Antisense oligonucleotide blockade of connexin expression during embryonic bone formation: evidence of functional compensation within a multigene family.

Abstract

Prior studies in our laboratory demonstrated the presence of gap junction proteins (connexins) throughout intramembranous bone formation [Minkoff et al. (1994) Anat Embryol 190:231-241]. In addition, two members of the connexin family of gap junction proteins, connexin 43 (Cx43; Gj alpha 1) and connexin 45 (Cx45; Gj alpha 6), were found by Civitelli et al. [1993; J Clin Invest 91:1888-1896] to be associated, specifically, with osteogenesis. Recently, however, a null mutation in the gene encoding Gj alpha 1 in mice has been produced by Reaume et al. [1995; Science 267:1831-1834]. Gj alpha 1 null homozygotes survived to term but died at birth of heart abnormalities. Examination of the null homozygous embryos, surprisingly, did not reveal overt histological or anatomical abnormalities in any organ system other than the heart. In view of this, the present investigation was initiated in order to evaluate bone formation under conditions in which the expression of Gj alpha 1 and Gj alpha 6, the connexins specifically associated with osteogenesis, had been perturbed, individually as well as in combination. An in vitro system employing organ cultures of dissociated embryonic chick mandibular mesenchyme was employed. Mesenchyme was cultured in the presence and absence of sense and antisense oligodeoxynucleotides (ODN), ranging in length from 15 to 24 mer and containing sequences that included the initiation codon of Gj alpha 1 and of Gj alpha 6. In cultures of mesenchyme, grown for 6 to 13 days in the presence of the combined antisense ODNs to Gj alpha 1 and Gj alpha 6, bone formation was markedly reduced or absent. By contrast, in cultures grown in medium containing the combination of corresponding sense ODNs to both Gj alpha 1 and Gj alpha 6, bone formation was evident. In addition, when cultures were grown in the presence of antisense or sense ODNs to either Gj alpha 1 or Gj alpha 6, individually, bone formation was seen. Immunohistochemical analysis of connexin expression revealed intense immunoreactive signal to Gj alpha 1 and Gj alpha 6 in bone of the control explants, in which no ODNs were present; in those cultures in which either Gj alpha 1 and Gj alpha 6 antisense ODNs were present, however, the expression of the respective connexin protein was either significantly reduced or absent. Further, in those explants in which Gj alpha 1 expression was blocked, immunoreactive signal to Gj alpha 6 appeared to have been amplified in regions of developing bone. These results suggest that, in avian osteogenic tissue, when Gj alpha 1 protein expression has been impeded another related connexin protein (Gj alpha 6) may subserve the functions of the missing connexin. The findings of this study, therefore, support the hypothesis that, within the connexin gene family, functional compensation can occur.