Tissue Flows Are Tuned by Actomyosin-Dependent Mechanics in Developing Embryos

Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solidlike (less fluidlike) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solidlike properties than in wild-type embryos but less solidlike properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solidlike properties in tissues that are relatively isotropic, whereas high-tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

Figure 1

Optogenetic manipulation of actomyosin tunes cell rearrangement rates and tissue flow during Drosophila body axis elongation. (a) Schematic of optogenetic tools. CIBN is targeted to the cell membrane. The light-sensitive PHR domain of CRY2 is fused to upstream myosin II regulators of the Rho/Rho-kinase pathway, RhoGEF2 (GEF) or RhoGAP71E (GAP). Under blue light illumination, CRY2 dimerizes with CIBN and the regulators accumulate at the cell membrane. (b) Number of cell rearrangements initiated in control, optoGEF, and optoGAP embryos, including both T1 processes and higher-order rosette rearrangement events. (c) Elongation of the germband tissue during convergent extension quantified by PIV analysis of time-lapse confocal movies of embryos. The length of the tissue was normalized to the value at $t=0$. (d) Instantaneous rate of rearrangements per cell per minute. (e) Correlation between the instantaneous rates of tissue elongation and cell rearrangement. Each data point represents the mean between embryos at an individual time point. The $\mathrm{mean}\pm \mathrm{SEM}$ between embryos is shown, with $n=3\text{–}5$ embryos per genotype. (f) AP cell edge contraction rate, (g) vertex resolution rate, and (h) new edge formation rate during cell rearrangements in the germband tissue of photoactivated embryos. Each data point represents a cell rearrangement initiated by contraction of an AP edge. There are $n=10\text{–}25$ edges per embryo from three to five embryos ($n=48\text{–}69$ edges per genotype). (i) Orientation of newly formed cell edges during cell rearrangements. While most of the new edges in control embryos correspond to edges oriented parallel to the anterior-posterior axis of the embryo (orientation $0\text{–}{30}^{\circ }$), optoGEF embryos show an increase in edges with diagonal orientation (${30}^{\circ }\text{–}{60}^{\circ }$), $n=3\text{–}5$ embryos per genotype, 10–25 junctions per embryo. For details see Sec. 4.

Figure 2

Cell packings and tissue mechanics in optogenetically manipulated germband tissue. (a) Stills from confocal movies showing cell packings during axis elongation: anterior, left; ventral, down; bar, $10\mu \mathrm{m}$. See also Movies S1–S3 [37]. (b) Average cell shape index $\overline{p}$ during convergent extension in photoactivated control, optoGEF, and optoGAP embryos. The $\mathrm{mean}\pm \mathrm{SEM}$ between embryos is shown, with $n=3\text{–}5$ embryos per genotype. (c) Cell shape alignment index $Q$ during convergent extension in photoactivated control, optoGEF, and optoGAP embryos. The $\mathrm{mean}\pm \mathrm{SEM}$ between embryos is shown, with $n=3\text{–}5$ embryos per genotype. The relationship between the corrected average cell shape index and cell shape alignment in the germband is shown for (d) control, (e) optoGEF, and (f) optoGAP embryos. The corrected cell shape index and cell shape alignment can be used to predict tissue mechanical behavior using an anisotropic vertex model [13]. Each data point represents the mean between embryos at an individual time point, with $n=3\text{–}5$ embryos per genotype. The solid line represents the predicted solid-fluid transition line and the shaded area represents the range of predicted values for the transition from prior vertex model simulations (see Sec. 4). (g) Distance to the predicted solid-fluid transition line, evaluated during the rapid phase of tissue elongation ($t=15\min$). (h) Relationship between cell rearrangement rate and predicted solid-fluid properties.

Figure 3

Patterns of actomyosin-generated tension at cell junctions in optogenetically manipulated germband tissue are linked with tissue fluidity. (a) Ratio of mean myosin intensities at AP compared to at DV junctions before and during tool activation beginning at $t=0$ (duration of blue light exposure indicated by blue line). Ratios for each embryo at each time point were calculated, and the $\mathrm{mean}\pm \mathrm{SEM}$ between embryos is shown, with $n=3\text{–}4$ embryos and more than 10 cells per embryo. See also Movie S4 [37]. (b) Shown on the left are ablations at AP cell junctions in optogenetically manipulated germband tissue. Cells are shown before (left) and 1 min after (right) ablation: myosin II (magenta) and CIBN-pmGFP (green). Arrowheads indicate the vertices connected to the junction in which the cut was made: anterior, left; ventral, down; image size, $20\times 20\mu {\mathrm{m}}^2$. Shown on the rights are maps of displacements in the surrounding tissue in the 30 s following AP ablations. The tissue in a 174-$\mu {\mathrm{m}}^2$ region surrounding the ablation point (red mark) was analyzed by PIV analysis following ablation. Radial plots show the mean total displacement after AP ablation, averaged over the regions in ${60}^{\circ }$ angular bins, along the anterior, posterior, dorsal, or ventral tissue axes, $n=3\text{–}11$ ablations per condition. (c) Radial plots showing the mean total displacement following DV ablations. (d) Initial vertex retraction velocities after ablation. The $\mathrm{mean}\pm \mathrm{SEM}$ between junctions is shown, with $n=10\text{–}15$ AP and 3–10 DV junctions per condition. (e) Mechanical anisotropy was reduced in optoGEF and optoGAP embryos and correlates with myosin planar polarity at 5 min after optogenetic activation. Mechanical anisotropy was calculated as the ratio of mean AP to mean DV peak retraction velocities, with $n=3\text{–}15$ edges per condition. (f) Relationship between predicted fluid-solid tissue properties and tension anisotropy. As a readout of anisotropy, the ratio of AP to DV peak retraction velocities after ablation of junctional myosin was used. (g) Relationship between tissue elongation rate and tension anisotropy. Dashed lines are a guide to the eye. Photoactivated control embryos are shown by a black circle, optoGEF by a magenta circle, and optoGAP by an orange circle.

Figure 4

Levels of actomyosin-generated tension at the medial-apical domain in optogenetically manipulated germband tissue are linked with tissue fluidity. (a) Mean myosin intensity in the medial-apical domain of cells over time before and during tool activation beginning at $t=0$, with $n=3\text{–}4$ embryos and more than 10 cells per embryo. (b) Relationship between tissue mechanical properties and overall level of tissue tension in photoactivated embryos. As a readout of overall tension levels, the average magnitude of tissue retraction velocity after medial ablation was used. (c) Relationship between tissue elongation rate and overall level of tissue tension. The dashed line is a guide to the eye. (d) Ablations in the medial-apical domain of cells in optogenetically manipulated germband tissue, for cells before (left) and 1 min after (right) ablation, showing myosin II (magenta) and CIBN-pmGFP (green). Arrowheads indicate the vertices connected to the cell in which the cut was made: anterior, left; ventral, down; image size, $20\mu \mathrm{m}$. (e) Maps of displacements in the surrounding tissue in the 30 s following ablations. The tissue in a 174-$\mu {\mathrm{m}}^2$ region surrounding the ablation point (red mark) was analyzed by PIV analysis following ablation. Radial plots show the mean total displacement, averaged over the regions in ${60}^{\circ }$ angular bins, along the anterior, posterior, dorsal, or ventral tissue axes, with $n=3\text{–}11$ ablations per condition.