Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking

Abstract

Many developmental processes break left-right (LR) symmetry with a consistent handedness. LR asymmetry emerges early in development, and in many species the primary determinant of this asymmetry has been linked to the cytoskeleton. However, the nature of the underlying chirally asymmetric cytoskeletal processes has remained elusive. In this study, we combine thin-film active chiral fluid theory with experimental analysis of the C. elegans embryo to show that the actomyosin cortex generates active chiral torques to facilitate chiral symmetry breaking. Active torques drive chiral counter-rotating cortical flow in the zygote, depend on myosin activity, and can be altered through mild changes in Rho signaling. Notably, they also execute the chiral skew event at the 4-cell stage to establish the C. elegans LR body axis. Taken together, our results uncover a novel, large-scale physical activity of the actomyosin cytoskeleton that provides a fundamental mechanism for chiral morphogenesis in development.

Keywords: C. elegans; L/R symmetry breaking; Wnt signaling; active torques; actomyosin cortex; biophysics; hydrodynamics; structural biology.

Figure 1.. Chiral flow depends on myosin activity.

(A) Sketch of a C. elegans embryo. Curved arrows illustrate chiral counter-rotating flow in the anterior (A, red) and posterior (P, green) half of the embryo, respectively. (B) Time-averaged cortical flow field (arrows) at the bottom surface of a representative C. elegans embryo viewed from the outside of the embryo in this and all other images. Arrow colors indicate y-velocity. Scale bar, 5 μm. Velocity scale arrow, 20 μm/min. (C) Histogram of instantaneous chiral counter-rotation velocity $v_c={\langle v_y\rangle }_P-{\langle v_y\rangle }_A$, where ${\langle v_y\rangle }_A$ $\left({\langle v_y\rangle }_P\right)$ is the average of the y-component of the velocity v over the left (right) shaded area in (B), for non-RNAi (858 frames from 25 embryos; gray) and mlc-4 (RNAi) (8 hrs; 223 frames from 7 embryos; beige). Dashed vertical lines indicate mean v_c. (D) y-velocity v_y along the AP axis averaged over 18 vertical stripes as indicated, for non-RNAi (black, averaged over 25 embryos) and mlc-4 (RNAi) (beige, averaged over 7 embryos). Error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.04165.003

Figure 1—figure supplement 1.. Similar flow fields were obtained when imaging actin as compared to imaging myosin Video 8.

Quantification of chiral flow for myosin and actin using a dual-colored transgenic line (SWG003). (A) Histogram of instantaneous chiral counter-rotation velocity $v_c={\langle v_y\rangle }_P-{\langle v_y\rangle }_A$, where ${\langle v_y\rangle }_A$$\left({\langle v_y\rangle }_P\right)$is the average of the y-component of the velocity v over the left (right) shaded area in Figure 1B, for NMY-2::GFP (245 frames from 6 embryos; gray) and Lifeact::tagRFP-T (245 frames from 6 embryos; beige). Dashed vertical lines indicate mean v_c. (B) y-velocity v_y along the AP axis averaged over 18 vertical stripes, as indicated in Figure 1B, for NMY-2::GFP (black, averaged over 6 embryos) and Lifeact::tagRFP-T (beige, averaged over 6 embryos). Error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.04165.004

Figure 2.. The cortex actively generates torques.

(A) Left, myosin heads consume ATP to pull (Kron and Spudich, 1986) and twist (Sase et al., 1997; Beausang et al., 2008) actin filaments, leading to the generation of a force dipole (top, magenta) and a torque dipole (bottom, beige). Right, these can generate an active tension and an active torque density at larger scales, causing an isolated piece of cortex to contract (top) and rotate (bottom). Gray surface, membrane; cube with wire frames, non-contracted (non-rotated) piece of cortex; magenta (beige) cubes, contracted (rotated) piece of cortex. The gray arrow points from the outside to the inside of the cell and the rotation is clockwise when viewed from the outside. (B) Top, myosin intensity (blue markers) and velocity profiles (magenta markers, AP flow velocity v_x; beige markers, y-velocity v_y) along the AP axis (Figure 1B,D) for the non-RNAi condition (averaged over 25 embryos). Error bars, SEM. Magenta and beige curves, respective theoretical velocity profiles (c = 0.58 ± 0.09). Bottom, sketch of a C. elegans embryo with clockwise active torques in beige (as viewed from the outside of the embryo). A gradient in myosin concentration along the AP axis (see plot above) leads to a gradient in active torques (shown here with varying sizes of the clockwise torques), resulting in a chiral flow (red and green arrows) orthogonal to the gradient. DOI: http://dx.doi.org/10.7554/eLife.04165.007

Figure 2—figure supplement 1.. Myosin distribution is azimuthally symmetric.

