Panel A: This panel contains fluorescence microscopy images of embryos overexpressing different RalA constructs. The images are labeled as RalA-WT, RalA-G20V, and RalA-S25N. Each row shows three images: one with mClover2 fluorescence, one with anti-Vasa staining, and one merged image with DAPI staining. The scale bar is 50 micrometers. Panel B: This panel shows a scatter plot with the number of primordial germ cells (PGCs) per embryo on the y-axis. The x-axis labels are RalA WT, RalA G20V, and RalA S25N. The plot includes statistical annotations indicating significant differences. Panel C: This panel contains fluorescence microscopy images of control and Rgl overexpressing embryos. Each row shows three images: one with mClover2 fluorescence, one with anti-Vasa staining, and one merged image with DAPI staining. The scale bar is 50 micrometers. Panel D: This panel shows a scatter plot with the number of PGCs per embryo on the y-axis. The x-axis labels are mClover2 and mClover2:Rgl. The plot includes statistical annotations indicating significant differences.
(related to Fig. 3): Increasing RalA activity mildly antagonizes PGC formation. (A) Embryos from mothers overexpressing mClover2-tagged RalA-WT, RalA-G20V (constitutively active), and RalA-S25N (dominant negative) were immunostained with anti-mClover2 and anti-Vasa to confirm expression of the construct and stain PGCs for counting. Expression of the respective UASp constructs was driven by two copies of maternal-tubulin-GAL4-VP16 for optimal expression during late oogenesis. Images depict maximum-intensity projections spanning the region of PGC formation. Scale bar = 50 µm. (B) PGCs in nuclear cycle 13–14 embryos from mothers of the indicated genotype were counted and plotted from A. Overexpression of RalA-WT was used as a control. Bars represent the mean ± standard deviation. (n > 20, Mann–Whitney test). (C) Embryos from mothers overexpressing mClover2-tagged Rgl were immunostained with anti-mClover2 and anti-Vasa to confirm expression of the construct and stain PGCs for counting. Expression of the UASp construct was driven by two copies of maternal-tubulin-GAL4-VP16 for optimal expression during late oogenesis. Images depict maximum intensity projections spanning the area of PGC formation. Scale bar = 50 µm. (D) The number of PGCs in nuclear cycle 13–14 embryos from mothers of the indicated genotype was counted and plotted from B. Overexpression of germline-targeted mClover2 was used as a control. Bars represent the mean ± standard deviation. (n > 15, Mann–Whitney test).