Panel A: Fluorescence microscopy images showing F-Actin (green) and nuclei (magenta) in embryos from germ cell-less heterozygous and germ cell-less homozygous mutant mothers at different time points. The images are maximum intensity projections of a 20 micrometer section. Panel B: Violin plot comparing the membrane-to-cytoplasm ratio of F-Actin fluorescence in embryos from germ cell-less heterozygous and germ cell-less homozygous mutant mothers. Panel C: Orthogonal views of embryos showing F-Actin (green) and nuclei (magenta) 150 seconds before nuclear cycle 10 metaphase in embryos from germ cell-less heterozygous and germ cell-less homozygous mutant mothers. Panel D: Fluorescence microscopy images showing Myosin II (green) and nuclei (magenta) in embryos from germ cell-less heterozygous and germ cell-less homozygous mutant mothers at different time points. The images are maximum intensity projections of a 20 micrometer section. Panel E: Violin plot comparing the membrane-to-cytoplasm ratio of Myosin II fluorescence in embryos from germ cell-less heterozygous and germ cell-less homozygous mutant mothers. Panel F: Orthogonal views of embryos showing Myosin II (green) and nuclei (magenta) 150 seconds before nuclear cycle 10 metaphase in embryos from germ cell-less heterozygous and germ cell-less homozygous mutant mothers. Panel G: Schematic diagram illustrating the segmentation of embryos. Panel H: Fluorescence microscopy image showing Myosin 2 (magenta) in an NC11-12 embryo, oriented with its anterior to the left and posterior to the right. The image shows a single plane roughly in the middle of the embryo. Panel I: Line plot showing the normalized Myosin II fluorescence intensity across the circumference of the embryo cortex.
Torso/PI3K activity decreases myosin II recruitment to the pole bud membrane. (A) Embryos from gcl+/− and gcl−/− mothers also expressing the F-actin marker moe[ABD]:GFP (green) and the nuclear marker His2AV:RFP (magenta) were posteriorly mounted and live imaged. Images are maximum intensity projections of a 20 µm section. The 300 s time point represents nuclear cycle 10 metaphase. Scale bar = 20 µm. (B) Membrane and cytoplasm were segmented from posterior-mounted embryo movies 150 s before nuclear cycle 10 metaphase, and the membrane-to-cytoplasm ratio of F-Actin fluorescence was plotted. (n = 6 embryos for gcl+/− maternal genotype, n = 5 embryos for gcl−/− maternal genotype, Mann–Whitney test). (C) Orthogonal view of embryos from gcl+/− and gcl−/− mothers also expressing the F-actin marker moe[ABD]:GFP (green) and the nuclear marker His2AV:RFP (magenta) 150 s before nuclear cycle 10 metaphase. Scale bar = 10 µm. (D) Embryos from gcl+/− and gcl−/− mothers also expressing a marker for the regulatory light chain of non-muscle myosin II, sqh:3xGFP (green) and the nuclear marker His2AV:RFP (magenta) were posteriorly mounted and live imaged. Images are maximum intensity projections of a 20 µm section. The 300-s time point represents nuclear cycle 10 metaphase. Scale bar = 20 µm. (E) Membrane and cytoplasm were segmented from posterior-mounted embryo movies 150s before nuclear cycle 10 metaphase, and the membrane-to-cytoplasm ratio of myosin II fluorescence was plotted. (n = 6 embryos for gcl+/− maternal genotype, n = 5 embryos for gcl−/− maternal genotype, Mann–Whitney test). (F) Orthogonal view of embryos from gcl+/− and gcl−/− mothers also expressing a marker for the regulatory light chain of non-muscle II, sqh:3xGFP (green) and the nuclear marker His2AV:RFP (magenta) 150 s before nuclear cycle 10 metaphase. Scale bar = 10 µm. (G) Schematic of embryo segmentation for the quantification in I. (H) Nuclear cycle 11–12 embryos from mothers expressing a marker for the regulatory light chain of non-muscle myosin II, sqh:mScarlet-I (magenta), were fixed and laterally mounted. The embryo shown is oriented with its anterior (A) to the left and its posterior (P) to the right. The image shows a single plane roughly in the middle of the embryo. Scale bar = 100 µm. (I) Plot of myosin II fluorescence intensity across the circumference of the embryo cortex, normalized to the average fluorescence intensity of the cleavage furrows per embryo. n = 25 embryos.