(A) Left, representative image of the actomyosin cortex labeled with GFP-tagged NMY-2 (gray, myosin) at 30% cortical retraction. Scale bar, 5 μm. Right, histogram of the difference in spatially averaged myosin fluorescence intensity between the posterior and anterior halves of the embryo, quantified from the respective shaded regions for non-RNAi embryos (N = 250 frames from 25 embryos; gray). Dashed line, mean of the difference in myosin intensity. Only the last 50 s of cortical flow was utilized from each video for generating this histogram. (B) Left, representative image of the actomyosin cortex labeled with GFP-tagged NMY-2 (gray, myosin). Right, histogram of the difference in spatially averaged myosin fluorescence intensity between the top and bottom halves of the embryo in the posterior from the respective shaded regions for non-RNAi embryos (N = 858 frames from 25 embryos; gray). Dashed line, mean of the difference in myosin intensity. The entire cortical flow period was utilized from each video for generating this histogram. DOI: http://dx.doi.org/10.7554/eLife.04165.008

Figure 3.. Ratio of active torque to active tension is modulated by Rho.

(A) Chiral counter-rotation velocity v_c (top), AP velocity v_x (middle), and chirality index c (bottom) for non-RNAi (gray), mlc-4 (4, 6, 8 hrs RNAi), ect-2 (4, 6, 8 hrs RNAi), and rga-3 (3, 5, 40 hrs RNAi). Error bars, error of the mean with 99% confidence. Yellow bars, significant difference to non-RNAi condition; brown bars, no significant difference. (B) Histogram of instantaneous chiral counter-rotation velocity v_c for mlc-4 (left; 6 hrs; 235 frames from 7 embryos), ect-2 (middle; 6 hrs; 338 frames from 9 embryos), and rga-3 (right; 5 hrs; 402 frames from 10 embryos) RNAi. Gray histograms, non-RNAi condition. Dashed lines, mean v_c. (C) Respective time-averaged cortical flow field (arrows) of representative embryos (gray, myosin). Arrow colors indicate y-velocity v_y. Scale bar, 5 μm. Velocity scale arrow, 20 μm/min. (D) Respective average myosin intensity (blue markers) and velocity profiles (magenta markers, AP flow velocity v_x; beige markers, y-velocity v_y) along the AP axis for each RNAi condition. Error bars, SEM. Magenta and beige curves, respective theoretical velocity profiles. Dashed lines, non-RNAi theoretical velocity profiles. DOI: http://dx.doi.org/10.7554/eLife.04165.009

Figure 3—figure supplement 1.. Chiral counter-rotation velocity v_c for mlc-4, ect-2, and rga-3 RNAi.

Each graph presents the instantaneous chiral counter-rotation velocity v_c histogram for the RNAi condition specified (beige). The histogram from the non-RNAi condition is shown in gray. Downward arrows indicate a significant decrease and upward arrows indicate a significant increase in v_c compared to the non-RNAi condition (Wilcoxon rank sum test with 99% confidence). The number of hours of RNAi is as indicated. DOI: http://dx.doi.org/10.7554/eLife.04165.010

Figure 3—figure supplement 2.. AP velocity v_x for mlc-4, ect-2, and rga-3 RNAi.

Each graph presents the instantaneous AP velocity v_x histogram for the RNAi condition specified (magenta). The histogram from the non-RNAi condition is shown in gray. Downward arrows indicate a significant decrease and upward arrows indicate a significant increase in v_x compared to the non-RNAi condition (Wilcoxon rank sum test with 99% confidence). The number of hours of RNAi is as indicated. DOI: http://dx.doi.org/10.7554/eLife.04165.011

Figure 3—figure supplement 3.. Theoretical velocity profiles for mlc-4, ect-2 ,and rga-3 RNAi.

Each graph presents the respective average myosin intensity (blue markers) and velocity profiles (magenta markers, AP flow velocity v_x; beige markers, y-velocity v_y) along the AP axis for each RNAi condition specified. Error bars, SEM. Magenta and beige curves, respective theoretical velocity profiles. Dashed lines, non-RNAi theoretical velocity profiles. The number of hours of RNAi is as indicated. See respective Videos 2–5. DOI: http://dx.doi.org/10.7554/eLife.04165.012

Figure 3—figure supplement 4.. Comparison of cortical structure through imaging GFP-tagged NMY-2.

(A) Representative images for non-RNAi, 4, 6, 8, and 11 hrs of mlc-4 (RNAi) are shown. Cortical structure is disrupted by 8 hrs of mlc-4 (RNAi) (magnified view with characteristic foci size obtained from spatial myosin fluorescence intensity–intensity correlation, compare to non-RNAi). (B) Representative images for non-RNAi, 4, 6, 8, and 11 hrs of ect-2 (RNAi). Cortical structure is disrupted by 8 hrs of ect-2 (RNAi) (magnified view with characteristic foci size, compare to non-RNAi). (C) Representative images for non-RNAi, 3, 5, and 40 hrs of rga-3 (RNAi). Cortical structure is disrupted by 5 hrs of rga-3 (RNAi) (magnified view with characteristic foci size, compare to non-RNAi). Scale bars, 10 μm. Error bars, error of the mean with 99% confidence. DOI: http://dx.doi.org/10.7554/eLife.04165.013

Figure 4.. Active torques participate in L/R body axis establishment.

