Panel A: A timeline diagram showing the collection and activation process of embryos. Key events include egg laying, pole bud formation, PGC formation, and somatic cellularization, marked at specific minutes. Panel B: A series of microscopy images showing embryos expressing His2AV:RFP alone or with OptoSos under blue-light activation. The images highlight the pole bud nuclei and the absence of a group of rounded nuclei in embryos expressing both His2AV:RFP and OptoSos. Panel C: Microscopy images of representative embryos under different conditions, immunostained with anti-Vasa, F-actin, and DNA. Panel D: A bar graph showing the percentage of embryos with different numbers of PGCs under various conditions (w1118 Dark, w1118 Light, OptoSos Dark, OptoSos Light).
The RasGEF activity of Sos is sufficient to inhibit PGC formation, but not pole bud formation. (A) Timeline for optogenetic Ras activation. (B) Embryos from mothers expressing His2AV:RFP alone or His2AV:RFP together with OptoSos were imaged under blue-light activation in a region of interest (outlined with a dotted white oval). Arrowheads indicate the pole bud nuclei that protrude against the posterior membrane. In embryos from mothers that express His2AV:RFP alone, a group of rounded nuclei was observed adjacent to the layer of elongated somatic nuclei. This group of rounded nuclei was not observed in embryos from mothers expressing both His2AV:RFP and the OptoSos construct. The first three rows show z-plane images, while the bottom row shows a maximum-intensity projection. Embryos are cycle 9–10. Scale bar = 50 μm. (C) Representative embryo for each indicated condition. Fixed embryos were immunostained with anti-Vasa (Green). F-actin (gray) outlines the membrane. DNA (blue). (D) Embryos from WT (w1118) or OptoSos-expressing mothers were either kept in the dark (dark) or activated with blue light at the posterior pole (light). After the 100-min activation cycle, embryos were fixed and stained with an antibody against Vasa to assess PGC formation. The percentage of embryos with the indicated number of PGCs was scored. N = 20 embryos for w1118 dark, n = 14 embryos for w1118 light, n = 12 embryos for OptoSos dark, and n = 20 embryos for OptoSos light.