Fig 3. Ovarian ecdysteroid biosynthesis controls a mating-induced increase in GSC numbers.

(A, D) Ecdysteroid levels in virgin (v) and mated (m) females in the ovarian somatic cell-specific (escort cells and follicle cells) nvd RNAi female flies. (A) c587-GAL4 driver was crossed with control or UAS transgene strains as indicated. UAS-nvd-Bm [wt] and UAS-nvd-Bm [H190A] overexpressed the wild-type form and enzymatic inactive form of Bombyx mori nvd transgenes, respectively. (D) nvd RNAi female flies were fed food supplemented with ethanol (EtOH; for control) and 7-dehydrocholesterol (7dC). (B, E and F) Frequencies of germaria containing one, two, and three GSCs (left y axis), and average number of GSCs per germarium (right y axis) of follicle cell-specific nvd RNAi animals with or without the B. mori nvd transgene (B), the ovarian somatic cell-specific EcR RNAi female flies and transheterozygous mutants for EcR (EcR^A483T and EcR^M554fs, mutants in the predicted ligand- binding domain) (E), ovarian somatic cell-specific nvd and sad RNAi female flies that were fed food supplemented with EtOH (for control), 7dC and 20E (F). (C) Frequency of mitotic GSCs was counted by staining with anti-phospho-histone H3 in nvd RNAi female flies. Values are represented as the mean with standard error of the mean in A and B. The numbers of samples examined are indicated in parentheses in A and D. The numbers of germaria analyzed are shown inside bars in B, C, E and F. For statistical analysis, Dunnett’s test was used for A and D. A Mann-Whitney U test was used for B, E and F. Chi-square analysis was used for C. **P ≤ 0.01, *P ≤ 0.05, n.s., non-significant (P > 0.05).