Cerberus-Nodal-Lefty-Pitx signaling cascade controls left<i>-</i>right asymmetry in amphioxus.

Abstract

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by <i>Nodal</i> antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that <i>Cerberus</i>, <i>Nodal</i>, <i>Lefty</i>, and their target transcription factor <i>Pitx</i> are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of <i>Cerberus</i> leads to ectopic right-sided expression of <i>Nodal</i>, <i>Lefty</i>, and <i>Pitx</i>, whereas overexpression of <i>Cerberus</i> represses their left-sided expression. Overexpression of Nodal in turn represses <i>Cerberus</i> and activates <i>Lefty</i> and <i>Pitx</i> ectopically on the right side. We also show Lefty represses <i>Nodal</i>, whereas <i>Pitx</i> activates <i>Nodal</i> These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with <i>Nodal</i> and combine to establish asymmetric <i>Pitx</i> expression. <i>Cerberus</i> and <i>Lefty</i> are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.