Asymmetric distribution of dynamic calcium signals in the node of mouse embryo during left–right axis formation

Abstract

In the node of mouse embryo, rotational movements of cilia generate an external liquid flow known as nodal flow, which determines left-right asymmetric gene expression. How nodal flow is converted into asymmetric gene expression is still controversial, but the increase of Ca(2+) levels in endodermal cells to the left of the node has been proposed to play a role. However, Ca(2+) signals inside the node itself have not yet been described. By our optimized Ca(2+) imaging method, we were able to observe dynamic Ca(2+) signals in the node in live mouse embryos. Pharmacological disruption of Ca(2+) signals did not affect ciliary movements or nodal flow, but did alter the expression patterns of the Nodal and Cerl-2 genes. Quantitative analyses of Ca(2+) signal frequencies and distributions showed that during left-right axis establishment, formerly symmetric Ca(2+) signals became biased to the left side. In iv/iv mutant embryos that showed randomized laterality due to ciliary immotility, Ca(2+) signals were found to be variously left-sided, right-sided, or bilateral, and thus symmetric on average. In Pkd2 mutant embryos, which lacked polycystin-2, a Ca(2+)-permeable cation channel necessary for left-right axis formation, the Ca(2+) signal frequency was lower than in wild-type embryos. Our data support a model in which dynamic Ca(2+) signals in the node are involved in left-right patterning.

Paraxial <b><i>Nodal</i></b> Expression Reveals a Novel Conserved Structure of the Left-Right Organizer in Four Mammalian Species

“…Starting on the basis of an initially symmetrical paraxial nodal mRNA domain, ciliary flow causes inhibition of the right-sided activity of the nodal protein by inducing asymmetry of cerl-2 at the start of somitogenesis [Marques et al, 2004;Oki et al, 2009;Takao et al, 2013], which is followed by maintenance of nodal asymmetry through SELI [Nakamura et al, 2006]. In contrast, the paramedian nodal mRNA domain in pigs and cattle is initially asymmetrical and occurs at the early notochord formation stage (stage 5).…”

Cilia and coordination of signaling networks during heart development

“…117 A third model suggests that the nodal flow initiates a Ca 2+ influx in crown cells on the left side of the node, via activation of the polycystins (PC2 and the PC1 homolog, PC1 like 1) in mechanosensory, immotile primary cilia. 7,9,118,119 In support of the latter model, the asymmetric expression of Ablim1 at the node during the mid-headfold stage requires both a nodal flow and PC2, but the details of this relationship have not been resolved. 114 Further, it was recently shown that glycosylation of the Notch receptor in the crown cells by the N-acetylgalactosamine-type O-glycosylation enzyme GALNT11 is essential for the optimal ratio between motile and immotile cilia at the mouse node, in addition to correct Pitx2 expression in Xenopus embryos.…”

Roles of Motile and Immotile Cilia in Left-Right Symmetry Breaking

“…Pkd1l1 may be responsible for sensing of the flow signal and regulating Ca 2+ channel activity of Pkd2. While oscillations of Ca 2+ signaling with subtle L>R asymmetry were detected in the node [18], direct observation of L-R asymmetric Ca 2+ signaling in crown cells has not been successful [13]. A long-standing question since the discovery of nodal flow concerns the action of the flow.…”