Gap junctions are involved in the early generation of left-right asymmetry

Invariant left-right asymmetry of the visceral organs is a fundamental feature of vertebrate embryogenesis. While a cascade of asymmetrically expressed genes has been described, the embryonic mechanism that orients the left-right axis relative to the dorsoventral and anteroposterior axes (a prerequisite for asymmetric gene expression) is unknown. We propose that this process involves dorsoventral differences in cell-cell communication through gap junctions composed of connexin proteins. Global modulation of gap junctional states in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of heart, gut, and gall bladder. Greatest sensitivity was observed between st. 5 and st. 12, well before the onset of organogenesis. Moreover, heterotaxia was also induced following microinjection of dominant negative and wild-type connexin mRNAs to modify the endogenous dorsoventral difference in junctional communication. Heterotaxia was induced by either blocking gap junction communication (GJC) dorsally or by introducing communication ventrally (but not the reverse). Both connexin misexpression and exposure to GJC-modifying drugs altered expression of the normally left-sided gene XNR-1, demonstrating that GJC functions upstream of XNR-1 in the pathway that patterns left-right asymmetry. Finally, lineage analysis to follow the progeny of microinjected cells indicated that they generally do not contribute the asymmetric organs. Together with the early sensitivity window, this suggests that GJC functions as part of a fundamental, early aspect of left-right patterning. In addition, we show that a potential regulatory mutation in Connexin43 is sufficient to cause heterotaxia. Despite uncertainty about the prevalence of the serine364 to proline substitution reported in human patients with laterality defects, the mutant protein is both a mild hypomorph and a potent antimorph as determined by the effect of its expression on left-right patterning. Taken together, our data suggest that endogenous dorsoventral differences in GJC within the early embryo are needed to consistently orient left-right asymmetry.