Panel A shows fluorescence microscopy images comparing Ras (anti-EGFP staining), anti-Vasa, and merged with DAPI signals across control and Ras RNAi embryos expressing Ras variants. Panel B shows fluorescence microscopy images comparing Ras (anti-EGFP staining), anti-Vasa, and merged with DAPI signals in gcl mutant ras RNAi embryos expressing Ras variants. Panel C shows a schematic diagram illustrating constitutive Ras activation variants and their downstream signaling pathways through Raf, RalA, and PI3K. Panel D shows a scatter plot quantifying primordial germ cells per embryo across ras RNAi and Ras-EGFP variant conditions. Panel E shows a scatter plot quantifying primordial germ cells per embryo in gcl mutant ras RNAi embryos expressing Ras-EGFP variants.
Ras effector loop mutations that activate PI3K significantly decrease PGC formation, while Raf-activating mutations have no effect on PGC formation. (A) Embryos derived from mothers depleted of endogenous Ras by Ras RNAi and expressing RNAi-insensitive EGFP-tagged Ras, Ras-S35, Ras-G37, and Ras-C40 were immunostained with anti-EGFP and anti-Vasa to confirm expression of the construct and stain PGCs for counting. Expression of the UASp constructs was driven in females by two copies of maternal-tubulin-GAL4-VP16 for optimal expression during late oogenesis. Images depict maximum intensity projections spanning area of PGC formation. (B) Embryos from gcl−/− mothers co-expressing Ras RNAi and RNAi-insensitive EGFP-tagged Ras, Ras-S35, Ras-G37, and Ras-C40 were immunostained with anti-EGFP and anti-Vasa to confirm expression of the construct and stain PGCs for counting. Expression of the UASp constructs was driven by maternal-tubulin-GAL4-VP16 for optimal expression during late oogenesis. Images depict maximum-intensity projections spanning the area of PGC formation. (C) Model depicting downstream pathways constitutively activated by Ras effector loop mutations. (D) PGCs in nuclear cycle 13–14 embryos from mothers of the indicated genotype were counted and plotted from A. The maternal genotype of control embryos is w1118. Bars represent the mean ± standard deviation. (n > 15, Mann–Whitney test). (E) PGCs in nuclear cycle 13–14 embryos from mothers of the indicated genotype were counted and plotted from B. The maternal genotype of control embryos is w1118. Bars represent the mean ± standard deviation. (n > 10, Mann–Whitney test).