Fig. 4.

Effects of dfoxo removal on the female fecundity. (A–D) Average number of eggs laid per 7-day-old female or after 7 days of RU486 treatment. Data are presented as the mean number of eggs laid per female over a 24-h period ± SEM. Eggs were counted from ten separate vials, and each vial contained ten females. (A) Females with constitutive and ubiquitous expression of the dominant negative insulin receptor. In both wild-type and dfoxo− backgrounds, daGAL > UAS-InRDN females laid significantly fewer eggs than both daGAL/+ and UAS-InRDN/+ controls (**P <0.05, t-test). There was no significant difference between daGAL > UAS-InRDN and daGAL > UAS-InRDN dfoxo− flies. (B) Females with late ablation of the median neurosecretary cells. In both wild-type and dfoxo− backgrounds, InsP3GAL > UAS-rpr laid significantly fewer eggs than both InsP3GAL/+ and UAS-rpr/+ controls (**P <0.05, t-test). (C) Females induced to ubiquitously overexpress the dominant negative insulin receptor by feeding RU486-containing food from day 3 of adulthood. In both wild-type and dfoxo− backgrounds, daGS > UAS-InRDN females induced with RU486 (+RU486) laid significantly fewer eggs uninduced controls (−RU486) (**P <0.05, t-test). (D) Females induced to ubiquitously overexpress dominant negative Dp110 by feeding RU486-containing food from day 3 of adulthood. In both wild-type and dfoxo− backgrounds, daGS > UAS-Dp110DN females induced with RU486 (+RU486) laid significantly fewer eggs uninduced controls (−RU486) (**P <0.05, t-test). (E) dFOXO protein expression in the germline. Western blot analysis of dFOXO protein expression in ovaries overexpressing two independent dFOXO transgenes (dfoxo-p8 and dfoxo-p13) within the germline under the control of the mataGAL4 driver. Blots were probed with anti-tubulin as a control for protein loading. Ovaries from these females overexpressing dFOXO protein in the germline look structurally wild-type, but egg production is significantly reduced. Eggs were collected from 7-day-old females over a 24-h period and counted. Data are presented as the mean number of eggs laid per female over these 24-h periods ± SEM (**P <0.05, anova). (F) Quantitative RT-PCR analysis of yolkless mRNA expression in female flies of the indicated genotypes normalized to actin5C. Data are presented as means ± SEM (n = 5). yolkless expression is upregulated in daGAL > UAS-InRDN females compared with controls (**P <0.05, t-test) in both wild-type and dfoxo− backgrounds.