Figure 4Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (A) Still frames of the time lapse membrane (GAP43::mCherry) recording are shown at the initial time point (top), after 6.5 min (middle), and after 13 min (bottom) of photo-activation. Cells in the non-activated region (green) underwent normal apical constriction. In the activated region, two populations of cells were observed, one that constricted (purple) and one that did not constrict (red). Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells. Cells that constricted less were considered to be defective in apical constriction and are referred to as non-constricting. Scale bar 20 µm. Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (B) All cells show pulsatile behavior. Graphs illustrate the area of three representative cells in each category (non-activated region: top; activated region with constricting cells: middle; activated region with non-constricting cells: bottom) over the course of one experiment. Yellow circles indicate the position of automatically identified local maxima used for further analyses.Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (C, D) (C) Diagram showing the extent of ratchet-like constrictions. The mean difference in apical area between adjacent peaks (relaxed cell surface) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse. Cells in the non-activated region (NR) constricted on average more than 5 µm2, which is characteristic for ratchet-like constriction. Non-constricting cells in the activated region (AR, non-con.) displayed a mean difference in apical area per pulse of about 0, reflecting non-ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size. Constricting cells in the activated region (AR, con.) show an intermediate behavior with less efficient constrictions than in the NR. Data points are significantly different as determined by ANOVA (F(2,539)=72.04, p=1.9e-28) and post-hoc Tukey test. (D) The pulsation period (T). All cells pulse with a period of about 90 s. There were no statistically significant differences between group means as determined by one-way ANOVA (F(2,540)=1.438, p=0.24). (C and D) Red dots indicate the mean values and the error bars represent the standard deviation. More than 500 pulses (nNR = 119, nAR, non-con. = 128, nAR, con = 295) of more than 100 cells were analyzed. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (E) Diagram showing the prevalence of cells in the photo-activated region that constricted (con.) and that did not constrict (Non-con.). More than 4/5 of the cells did not constrict to half of the initial area (N = 5 embryos; n: 407 cells; Cohen's D: d = 10). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (F,G) Graphs showing the average apical area (F) and anisotropy (G) of cells in the non-activated region (green), in the activated region that constricted (purple) and that did not constrict (red). The straight line indicates the mean value at a given time-point, the semi-transparent colored area indicated the respective standard deviation. Cells in the non-activated region constricted efficiently and progressively acquired anisotropy. Constricting cells in the activated region constricted slower in an isotropic manner. Non-constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy (nNR = 34, nAR, non-con. = 20, nAR, con = 59).