Ssected ovaries and testis were fixed for 10 minutes in 4 paraformaldehyde (Sigma) diluted in Grace’s medium (Lonza Walkersville Inc., USA), and washed three times in PBT (16PBS, 0.1 Triton-x100, 1 mg/ml BSA). Primary antibodies were diluted in PBT as follows: mouse anti-Hts (1:20, 1B1, Developmental Studies Hybridoma Bank, USA), mouse anti-FasIII (1:50, Developmental Studies Hybridoma Bank, USA), guinea pig anti-Tj (1:1000 [29]), rabbit anti-C(3)G (1:3000, gift from M. Lilly), mouse anti-USP (1:200, gift from R. Barrio) and guinea pig anti-Fax (1:1000, described below). Primary antibodies were incubated overnight at 4uC, washed three times in PBT then incubated overnight at 4uC in secondary antibodies at a dilution of 1:2000. 
Secondary antibodies were generated in goat against mouse, guinea pig and rabbit and conjugated with Alexa Fluor 488, 568 and 633 (Invitrogen, USA). Stained tissues were washed twice in PBT and once in PBT with 50 ng/ml DAPI then mounted in Vectashield mounting medium (Vector Labs). Antifailed axon connections (Fax) was produced by Covance, USA by raising antibodies in guinea pigs against Fax isoform A amino acids 97-294 as described [40]. Confocal images were acquired using a 63x (NA 1.32) PanApo lens and Leica TCS SP5 confocal microscope.Materials and Methods Drosophila StocksExperiments were usually conducted on flies 4-? days old, raised under standard conditions on yeast/cornmeal/molasses/ agar medium. Adult flies were fed yeast paste every third day. c587-GAL4 is described by [38]. The FLP-out MedChemExpress 166518-60-1 strain: hsFlp; Tub FRT-CD2-FRT-GAL4 UAS-GFP was a gift from G. Struhl. Lines expressing EcR RNAi (37059), Usp RNAi (16893), and E75 RNAi (44851) were obtained from the Vienna RNAi Stock Center. EcR RNAi was expressed in the presence of UAS-dcr2 to increase the strength of knock down. All other stocks were obtained from the Bloomington Stock Center.Analyses of Phenotypic Effects on Ovarian Cells Genetic Disruption of Ecdysone SignalingCrosses generating flies of the genotypes c587 GAL4; UAS-USP RNAi/gal80ts, c587 GAL4;gal80ts;UAS-E75 RNAi, c587;UASEcR RNAi/gal80ts; UAS-dcr2 and c587 GAL4;UAS-EcR.BOvaries and testis were stained with anti-Hts, anti-FasIII and anti-Tj antibodies. Anti-Hts labels an endoplasmic reticulum-like structure present within germ cells (called a fusome in 2- to 16-cell cysts and a spectrosome in GSCs and CBs) whose number ofSteroid Signaling Mediates Female GametogenesisFigure 5. Ecdysone signaling is not required for GSC maintenance, early germ cell development, or somatic cell shape in the testis. A) The number of male GSCs was counted after the order BI 78D3 indicated periods following a shift to 29uC to compromise ecdysone signaling as indicated. Error bars indicate s.d. B) c587 alone 29uC day 8; C) c587::USP RNAi 29uC day 8; D) c587::EcR RNAi 29uC day 8; E) c587::E75 RNAi 29uC day 8; F) ecd1 29uC day 4. (B9 9: enlarged regions). GSCs outlined in B9, C9, D9, E9 and F9 and asterisk position of hub cells. Green: somatic cells (anti-Tj) magenta: cellSteroid Signaling Mediates Female Gametogenesismembranes and fusome (anti-hts and anti-FasIII). Scale bar: 10 mm. G, H) EM analysis of ecd1 cysts from males kept G) at 18uC, or H) 29uC day 8. Magenta: pseudocolour (germ cells within a single cyst), green: pseudocolour (cyst cells 
