Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node

Invariant patterning of left-right asymmetry during embryogenesis depends u pon a cascade of inductive and repressive interactions between asymmetrical ly expressed genes. Different cascades of asymmetric genes distinguish the left and right sides of the embryo and are maintained by a midline barrier. As such, the left and right sides of an embryo can be viewed as distinct a nd autonomous fields. Here we describe a series of experiments that indicat e that the initiation of these programs requires communication between the two sides of the blastoderm. When deprived of either the left or the right lateral halves of the blastoderm, embryos are incapable of patterning norma l left-right gene expression at Hensen's node. Not only are both flanks req uired, suggesting that there is no single signaling source for LR pattern, but the blastoderm must be intact. These results are consistent with our pr eviously proposed model in which the orientation of LR asymmetry in the fro g, Xenopus laevis, depends on large-scale partitioning of LR determinants t hrough intercellular gap junction channels (M. Levin and M. Mercola (1998) Developmental Biology 203, 90-105), Here we evaluate whether gap junctional communication is required for the LR asymmetry in the chick, where it is p ossible to order early events relative to the well-characterized left and r ight hierarchies of gene expression. Treatment of cultured chick embryos wi th lindane, which diminishes gap junctional communication, frequently unbia sed normal LR asymmetry of Shh and Nodal gene expression, causing the norma lly left-sided program to be recapitulated symmetrically on the right side of the embryo. A survey of early expression of connexin mRNAs revealed that Cx43 is present throughout the blastoderm at Hamburger-Hamilton stage 2-3, prior to known asymmetric gene expression. Application of antisense oligod eoxynucleotides or blocking antibody to cultured embryos also resulted in b ilateral expression of Shh and Nodal transcripts. Importantly, the node and primitive streak at these stages lack Cx43 mRNA, This result, together wit h the requirement for an intact blastoderm, suggests that the path of commu nication through gap junction channels circumvents the node and streak. We propose that left-right information is transferred unidirectionally through out the epiblast by gap junction channels in order to pattern left-sided Sh h expression at Hensen's node.