Roles of Motile and Immotile Cilia in Left-Right Symmetry Breaking
Our body possesses three body axes, anteroposterior, dorsoventral, and left-right (L-R) axes. L-R asymmetry is achieved by three consecutive steps: symmetry breaking at the node, differential patterning of the lateral plate by a signaling molecule Nodal, and finally situs-specific organogenesis. Breaking of L-R symmetry in the mouse embryo takes place in the ventral node, where two types of cilia are found. Whereas centrally located motile cilia generate a leftward fluid flow, peripherally located immotile cilia sense a flow-dependent signal. Although Ca2+ signaling is implicated in flow sensing, it is still not clear what triggers Ca2+ signaling, a determinant molecule transported by the flow or mechanical force induced by the flow.
KeywordsCilia Fluid flow Laterality Left-right asymmetry
The initial breaking of L-R symmetry, which occurs in or near the node and at the late neural-fold stage
Transfer of an L-R-biased signal(s) from the node to the lateral plate mesoderm (LPM) , which leads to L-R asymmetric expression of signaling molecules such as the transforming growth factor-β (TGF-β)-related proteins Nodal and Lefty on the left side of the LPM
L-R asymmetric morphogenesis of visceral organs induced by these signaling molecules
7.2 Symmetry Breaking by Motile Cilia and Fluid Flow
How is the unidirectional fluid flow generated by rotational movement of the cilia? Hydrodynamic principles predict that the cilia can generate a unidirectional flow if they are tilted toward a specific direction. When the cilia move closer to the surface, the movement of fluid near the surface will be restricted as a result of the “no-slip boundary effect.” Conversely, when the cilia move away from the surface, they move the neighboring fluid more effectively. If cilia are tilted toward the posterior side, they will be moving toward the right when they come close to the surface and toward the left when they are far from the surface, thus generating a leftward flow. Observation of these rotating cilia by high-speed video microscopy revealed that they are indeed tilted posteriorly at an average angle of 30° [4, 5]. Recent evidence  suggests that, in addition to the “no-slip boundary effect,” intrinsic asymmetry in rotational stroke may also help generating the unidirectional flow.
How is A-P information translated into the posterior tilt of the node cilia? Given its similarity to positioning of the hair in the Drosophila wing, a mechanism resembling the planar cell polarity (PCP) pathway involving noncanonical Wnt signaling  seems to underlie positioning of the node cilia. Thus, some of the PCP core proteins such as Prickle2 and Vangl1 are localized to the anterior side of node cells [8, 9], whereas Dvl protein is localized to the posterior side  (Fig. 7.3). However, it remains unknown what positional cue is responsible for the polarized localization of these PCP core proteins.
7.3 Sensing of the Fluid Flow by Immotile Cilia
Sensing of the fluid flow by immotile cilia requires a Ca2+ channel composed of Pkd2  and Pkd1l1 [16, 17]. Indeed, several Ca2+ signaling blockers have been shown to disrupt asymmetric gene expression in crown cells . In particular, the effects of GdCl3 [an inhibitor of stretch-sensitive transient receptor potential (TRP) channels], 2-ABP [an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor], and thapsigargin (an inhibitor of Ca2+-dependent ATPase activity in the endoplasmic reticulum) suggest involvement of a TRP-type channel such as Pkd2 and the IP3 receptor in the sensing of nodal flow. A mutation in Pkd2 that disrupts the ciliary localization of the encoded protein results in L-R defects similar to those of Pkd2−/−embryos [13, 16], suggesting that Pkd2, together with Pkd1l1, functions in the ciliary compartment of crown cells. Whereas Pkd2 encodes a Ca2+ channel with a short extracellular domain, Pkd1l1 possesses a much larger extracellular domain at its amino terminus. Pkd1l1 may be responsible for sensing of the flow signal and regulating Ca2+ channel activity of Pkd2. While oscillations of Ca2+ signaling with subtle L>R asymmetry were detected in the node , direct observation of L-R asymmetric Ca2+ signaling in crown cells has not been successful .
7.4 Readouts of the Flow
L-R asymmetry of Cerl2 expression is generated at a posttranscriptional level , by degradation of Cerl2 mRNA via its 3′ untranslated region. Preferential decay of Cerl2 mRNA on the left is initiated by the leftward flow and further enhanced by the operation of Wnt-Cerl2 interlinked feedback loops, in which Wnt3 upregulates Wnt3 expression and promotes Cerl2 mRNA decay, whereas Cerl2 promotes Wnt degradation. Mathematical modeling and experimental data suggest that these feedback loops behave as a bistable switch that is able to amplify in a noise-resistant manner a small bias conferred by fluid flow.
7.5 Future Directions
Although rapid progress has been made in the last 20 years, many important questions remain unanswered. Firstly, how is A-P information translated into the posterior tilt of node cilia? Namely, what is the nature of the A-P information that polarizes node cells along the A-P axis? Secondly, how is the direction of rotation determined for node cilia? Thirdly, how does the nodal flow work? How do motile cilia sense the flow? Do they sense a signaling molecule that is transported by the flow or sense mechanical force? Fourthly, what is the precise role of Ca2+ signaling? How does Ca2+ signaling induce degradation of Cerl2 mRNA? Finally, to what extent is the mechanism for breaking of L-R symmetry conserved among species? L-R symmetry breaking does not appear to depend on cilia in Drosophila and snail . Further development of various approaches (including genetic, cellular, biophysical, and mathematical) will be necessary to answer these questions.
I thank current and former members of my laboratory for discussion as well as for providing illustrations. The work performed in my laboratory has been supported by CREST, Japan Science and Technology Corporation (JST), and by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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