Effect of systemic calcium deficiency on the expression of transforming growth factor-beta in chick embryonic calvaria.

The developmental process of intramembranous ossification involves bone formation directly from mesenchymal differentiation without a cartilage intermediate. We have previously observed that systemic calcium deficiency in the developing chick embryo, produced by long-term shell-less culture, results in the appearance of chondrocyte-like cells in the calvarium, a parietal bone which normally develops via intramembranous ossification. This investigation aims to analyze the mechanism underlying this calcium deficiency-related, aberrant appearance of cartilage phenotype in the chick embryonic calvarium. In view of the reported involvement of transforming growth factor beta (TGF-beta) in osteogenesis and chondrogenesis, we have examined and compared here the expression of TGF-beta in the chick embryonic calvaria of normal (in ovo development, NL), shell-less (SL), and calcium-supplemented SL (SL+Ca) embryos. TGF-beta expression was analyzed at the mRNA level by blot and in situ cDNA hybridization, and at the protein level by immunohistochemistry and immunoblotting. The results presented here indicate that: 1) TGF-beta is expressed in the chick embryonic calvarium by both periosteal cells and osteocytes, as revealed by in situ hybridization and immunohistochemistry; 2) TGF-beta expression is significantly increased in SL calvarium compared to NL calvarium, at both protein and mRNA levels; 3) the number of TGF-beta expressing cells increases in the SL calvarium, particularly along the central, subcambial core region of the bone; and 4) exogenous calcium repletion to the SL embryo affects the expression of TGF-beta such that the pattern approaches that in the NL embryo. Taken together, these results indicate that altered TGF-beta expression accompanies the aberrant appearance of cartilage phenotype caused by systemic calcium deficiency. We postulate that normal cellular differentiation along the osteogenic pathway during embryonic intramembranous ossification is crucially dependent on regulated TGF-beta expression.