in contact with the pseudocoloured cyst). Scale bar: 2 mm. doi:10.1371/journal.pone.0046109.gbranches corresponds to cyst size. Anti-Hts staining additionally allows determination of GSC.Ssected ovaries and testis were fixed for 10 minutes in 4 paraformaldehyde (Sigma) diluted in Grace’s medium (Lonza Walkersville Inc., USA), and washed three times in PBT (16PBS, 0.1 Triton-x100, 1 mg/ml BSA). Primary antibodies were diluted in PBT as follows: mouse anti-Hts (1:20, 1B1, Developmental Studies Hybridoma Bank, USA), mouse anti-FasIII (1:50, Developmental Studies Hybridoma Bank, USA), guinea pig anti-Tj (1:1000 [29]), rabbit anti-C(3)G (1:3000, gift from M. Lilly), mouse anti-USP (1:200, gift from R. Barrio) and guinea pig anti-Fax (1:1000, described below). Primary antibodies were incubated overnight at 4uC, washed three times in PBT then incubated overnight at 4uC in secondary antibodies at a dilution of 1:2000. Secondary antibodies were generated in goat against mouse, guinea pig and rabbit and conjugated with Alexa Fluor 488, 568 and 633 (Invitrogen, USA). Stained tissues were washed twice in PBT and once in PBT with 50 ng/ml DAPI then mounted in Vectashield mounting medium (Vector Labs). Antifailed axon connections (Fax) was produced by Covance, USA by raising antibodies in guinea pigs against Fax isoform A amino acids 97-294 as described [40]. Confocal images were acquired using a 63x (NA 1.32) PanApo lens and Leica TCS SP5 confocal microscope.Materials and Methods Drosophila StocksExperiments were usually conducted on flies 4-? days old, raised under standard conditions on yeast/cornmeal/molasses/ agar medium. Adult flies were fed yeast paste every third day. c587-GAL4 is described by [38]. The FLP-out strain: hsFlp; Tub FRT-CD2-FRT-GAL4 UAS-GFP was a gift from G. Struhl. Lines expressing EcR RNAi (37059), Usp RNAi (16893), and E75 RNAi (44851) were obtained from the Vienna RNAi Stock Center. EcR RNAi was expressed in the presence of UAS-dcr2 to increase the strength of knock down. All other stocks were obtained from the Bloomington Stock Center.Analyses of Phenotypic Effects on Ovarian Cells Genetic Disruption of Ecdysone SignalingCrosses generating flies of the genotypes c587 GAL4; UAS-USP RNAi/gal80ts, c587 GAL4;gal80ts;UAS-E75 RNAi, c587;UASEcR RNAi/gal80ts; UAS-dcr2 and c587 GAL4;UAS-EcR.BOvaries and testis were stained with anti-Hts, anti-FasIII and anti-Tj antibodies. Anti-Hts labels an endoplasmic reticulum-like structure present within germ cells (called a fusome in 2- to 16-cell cysts and a spectrosome in GSCs and CBs) whose number ofSteroid Signaling Mediates Female GametogenesisFigure 5. Ecdysone signaling is not required for GSC maintenance, early germ cell development, or somatic cell shape in the testis. A) The number of male GSCs was counted after the indicated periods following a shift to 29uC to compromise ecdysone signaling as indicated. Error bars indicate s.d. B) c587 alone 29uC day 8; C) c587::USP RNAi 29uC day 8; D) c587::EcR RNAi 29uC day 8; E) c587::E75 RNAi 29uC day 8; F) ecd1 29uC day 4. (B9 9: enlarged regions). GSCs outlined in B9, C9, D9, E9 and F9 and asterisk position of hub cells. Green: somatic cells (anti-Tj) magenta: cellSteroid Signaling Mediates Female Gametogenesismembranes and fusome (anti-hts and anti-FasIII). Scale bar: 10 mm. G, H) EM analysis of ecd1 cysts from males kept G) at 18uC, or H) 29uC day 8. Magenta: pseudocolour (germ cells within a single cyst), green: pseudocolour (cyst cells in contact with the pseudocoloured cyst). Scale bar: 2 mm. doi:10.1371/journal.pone.0046109.gbranches corresponds to cyst size. Anti-Hts staining additionally allows determination of GSC.