Cilia-driven asymmetric Hedgehog signalling determines the amphioxus left-right axis by controlling Cerberus/Dand5 expression

Abstract

Cilia rotation-driven nodal flow is critical for the left-right (L-R) break in symmetry in most vertebrates. However, the mechanism by which the flow signal is translated to asymmetric gene expression has been insufficiently addressed. Here, we show that Hh signalling is asymmetrically activated (L<R) in the region where initial asymmetric Dand5 (also called Cerberus) expression is detected. Upregulation of Hh signalling on the left side of wild-type embryos induces ectopic Dand5 expression on the left side, and the unilateral recovery of Hh signalling in Hh homozygous mutants induces Dand5 expression in the Hh signal recovery side. Immunofluorescence analysis results revealed that Hh fusion protein is asymmetrically enriched in the anterior-right paraxial mesoderm at the early neurula stage. Inhibiting embryonic cilia motility by methylcellulose blocks right-side Hh protein enrichment and randomizes Dand5 expression and organ positioning along the L-R axis. These findings present a model showing that cilia movement is crucial for the symmetry breaks in amphioxus through asymmetric Hh protein transport. The resultant asymmetric Hh signalling provides a clue into the induction of asymmetric Dand5 expression.