(A) A schematic of the skew angle measurement in the AP–LR plane. Gray dashed line, initial nuclei position; black dashed line, skewed nuclei position; beige arrows, direction of cortical flow on the dorsal surface (Video 6). To the right are the chiral skew angles of ABa for non-RNAi (gray), ect-2 (RNAi) (4.5 hrs) and rga-3 (RNAi) (4.5 hrs) in the AP–LR plane. Gray circles, skew angle in individual videos; shaded areas, SEM; green horizontal lines, mean skew angle; red horizontal lines, median skew angle; yellow shaded areas, knockdown conditions with a significant difference (95% confidence with the Wilcoxon rank sum test) from the non-RNAi condition. (B) Chiral counter-rotation velocity v_c for non-RNAi (gray), ect-2 (RNAi) (4.5 hrs) and rga-3 (RNAi) (4.5 hrs) quantified at the 4-cell stage during ABa cytokinesis. Note that one outlier was removed for computing mean v_c for rga-3 (RNAi). The expected flow profiles from our theoretical description, given a stripe of high myosin activity (corresponding to the cleavage plane), is shown in Figure 4—figure supplement 5. (C) Overall chirality index c, for non-RNAi (gray) and for Wnt signaling genes (40 hrs RNAi) that impact the establishment of the L/R body axis. Interestingly, gsk-3 not only results in a reduced chiral counter-rotation velocity but also in an increased AP velocity (Figure 4—figure supplements 2–4). Error bars, error of the mean with 99% confidence. Yellow bars, significant difference to non-RNAi condition; brown bars, no significant difference. DOI: http://dx.doi.org/10.7554/eLife.04165.017

Figure 4—figure supplement 1.. Chiral skew quantifications during bilateral symmetry breaking of the organism.

(A) A schematic of the skew angle measurement in the AP–LR plane. Gray dashed line, initial nuclei position; black dashed line, skewed nuclei position; beige arrows, direction of cortical flow on the dorsal surface (ABa counter-rotating flow shown in Video 6). Below, skew angles of ABa and ABp for non-RNAi (gray), ect-2 (RNAi) (4.5 hrs) and rga-3 (RNAi) (4.5 hrs) in the AP–LR plane. Gray circles, skew angle in individual videos; shaded areas, SEM; green horizontal lines, mean skew angle; red horizontal lines, median skew angle; yellow shaded areas, knockdown conditions with a significant difference (95% confidence with the Wilcoxon rank sum test) from the non-RNAi condition. (B) Same as (A) with the skew determined in the DV–LR plane. (C) Skew calculated without projections on to a particular plane. DOI: http://dx.doi.org/10.7554/eLife.04165.018

Figure 4—figure supplement 2.. Chiral counter-rotation velocity v_c for RNAi of Wnt signaling genes.

Each graph presents the instantaneous chiral counter-rotation velocity v_c histogram for the RNAi condition specified (beige). The histogram from the non-RNAi condition is shown in gray. Downward arrows indicate a significant decrease and upward arrows indicate a significant increase in v_c compared to the non-RNAi condition (Wilcoxon rank sum test with 99% confidence). The number of hours of RNAi is as indicated. DOI: http://dx.doi.org/10.7554/eLife.04165.019

Figure 4—figure supplement 3.. AP velocity v_x for RNAi of Wnt signaling genes.

Each graph presents the instantaneous AP velocity v_x histogram for the RNAi condition specified (magenta). The histogram from the non-RNAi condition is shown in gray. Downward arrows indicate a significant decrease and upward arrows indicate a significant increase in v_x compared to the non-RNAi condition (Wilcoxon rank sum test with 99% confidence). The number of hours of RNAi is as indicated. DOI: http://dx.doi.org/10.7554/eLife.04165.020

Figure 4—figure supplement 4.. Theoretical velocity profiles for RNAi of Wnt signaling genes.

Each graph presents the respective average myosin intensity (blue markers) and velocity profiles (magenta markers, AP flow velocity v_x; beige markers, y-velocity v_y) along the AP axis for each RNAi condition specified. Error bars, SEM. Magenta and beige curves, respective theoretical velocity profiles. Dashed lines, non-RNAi theoretical velocity profiles. The number of hours of RNAi is as indicated. See Video 7. DOI: http://dx.doi.org/10.7554/eLife.04165.021

Figure 4—figure supplement 5.. Theoretical velocity profiles for a stripe of high myosin activity.

The graph presents theoretical axial velocity v_x (magenta) and chiral velocity v_y (beige) profiles given a stripe of high myosin activity (blue). The gradient in myosin activity will then lead to axial flows along the gradient and directed towards the stripe as well as chiral flows orthogonal to the gradient leading to counter-rotations. DOI: http://dx.doi.org/10.7554/eLife.04165.022