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Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.

Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R - J. Cell Biol. (2000)

Bottom Line: We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T.Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells.Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Bone and Mineral and Infectious Diseases, Department of Internal Medicine, Washington University School of Medicine, Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA.

ABSTRACT
Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987-995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249-2258). Here, using the Cx43- mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43- animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

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Axial development in Cx43−/− embryos. Lateral view of the thoracic vertebrae stained with alizarin red/alcian blue of (a and b) E15.5 and (c) E16.5 (a and d) wild-type embryos and (b, c, and e) Cx43−/− homozygous mutants. (b and c) Note the delayed ossification of the vertebrae and ribs and the thinner and deformed ribs in the mutant animals. Posterior view of the head and thorax of alizarin red–stained (d) wild-type and (e) Cx43−/− embryos at E18.5 showing a similar ossification of the vertebrae, limbs, and exoccipital bones, but delayed ossification of the parietal, interparietal, and supraoccipital bones. Note that the two ossification centers of the supraoccipital bone have not fused in the Cx43−/−.
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Figure 5: Axial development in Cx43−/− embryos. Lateral view of the thoracic vertebrae stained with alizarin red/alcian blue of (a and b) E15.5 and (c) E16.5 (a and d) wild-type embryos and (b, c, and e) Cx43−/− homozygous mutants. (b and c) Note the delayed ossification of the vertebrae and ribs and the thinner and deformed ribs in the mutant animals. Posterior view of the head and thorax of alizarin red–stained (d) wild-type and (e) Cx43−/− embryos at E18.5 showing a similar ossification of the vertebrae, limbs, and exoccipital bones, but delayed ossification of the parietal, interparietal, and supraoccipital bones. Note that the two ossification centers of the supraoccipital bone have not fused in the Cx43−/−.

Mentions: The axial skeleton also revealed retarded ossification in these mutants. At E15.5, only the cartilaginous vertebrae and ribs were present in mutant animals; normally, the pedicles and laminae of the vertebrae and the dorsal half of the ribs are ossified at this stage (Fig. 5, a and b). At E16.5, areas of endochondral ossification were visible in the pedicles and laminae of the thoracic vertebrae and in the dorsal aspect of the ribs of Cx43- littermates (Fig. 5 c). The ribs of homozygous mutants appeared jagged-shaped and slightly thinner, and the thoracic cage was more brittle than in normal mice. A comparison of the axial skeleton of mutant and wild-type animals, between E15.5 and E18.5, revealed a delay of endochondral ossification of ∼1–2 d (Fig. 5, a–e). Thus, the developmental abnormalities in Cx43-deficient animals are not restricted to skeletal elements of neural crest origin. They generally involve many mesoderm-derived skeletal segments. Whereas ossification of the calvarium was still retarded and incomplete at E18.5 (Fig. 5d and Fig. e) and at birth (see above), the axial skeleton appeared normally mineralized in newborn Cx43−/− animals at these stages. Accordingly, ossification of the ribs and vertebral laminae were essentially complete at E18.5, though the ribs remained deformed, with a jagged appearance in the homozygous mutants compared with control littermates (Fig. 5d and Fig. e). At birth, there were no differences in number, size, or spacing of the vertebrae and ribs (data not shown), except for the rib deformities just described.


Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.

Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R - J. Cell Biol. (2000)

Axial development in Cx43−/− embryos. Lateral view of the thoracic vertebrae stained with alizarin red/alcian blue of (a and b) E15.5 and (c) E16.5 (a and d) wild-type embryos and (b, c, and e) Cx43−/− homozygous mutants. (b and c) Note the delayed ossification of the vertebrae and ribs and the thinner and deformed ribs in the mutant animals. Posterior view of the head and thorax of alizarin red–stained (d) wild-type and (e) Cx43−/− embryos at E18.5 showing a similar ossification of the vertebrae, limbs, and exoccipital bones, but delayed ossification of the parietal, interparietal, and supraoccipital bones. Note that the two ossification centers of the supraoccipital bone have not fused in the Cx43−/−.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169447&req=5

Figure 5: Axial development in Cx43−/− embryos. Lateral view of the thoracic vertebrae stained with alizarin red/alcian blue of (a and b) E15.5 and (c) E16.5 (a and d) wild-type embryos and (b, c, and e) Cx43−/− homozygous mutants. (b and c) Note the delayed ossification of the vertebrae and ribs and the thinner and deformed ribs in the mutant animals. Posterior view of the head and thorax of alizarin red–stained (d) wild-type and (e) Cx43−/− embryos at E18.5 showing a similar ossification of the vertebrae, limbs, and exoccipital bones, but delayed ossification of the parietal, interparietal, and supraoccipital bones. Note that the two ossification centers of the supraoccipital bone have not fused in the Cx43−/−.
Mentions: The axial skeleton also revealed retarded ossification in these mutants. At E15.5, only the cartilaginous vertebrae and ribs were present in mutant animals; normally, the pedicles and laminae of the vertebrae and the dorsal half of the ribs are ossified at this stage (Fig. 5, a and b). At E16.5, areas of endochondral ossification were visible in the pedicles and laminae of the thoracic vertebrae and in the dorsal aspect of the ribs of Cx43- littermates (Fig. 5 c). The ribs of homozygous mutants appeared jagged-shaped and slightly thinner, and the thoracic cage was more brittle than in normal mice. A comparison of the axial skeleton of mutant and wild-type animals, between E15.5 and E18.5, revealed a delay of endochondral ossification of ∼1–2 d (Fig. 5, a–e). Thus, the developmental abnormalities in Cx43-deficient animals are not restricted to skeletal elements of neural crest origin. They generally involve many mesoderm-derived skeletal segments. Whereas ossification of the calvarium was still retarded and incomplete at E18.5 (Fig. 5d and Fig. e) and at birth (see above), the axial skeleton appeared normally mineralized in newborn Cx43−/− animals at these stages. Accordingly, ossification of the ribs and vertebral laminae were essentially complete at E18.5, though the ribs remained deformed, with a jagged appearance in the homozygous mutants compared with control littermates (Fig. 5d and Fig. e). At birth, there were no differences in number, size, or spacing of the vertebrae and ribs (data not shown), except for the rib deformities just described.

Bottom Line: We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T.Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells.Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Bone and Mineral and Infectious Diseases, Department of Internal Medicine, Washington University School of Medicine, Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA.

ABSTRACT
Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987-995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249-2258). Here, using the Cx43- mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43- animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

Show MeSH
Related in: MedlinePlus