Downregulation of basal myosin-II is required for cell shape changes and tissue invagination (2018)
Figure 3

Figure 3. Basal myosin-II downregulation prior to tissue invagination is required for cell lengthening.

(A-C) Embryos co-expressing the optogenetic module and Sqh::mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo-activation of the cell base in the anterior (left) half of the embryo and confocal recording of the whole embryo. The experiment was started at the end of cellularization when initial accumulation of apical myosin-II was detected and terminated at different time points to record the plasma membrane signal (CIBN::GFP-pm) in the whole embryo using two-photon illumination. Still frames visualizing the plasma membrane (CIBN::GFP-pm) at a sub-apical plane at the initial time point of the experiment (A), 3 min (B) and 10 min (C) after initial photo-activation.

(D-I) The CIBN::GFP-pm signal was used to reconstruct cell shape in 3D as described in the STAR Methods. Representative examples (18 ≤ n ≤ 136) of reconstructed cell shapes in the non-activated region at the initial time point (D), 3 min (E) and 10 min (F) after photo-activation show that the cells undergo apical constriction and the characteristic transition from a columnar to a conic shape. In contrast, representative examples (83 ≤ n ≤ 133) of reconstructed cell shapes in the activated region at the initial time point (G), 3 min (H) and 10 min (I) reveal that photo-activated cells did not undergo stereotypic cell shape changes and remain columnar.

(J-M) Based on the reconstructed cell shapes in the non-activated (green) and activated region (red), cell length (J), apical/basal ratio (K), basal area (L) and volume (M) were quantified at different times after initial photo-activation of the cell base. (J) While cells in the non-activated region elongated from ~ 25 µm to ~ 40 µm, cells in the activated region showed impaired cell lengthening, both in terms of final cell length (<40 µm) and elongation speed. ANOVA result: F(7,666)=117.9, p=3.8e-112. Cohen's D > 0.87 between NR and AR (excluding t = 0 min). (K) The apical/basal (A/B) ratio is a parameter describing cell shape that corresponds to the ratio of the most apical volume divided by the most basal volume. A ratio of 1 indicates a columnar shape, a ratio < 1 indicates a conic shape and a high ratio > 1 indicates an inverted conic shape with an apical surface that is bigger than the basal surface. In the non-activated region (top-left graph) the A/B ratio decreased from ~ 1 at the initial time point to 0.5. In the activated region (top-right graph) the A/B ratio spread around a value of ~ 1 indicating persisting columnar cell shape. After 10 min of photo-activation the A/B ratio showed higher variance, spreading between ~ 0.5 and ~ 4.5 indicating the co-existence of cells acquiring different cell shapes. There were no statistically significant differences between group means within the activated cell dataset as determined by one-way ANOVA (F(3,384)=1.617, p=0.18). The cartoon illustrates the cell shapes represented by different A/B ratios. ANOVA result (all datapoints): F(7,666)=44.95, p=4e-52. Cohen's D > 0.73 between NR and AR (excluding t = 0 min). (L) The basal cell area of 27 µm pre-activation (pre A; t = 0 min) increased to 50 µm in the non-activated region (NR) while it remained constant in the activated region (AR) after 11 min. (M) Cell volume is conserved in the non-activated and activated region. Cell volume was normalized to the mean of all samples. An one-way ANOVA test was performed (F(7,666)=3.34, p=0.0017) followed by a post-hoc Tukey test and Cohen's D test (d<0.5) both revealing no significant difference between any sample pair. (J-M) 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 and the '+' symbol indicates an outlier. Notches indicate comparison intervals. Between nNR:11 min = 18 and nNR:0 min = 136 cells per sample with a total number of ntotal = 674 cells from at least two embryos per sample and altogether 9 embryos were analyzed. In all panels, **** indicates p ≤ 0.0001, *** indicates p ≤ 0.001, and n.s. indicates no statistically significant differences according to two-sample t-Test.