Mouse nodal immotile cilia sense bending direction for left-right determination: Mechanical regulation in initiation of symmetry breaking.

Abstract

How is the left-right asymmetry of a mammalian body determined? Cilia at the node of mouse early embryos play an essential role in symmetry breaking. While motile cilia located at the central region of the node generate the leftward nodal flow, immotile cilia located at the periphery of the node sense flow-dependent signals, and only L-side ones are activated. The cation channel Pkd2 localized at immotile cilia is required for sensing the flow and triggers Dand5 mRNA decay, however, it has long remained elusive how cilia sense the flow and why only the L-side ones are activated. Flow-dependent passive-motion of immotile cilia in 3-D: We measure the angle of L- and R-side immotile cilia in vivo. Under controlling nodal flow by UV, significant differences in bending direction were observed: the L-side cilia showed ventral bending (5.0±9.2°; n=21), whereas the R-side ones showed dorsal bending (4.2±7.4°; n=18). Manipulation of a single immotile cilium and observation of mRNA decay: In the absence of the flow, the significant Dand5 mRNA decay was induced at 2h after the mechanical stimulation by optical tweezers (53±26%; n=28), while it was not detected with Pkd2-/- embryo (110±14%; n=7). These results demonstrate that mechanical stimuli is sufficient to activate mRNA decay via the Pkd2 channel. Immotile cilia sense bending direction in a manner dependent on polarized localization of Pkd2: The localization of the Pkd2 channel was significantly biased toward the dorsal side (0.54±0.12; n=50). In the presence of nodal flow, the ventral bending of the L-side cilium imposes a strain of 0.014±0.013 (n=8) to the dorsal side of them, which is sufficient to activate dorsally localized Pkd2. These observations explain the differential response of immotile cilia to the directional flow.