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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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Development of the appendicular skeleton in Cx43−/− embryos. Front limbs at (a) E14.5, (b) clavicle at birth, and (c and d) front limbs at birth were stained with alizarin red/alcian blue. Note, the delayed mineralization of the scapula and the long limb bones (humerus, radius, and cubitus) in the mutant embryos at (a) E14.5 compared with (c and d) normal birth. Also, note the lack of both intramembranous and endochondral ossification of the clavicle, present only as a small cartilaginous template in the (a, arrows) Cx43−/− animals at E14.5. However, the clavicles appeared normal at (b) birth. (e) Longitudinal sections of safranin O/fast green–stained femurs of wild-type and Cx43−/− neonates. The size and morphology of the growth plate is apparently normal, as is the primary spongiosa in the homozygous mutant bone.
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Figure 6: Development of the appendicular skeleton in Cx43−/− embryos. Front limbs at (a) E14.5, (b) clavicle at birth, and (c and d) front limbs at birth were stained with alizarin red/alcian blue. Note, the delayed mineralization of the scapula and the long limb bones (humerus, radius, and cubitus) in the mutant embryos at (a) E14.5 compared with (c and d) normal birth. Also, note the lack of both intramembranous and endochondral ossification of the clavicle, present only as a small cartilaginous template in the (a, arrows) Cx43−/− animals at E14.5. However, the clavicles appeared normal at (b) birth. (e) Longitudinal sections of safranin O/fast green–stained femurs of wild-type and Cx43−/− neonates. The size and morphology of the growth plate is apparently normal, as is the primary spongiosa in the homozygous mutant bone.

Mentions: In the early stages of ossification (E14.5), a delay was also observed in the limbs of Cx43−/− embryos (Fig. 6 a). The limbs of the mutant animals were also smaller compared with wild-type littermates. Interestingly, at this stage, the clavicle was not ossified in the Cx43−/− mice, however, in normal mice, both the cartilaginous and membranous sections of the clavicle were already mineralized, for the most part (Fig. 6 a, arrows). However, these differences became less prominent with time, so that at birth the clavicle of Cx43−/− mice was completely ossified (Fig. 6 b) and the limbs were morphologically normal (Fig. 6c and Fig. d). Histological sections of long bones of homozygous mutant animals did not reveal detectable differences compared with wild-type bones (Fig. 6 e). The morphology and size of the growth plate was normal, as was mineralization of the primary spongiosa (Fig. 6 e).


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)

Development of the appendicular skeleton in Cx43−/− embryos. Front limbs at (a) E14.5, (b) clavicle at birth, and (c and d) front limbs at birth were stained with alizarin red/alcian blue. Note, the delayed mineralization of the scapula and the long limb bones (humerus, radius, and cubitus) in the mutant embryos at (a) E14.5 compared with (c and d) normal birth. Also, note the lack of both intramembranous and endochondral ossification of the clavicle, present only as a small cartilaginous template in the (a, arrows) Cx43−/− animals at E14.5. However, the clavicles appeared normal at (b) birth. (e) Longitudinal sections of safranin O/fast green–stained femurs of wild-type and Cx43−/− neonates. The size and morphology of the growth plate is apparently normal, as is the primary spongiosa in the homozygous mutant bone.
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Related In: Results  -  Collection

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

Figure 6: Development of the appendicular skeleton in Cx43−/− embryos. Front limbs at (a) E14.5, (b) clavicle at birth, and (c and d) front limbs at birth were stained with alizarin red/alcian blue. Note, the delayed mineralization of the scapula and the long limb bones (humerus, radius, and cubitus) in the mutant embryos at (a) E14.5 compared with (c and d) normal birth. Also, note the lack of both intramembranous and endochondral ossification of the clavicle, present only as a small cartilaginous template in the (a, arrows) Cx43−/− animals at E14.5. However, the clavicles appeared normal at (b) birth. (e) Longitudinal sections of safranin O/fast green–stained femurs of wild-type and Cx43−/− neonates. The size and morphology of the growth plate is apparently normal, as is the primary spongiosa in the homozygous mutant bone.
Mentions: In the early stages of ossification (E14.5), a delay was also observed in the limbs of Cx43−/− embryos (Fig. 6 a). The limbs of the mutant animals were also smaller compared with wild-type littermates. Interestingly, at this stage, the clavicle was not ossified in the Cx43−/− mice, however, in normal mice, both the cartilaginous and membranous sections of the clavicle were already mineralized, for the most part (Fig. 6 a, arrows). However, these differences became less prominent with time, so that at birth the clavicle of Cx43−/− mice was completely ossified (Fig. 6 b) and the limbs were morphologically normal (Fig. 6c and Fig. d). Histological sections of long bones of homozygous mutant animals did not reveal detectable differences compared with wild-type bones (Fig. 6 e). The morphology and size of the growth plate was normal, as was mineralization of the primary spongiosa (Fig. 6 e).

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