Antisense oligonucleotide blockade of connexin expression during embryonicbone formation: Evidence of functional compensation within a multigene family

Prior studies in our laboratory demonstrated the presence of gap junction p roteins (connexins) throughout intramembranous bone Formation [Minkoff et a l. (1994) Anat Embryol 190.231-241]. In addition, two members of the connex in Family of gap junction proteins, connexin 43 (Cx43; Gj alpha 1) and conn exin 45 (Cx45; Gj alpha 6), were found by Civitelli et al. [1993; J Clin In vest 91 :1888-1896] to be associated, specifically, with osteogenesis; Rece ntly however, a null mutation in the gene encoding Gj alpha 1 in mice has b een 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 ov ert histological or anatomical abnormalities in any organ system other than the heart. In view of this, the present investigation was initiated in ord er to evaluate bone formation under conditions in which the expression of G j alpha I and Gj alpha 6, the connexins specifically associated with osteog enesis, had been perturbed, individually as well as in combination. An in v itro system employing organ cultures of dissociated embryonic chick mandibu lar mesenchyme was employed. Mesenchyme was cultured in the presence and ab sence of sense and antisense oligodeoxynucleotides (ODN), ranging in length from 15 to 24 mer and containing sequences that included the initiation co don 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 I an d Gj alpha 6, bone formation was markedly reduced or absent. By contrast, i n cultures grown in medium containing the combination of corresponding sens e ODNs to both Gj alpha I and Gj alpha 6, bone formation was evident. In ad dition, when cultures were grown in the presence of antisense or sense ODNs to either Gj alpha I or Gj alpha 6, individually, bone formation was seen. Immunohistochemical analysis of connexin expression revealed intense immun oreactive signal to Gj alpha I and Gj alpha 6 in bone of the control explan ts, in which no ODNs were present; in those cultures in which either Gj alp ha 1 or 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 I expression was blocked, imm unoreactive 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 conne xin protein (Gj alpha 6) may subserve the functions of the missing connexin . The findings of this study, therefore, support the hypothesis that, withi n the connexin gene family, functional compensation can occur. (C) 1999 Wil ey-Liss, Inc.