Panel A shows fluorescence microscopy images comparing GFP:Grp1 signal intensity in embryos from wild-type, torso mutant, and tsl mutant mothers. Panel B shows fluorescence microscopy images and orthogonal views of wild-type and torso mutant embryos at 0, 150, and 300 seconds. Panel C shows a line graph comparing the phosphatidylinositol 3,4,5-trisphosphate membrane-to-cytoplasm ratio over time in embryos of different maternal genotypes. Panel D shows a violin plot comparing the phosphatidylinositol 3,4,5-trisphosphate membrane-to-cytoplasm ratio between wild-type and torso mutants at 300 seconds.
PIP3 levels in the embryo are dependent on Torso dosage. (A) Embryos from WT, torsoHH/WK, and tsl3/4 mothers also expressing the PIP3 biosensor GFP:Grp1[PH] (fire LUT) were live-imaged to visualize PIP3 spatial distribution. Embryos are imaged prior to nuclear cycle 9, since the nuclei have not yet migrated to the cortex. Embryos are oriented with their anterior poles to the left and their posterior poles to the right. Images show a single plane roughly through the middle of the embryo. (B) Embryos from WT and torsoHH/WK mutant mothers also expressing the PIP3 biosensor GFP:Grp1[PH] (fire LUT) and nuclear marker His2AV:RFP (gray) were posteriorly mounted and live imaged at nuclear cycle 10. Images are maximum intensity projections of a 20 µm section. The 300-s time point represents nuclear cycle 10 metaphase. Scale bar = 20 µm. Orthogonal views are shown below maximum intensity projections. Scale bar = 10 µm. (C) Time-lapse measurement of PIP3 membrane-to-cytoplasm ratio in embryos from WT, gcl+/−, gcl−/−, and torsoHH/WK mothers. Bars represent the mean ± SEM. (D) The membrane-to-cytoplasm ratio of PIP3 fluorescence was plotted for embryos from WT and torsoHH/WK mutant mothers at nuclear cycle 10 metaphase (300 s timepoint). (n = 7 embryos from WT mothers, n = 7 embryos from torsoHH/WK mothers, Mann–Whitney test).