Figure 4. Different zones of localized contractility contribute to invagination inC. intestinalis.

A-D: Cross-sectional views of embryos treated with 100µM Blebbistatin during Step 1 (B) or Step 2 (D), fixed at the end of each step and phalloidin-stained. A, C: Controls for Step 1 and Step 2. E-H: Apical surface area (E, G) and apico-basal height (F, H) at early Step1, late Step 1 and late Step 2 in embryos treated with 100µM Blebbistatin (E, F) or 100µM Y-27632 (G, H). Dashed lines link data points at the onset, intermediate time points and end of each treatment. Measurements for paired WT controls shown in solid yellow lines. Asterisks in E-H indicate significant differences (asterisks indicate p < 0.05 for 2-tailed t-test); error bars indicate standard deviations. Numbers of embryos measured were (Blebbistatin: n≥7 for all measurements; Y-27632: n = 4 and n = 5 for Step 2 control and Y-treated embryos, respectively, n ≥ 7 for all other measurements). Embryos in panels G,H and E,F were fixed at a slightly different times during late step 2. I-R: Phalloidin (K,L), 1P–myosin (I,J, M-P) and 2P–Myosin (Q,R) staining in controls and in embryos treated with 100 µM Y-27632 for 30 minutes and fixed at the end of Step 1 (I-L) or Step 2 (M-R). I-N: frontal sections. O, P: horizontal sections along the lines indicated in M,N. Q, R: blow-up of sub-apical horizontal sections across the vegetal pole. Arrows indicate lateral boundaries of the endoderm plate. S: Comparison of phosphomyosin levels on the indicated surfaces of A 7.1 cells in controls (solid black lines) and in embryos treated with 100 µM Y-27632 for 30 minutes prior to fixation at end of either Step 1 or Step 2 (light dashed lines). Figure S3 shows the effects on invagination of microtubule depolymerization and dominant-negative inhibition of RhoA. Movie S5 documents apical expansion during Step 2 in Y-treated embryos.