Cbfa1 transgenic mice, all of the presented data in this paper are from mice that contain the runt domain with NH2-terminal domain of type I Cbfa1 except skeletal examinations, in which a mouse with runt domain only is presented. Bar, 2 mm.
Figure 4
Figure 4
DN effect of the truncated form of Cbfa1, which contains the runt domain with NH2-terminal domain of type I Cbfa1. 0.5 μg of Cbfa1 target gene reporter plasmid (p6XOSE2-Luc), 0.2 μg of control reference reporter plasmid (pRL-SV40), and the indicated amounts of expression vectors were cotransfected into chick sterna chondrocytes. Relative luciferase activity was calculated as described in Materials and Methods. Values are means of four wells. Similar results were obtained in two independent experiments.
Figure 3
Figure 3
Whole skeletons at E15.5. The skeletal system was also examined at E15.5 to compare type I (B) and type II Cbfa1 transgenic mice (C). (A) Wild-type mice at E15.5. Mineralization is advanced in both type I and II Cbfa1 transgenic mice in most skeletal components, including occipital bone, chondrocranium, ribs, vertebrae, and long bones, but the mineralization in type II Cbfa1 transgenic mice is more than that in type I Cbfa1 transgenic mice. The thoracic cage in type II Cbfa1 transgenic mice is smaller than that in type I Cbfa1 transgenic mice. Although fusion in joints and vertebrae is observed in both type I and II Cbfa1 transgenic mice, it is more advanced in type II Cbfa1 transgenic mice. Representative skeletons are shown. Bar, 1 mm.
Figure 6
Figure 6
Process of endochondral ossification in limbs. The forelimb (A) and hind limb (B) from β-galactosidase transgenic mice and the forelimbs from wild-type (C, E, H, K, N, and Q), Cbfa1 transgenic (D, F, I, L, O, and R), and DN-Cbfal transgenic mice (G, J, M, P, and S) were examined histologically. (A and B) β-Galactosidase staining in E11.5 (A) and E16.5 (B) transgenic mice expressing β-galactosidase by Col2a1 promoter/enhancer. Staining is observed in chondroprogenitor cells in precartilaginous condensation (A). Staining is observed in chondrocytes but very weakly in hypertrophic chondrocytes and the staining is absent in osteoblasts (B). (C and D) H-E staining at E12.5. Humerus, radius, and ulna are uninterrupted in Cbfa1 transgenic mice (D). (E–J) H-E staining at E15.5. In Cbfa1 transgenic mice, humerus, radius, and ulna remain fused and the growth plate is disorganized and mainly composed of hypertrophic chondrocytes in the fused region (F and I). In DN-Cbfa1 transgenic mice, ulna remains cartilaginous and is composed of small immature chondrocytes and no vascular invasion is observed (G and J). (H–J) Higher magnifications of boxed regions in E–G. (K–P) In situ hybridization using type II collagen (K–M) and type X collagen (N–P) antisense probes at E15.5. In Cbfa1 transgenic mice, type II collagen expression is restricted in epiphyses and a small part of the fused region (L), but type X collagen is widely expressed (O). In contrast, in DN-Cbfa1 transgenic mice, type II collagen expression is observed in a whole part of humerus, radius, and ulna (M), and no type X collagen expression is detected (P). (Q–S) von Kossa and H-E double staining at E18.5. Bones of Cbfa1 transgenic mice are heavily mineralized (R), whereas mineralization in DN-Cbfa1 transgenic mice is restricted in bone collars and calcified cartilage in diaphysis of ulna (S). Bars: (A–P) 100 μm; (Q–S) 500 μm.
Figure 5
Figure 5
Endochondral ossification in cartilaginous tissues. (A and B) Frontal sections of the nasal septum from wild-type (A) and Cbfa1 transgenic (B) mice at E18.5. In Cbfa1 transgenic mice, most chondrocytes are hypertrophic and half of the nasal septum is replaced by bone (B). (C and D) Sagittal sections of the vertebral column from wild-type (C) and Cbfa1 transgenic (D) mice at E18.5. Most of vertebral bodies are replaced by bone, and chondrocytes in intervertebral regions are hypertrophic and partly replaced by bone in Cbfa1 transgenic mice (D). (E–H) Sagittal sections of the thyroid (Th), cricoid (C), and tracheal (Tr) cartilages from wild-type (E and G) and Cbfa1 transgenic (F and H) mice at E18.5. In Cbfa1 transgenic mice, tracheal rings are fused, large areas in thyroid, cricoid, and tracheal cartilages are occupied by hypertrophic chondrocytes, and some regions are replaced by bone (F and H). (G and H) Higher magnifications of boxed regions in E and F, respectively. Arrowheads in F show the regions that are replaced by bone. (I–L) Sections of the Meckel's cartilage from wild-type (I and K) and Cbfa1 transgenic (J and L) mice at E18.5. In wild-type mice, Meckel's cartilage is intact, but most of it is replaced by bone in Cbfa1 transgenic mice. (K and L) Higher magnifications of boxed regions in I and J, respectively. All sections were stained with H-E. Bars, 100 μm.
