Cilia-driven asymmetric Hedgehog signalling determines the amphioxus left-right axis by controlling 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.

Molecular and cellular basis of left–right asymmetry in vertebrates

“…14 ). Like vertebrates, chordates such as amphioxus 145 , 146 ) and ascidians 147 ) deploy cilia and the Nodal-Pitx2 signaling pathway. Sea urchins also rely on motile cilia and the Nodal-Pitx2 pathway, 148 ) but, unlike vertebrates, Nodal specifies the future right side, whereas BMP acts on the left side.…”

Cardiac Development: A Glimpse on Its Translational Contributions

“…More recently, it has been demonstrated that asymmetric right sided cues directly by Prrx1 are crucial to direct rightward cardiac looping, independent of Pitx2 expression [168] configuring an enhanced epithelial-to-mesenchymal transition (EMT) in the right as compared to the left lateral plate mesoderm, a process that is modulated by distinct microRNAs [252]. [253][254][255][256][257][258][259][260][261]. As a consequence, cardiac looping takes place (panel A) and the heart display distinct left and right cardiac components that, if impaired, leads to heterotaxia (panel C).…”

“…Left-right symmetry break and epicardium development. Schematic representation of the conserved left-right symmetry break molecular cascade, emanating from the node and leading to Nodal and Pitx2 activation in the left lateral plate mesoderm (panel A) [253][254][255][256][257][258][259][260][261]. As a consequence, cardiac looping takes place (panel A) and the heart display distinct left and right cardiac components that, if impaired, leads to heterotaxia (panel C).…”

“…Our understanding of the molecular events leading to impaired left-right signaling has greatly increased over the last years. Primary events leading to symmetry break occurs at the node, as cilia triggered leftwards directional flow is established [253][254][255][256][257][258], a process that is rather conserved during evolution [259][260][261]. Impaired cilia function (i.e., primary cilia dyskinesia) underlies cardiac heterotaxia and also several associated syndromes such as Kartagener syndrome [262,263].…”

Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

“…Before injection, the Cas9 nuclease/sgRNA injection solution was incubated at 37 • C for 5 min in a thermal cycler to facilitate Cas9/sgRNA RNP complex formation. Microinjection of unfertilized eggs was carried out as previously described [13], while single blastomere injection at the two-cell stage was conducted as recently described [14]. Around 100-200 unfertilized eggs or 50 two-cell embryos were injected for each experiment.…”