Panel A: Two microscopy images of embryos from wild-type and germ cell-less homozygous mutant mothers. The embryos are shown in a single plane roughly through the middle, with their anterior poles to the left and posterior poles to the right. The color scale indicates the intensity of GFP:Grp1 signal. Panel B: Three sets of microscopy images are maximum-intensity projections of a 20-micrometer section, taken at 0, 150, and 300 seconds (NC10 metaphase) for embryos from germ cell-less homozygous and germ cell-less heterozygous mutant mothers. Panel C: A violin plot comparing the membrane-to-cytoplasm ratio of PIP2 fluorescence in embryos from germ cell-less homozygous mutant and germ cell-less heterozygous mutant mothers. The plot shows individual data points and the distribution of the ratios. Panel D: Orthogonal views of embryos from germ cell-less wild-type and germ cell-less homozygous mutant mothers expressing the PIP2 biosensor PLC[PH]:mCherry and nuclear marker His2AV:GFP, imaged 150 seconds before nuclear cycle 10 metaphase. Panel E: Three sets of microscopy images showing embryos from germ cell-less homozygous mutant and germ cell-less heterozygous mutant mothers expressing the PIP3 biosensor GFP:Grp1 and nuclear marker His2AV:RFP. The images are maximum-intensity projections of a 20-micrometer section, taken at 0 seconds, 150 seconds, and 300 seconds (NC10 metaphase). Panel F: A violin plot comparing the membrane-to-cytoplasm ratio of PIP3 fluorescence in embryos from germ cell-less homozygous mutant and germ cell-less heterozygous mutant mothers. The plot shows individual data points and the distribution of the ratios. Panel G: Orthogonal views of embryos from germ cell-less wild-type and germ cell-less homozygous mutant mothers expressing the PIP3 biosensor GFP:Grp1 and nuclear marker His2AV:RFP, imaged 150 seconds before nuclear cycle 10 metaphase.
PIP3 acts downstream of Torso and is inappropriately enriched in the posterior pole membrane when GCL is absent. (A) Embryos from WT and gcl−/− mothers also expressing the PIP3 biosensor GFP:Grp1[PH] (fire LUT) were live imaged on their side to visualize PIP3 spatial distribution. Embryos are less than an hour old, since nuclei have not yet migrated to the cortex. Scale bar = 100 µm. Lateral view of embryos 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. Scale bar = 100 µm. (B) Embryos from g+/− and gcl−/− mothers at embryonic nuclear cycle 10 expressing the PIP2 biosensor PLCγ[PH]:mCherry (fire LUT) and nuclear marker His2AV:GFP (gray) 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. (C) Membrane and cytoplasm were segmented from posterior-mounted embryo movies 150 s before nuclear cycle 10 metaphase, and the membrane-to-cytoplasm ratio of PIP2 fluorescence was plotted. (n = 6 embryos for g+/− maternal genotype, n = 8 embryos for gcl−/− maternal genotype, Mann–Whitney test). (D) Orthogonal view of embryos from g+/− and gcl−/− mothers expressing the PIP2 biosensor PLCγ[PH]:mCherry (fire LUT) and nuclear marker His2AV:GFP (gray) 150 s before nuclear cycle 10 metaphase. Scale bar = 10 µm. (E) Embryos from g+/− and gcl−/− mutant mothers also maternally expressing the PIP3 biosensor GFP:Grp1[PH] (fire LUT) and nuclear marker His2AV:RFP (gray) were posteriorly mounted and live imaged at embryonic 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. (F) Membrane and cytoplasm were segmented from posterior-mounted embryo movies 150 s before nuclear cycle 10 metaphase, and the membrane-to-cytoplasm ratio of PIP3 fluorescence was plotted. (n = 6 embryos for g+/− maternal genotype, n = 5 embryos for gcl−/− maternal genotype, Mann–Whitney test). (G) Orthogonal view of embryos from g+/− and gcl−/− mothers also expressing the PIP3 biosensor GFP:Grp1[PH] (fire LUT) and nuclear marker His2AV:RFP (gray) imaged 150 s before nuclear cycle 10 metaphase. Scale bar = 10 µm.