Figure 7
Figure 7
Chondrocyte maturation in DN-Cbfa1 transgenic mice. Maturational stage of chondrocytes in DN-Cbfa1 transgenic mice at E18.5 (A–C, E, G, and H) was examined with wild-type mice at E15.5 as positive control (D and F) by in situ hybridization using a type II collagen probe (B), PTH/PTHrPR probe (C and D), Ihh probe (E and F), and type X collagen probe (G and H). (A) H-E staining. In DN-Cbfa1 transgenic mice, a series of sections from the tibia is used in A–C, E, and G and a series of sections from the radius and ulna is used in H and Fig. 6 S. In wild-type mice, a series of sections from the tibia and fibula are used (D and F). In DN-Cbfa1 transgenic mice, most chondrocytes express type II collagen (B) but not PTH/PTHrPR (C), Ihh (E), or type X collagen (G). PTH/PTHrPR (D) and Ihh (F) are strongly expressed in prehypertrophic and early hypertrophic chondrocytes in wild-type mice. In DN-Cbfa1 transgenic mice, PTH/PTHrPR expression is restricted in osteoblastic cells in bone collar (C). A few type X collagen–positive cells are seen close to hematopoietic cells that invaded to inside the bone collar of radius and ulna in DN-Cbfa1 transgenic mice (H). Bar, 100 μm.
Figure 8
Figure 8
Expression of Cbfa1 and DN-Cbfa1 in cultured chondrocytes. Chick sterna chondrocytes were infected with avian retrovirus RCAS (A) encoding type I Cbfa1 (Cbfa1) or the runt domain with NH2-terminal domain of type I Cbfa1 (DN-Cbfa1). Control cultures (Control) were infected with RCAS (A) virus encoding vector alone. (A) Immunoblot of the introduced gene products. (B) Photographs of cultures 5 d after virus infection. (C) Histochemical analysis of alkaline phosphatase activity (APase) and matrix calcification (Alizarin-red). (D) Northern blot of type II collagen (Type II), type X collagen (Type X), and tenascin (TN). Expression of DN-Cbfa1 retained the small cell size (B) and high levels of tenascin and type II collagen expression (D), whereas it inhibited the induction of alkaline phosphatase (C), matrix calcification (C), and type X collagen (D), indicating that DN-Cbfa1 disturbed chondrocyte maturation and maintained it in an early developmental state.
Figure 9
Figure 9
GDF-5 expression in developing limbs. (A–D) Whole-mount in situ hybridization of hind limbs from wild-type (A and C) and Cbfa1 transgenic (B and D) mice using antisense GDF-5 probe. In wild-type mice, GDF-5 expression was detected in knee and metatarsophalangeal joints at E12.5 (A). Additional stripes of expression are seen in interphalangeal joints and between the developing rows of tarsals at E13.5 (not shown) and E14.5 (C), whereas no GDF-5 expression was detected in the knee (B and D) and its expression in digital rays first appeared at E14.5 in Cbfa1 transgenic mice (D). A and B, E12.5; C and D, E14.5. In addition, GDF-5 expression in metatarsophalangeal joints is weak and no expressions between the developing tarsals are seen in Cbfa1 transgenic mice at E14.5 (D). (E–G) In situ hybridization of GDF-5 using sections from forelimbs of wild-type (E) and Cbfa1 transgenic (F and G) mice at E12.5. GDF-5 expression is clearly localized to the joint regions of the shoulder and elbow in wild-type mice (E). In Cbfa1 transgenic mice, GDF-5 expression is detected in the shoulder (F), but not in the elbow (G) joints. S, scapula; H, humerus; R, radius; U, ulna. Bars: (A–D) 1 mm; (E–G) 100 μm.
Figure 10
Figure 10
Tenascin expression in developing limbs. (A–F) Immunohistochemistry of elbow joint regions (A–C) and shoulder joint regions (D–F) from wild-type (A and D), Cbfa1 transgenic (B and E), and DN-Cbfa1 transgenic (C and F) mice at E15.5 using antitenascin antibody. Tenascin is strongly expressed along the edge of epiphysis and perichondrium and in bone of wild-type mice (A and D), but not in presumptive joint regions of Cbfa1 transgenic mice (B and E). In DN-Cbfa1 transgenic mice, tenascin is expressed more abundantly in the humerus, radius, and ulna (C and F). (G–I) H-E staining in the shoulder joint regions from wild-type (G), Cbfa1 transgenic (H), and DN-Cbfa1 transgenic (I) mice at E15.5. R, radius; U, ulna; H, humerus; S, scapula. Bars: (A–F) 300 μm; (G–I) 50 μm.
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