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Fig 2
(A) Fluorescence images of stage III Tg(actb1:Utr-GFP) oocytes labeling F-actin during consecutive stages before maturation (stage III) and 30, 60, and 90 min after maturation induction with the DHP hormone. Black arrowhead denotes the GVBD onset. White arrow denotes the GCs surrounding the oocyte. The 35 μm × 35 μm white box demarcates the region for acquiring bulk actin intensity in S3B Fig. (B) Fluorescence image of a stage IV oocyte fixed, sectioned, and stained with Phalloidin to visualize and measure bulk F-actin gradient along the AV axis of the oocyte shown in (B’). (B’) Bulk actin intensity within cytoplasmic pockets normalized to its value at the AP along the AV axis, measured from fixed stage IV oocytes stained with phalloidin as in (B); (N = 3 experiments, n = 17 oocytes). See Table A in S2 Data for underlying data. (C) Fluorescence images of ooplasmic extract obtained from stage III Tg(actb1:Utr-GFP) oocytes labeling F-actin (gray) and exposed to Lysotracker to mark Ygs (magenta) during 30–75 min after maturation onset. The dashed box indicates the extract diameter used for acquiring the kymograph in (C’). The arrowheads indicate the F-actin and ooplasm enrichment. The white solid line demarcates the extract boundary. (C’) Kymograph acquired along the extract diameter in (C) as a function of time. The white dashed lines mark F-actin and ooplasmic flows, while the arrowheads indicate the F-actin and ooplasm enrichment. (D) Kymograph acquired along the AV axis of Tg(hsp:clip170-GFP) oocyte marking ooplasm as a function of time. White lines trace the flow of ooplasmic pockets over time. (D’) Ooplasmic flow profile along the AV axis, measured from the slopes of ooplasmic flow trajectories shown in (D). Normalized AV of 0 and 1 correspond to the animal and vegetal poles, respectively (N = 3, n = 17). See Table B in S2 Data for underlying data. (E) Fluorescence images of oocytes injected with 200 pg Utrophin-GFP mRNA to label F-actin during first and second meiosis corresponding to 135 and 170 min after maturation induction with the DHP hormone, respectively. White arrowheads mark actin comets forming within the blastodisc region on the surface of granules. (F) Fluorescence images of stage III oocytes injected with CyclinB-GFP mRNA before (stage III) and 30, 60, and 90 min after maturation onset. Arrowhead denotes the GVBD onset. The yellow line along the oocyte circumference (Circum) was used to acquire the intensity profiles plotted in (F’). (F’) Change in CyclinB signal normalized to its distribution at the time prior to GVBD, measured at 30 min (cyan) and 60 min (magenta) after GVBD along the oocyte circumference (the yellow line in F). Circumference of 0 and 800 μm correspond to the AP and VP, respectively (N = 2, n = 5). See Table C in S2 Data for underlying data. (G) Schematic summarizing the role of actin in ooplasmic flows and blastodisc formation. Bulk actin, initially stored within the GV, is released at the AP of the oocyte upon GVBD, thereby generating a local actin gradient. This actin gradient triggers bulk actomyosin flows towards the AP, which drags the ooplasm along, resulting in blastodisc formation. In addition, the blastodisc interface is maintained during first and second meiosis by actin comet-like structures forming on the surface of granules and—in analogy to previous observations in fertilized eggs [23]—preventing them from diffusing into the blastodisc region. Schematics in each panel demarcate the imaging plane used for obtaining the images in that panel. Error bars, SEM. AP, animal pole; AV, animal-vegetal; GC, granulosa cell; GV, germinal vesicle; GVBD, germinal vesicle breakdown; VP, vegetal pole; Ygs, yolk granules.