Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells

A chimeric construct, termed 3243H7, composed of fused portions of the rat gap junction proteins connexin32 (Cx32) and connexin43 (Cx43) has been shown to have selective dominant inhibitory activity when tested in the Xenopus oocyte pair system. Co-injection of mRNA coding for 3243H7 together with mRNAs coding for Cx32 or Cx43 completely blocked the development of channel conductances, while the construct was ineffective at blocking intercellular channel assembly when coinjected with rat connexin37 (Cx37). Injection of 3243H7 into the right anterodorsal blastomere of 8-cell-stage Xenopus embryos resulted in disadhesion and delamination of the resultant clone of cells evident by embryonic stage 8; a substantial number, although not all, of the progeny of the injected cell were eliminated from the embryo by stage 12. A second construct, 3243H8, differing from 3243H7 in the relative position of the middle splice, had no dominant negative activity in the oocyte pair assay, nor any detectable effects on Xenopus development, even when injected at four-fold higher concentrations. The 3243H7-induced embryonic defects could be rescued by coinjection of Cx37 with 3243H7. A blastomere reaggregation assay was used to demonstrate that a depression of dye-transfer could be detected in 3243H7-injected cells as early as stage 7; Lucifer yellow injections into single cells also demonstrated that injection of 3243H7 resulted in a block of intercellular communication. These experiments indicate that maintenance of embryonic cell adhesion with concomitant positional information requires gap junction-mediated intercellular communication.link_to_subscribed_fulltex

Connexins in the early development of the African clawed frog Xenopus laevis (Amphibia): The role of the connexin43 carboxyl terminal tail in the establishment of the dorso-ventral axis

Connexins are a family of related proteins identified in vertebrate forming gap junctions, which mediate cell-to-cell communication in early embryos, with an important role in establishing embryonic asymmetry and ‘communication compartments’. By in situ hybridization, immunocytochemistry, reverse transcriptase PCR (RT-PCR) and western blotting we show that a Cx43-like molecule is present in oocytes and embryos of the African clawed frog Xenopus laevis, with specific localization in the animal-vegetal axis. This specific distribution is suggestive for an important role for this protein in the establishment of the dorso-ventral axis. Antisense RNA and antibodies directed against rat carboxyl terminal tail of the Cx43 (CT-Cx43) and injected in 1-cell stage Xenopus embryos, induced pronounced alterations in nervous system development, with a severe ventralization phenotype. Coherently, the overexpression of CT-Cx43 produced a dorsalization of the embryos. In antisense treated embryos, the expression of the beta-catenin gene is eliminated from the Nieuwkoop center, the pattern expression of the Chordin, Xnot and Xbra is modified, with no effect in expression of the Goosecoid gene. In CT-Cx43 mRNA treated embryos the pattern of expression of the beta-catenin, Chordin, Goosecoid, Xnot and engrailed-2 genes is modified. The expression of beta-catenin is increased in the Nieuwkoop center, the expression pattern of Chordin and Goosecoid is expanded to the posterior neural plate and engrailed-2 presents ectopic expression in the ventral region. Taken together our data suggest a role for CT-Cx43 as a maternal determinant with a critical function in the formation of the dorso-ventral axis in Xenopus laevis. The Cx43 may be one of the earliest markers of the dorso-ventral axis in these embryos and could possibly be acting through regionalization of factors responsible for the establishment of this axis