Mifepristone

Drosophila strains.
All the target transgenes
for over-expression (Table 1) were obtained from Bloomington Drosophila Stock Center. The ubiquitous Geneswitch driver lines Act-GS-255B and
Act-GS-255A contain multiple copies of a P element construct in which
expression of the Geneswitch cDNA is under the control of the tissue-general Actin5C promoter [16 (link)]. The UAS-ultraGFP strain contain multiple copies of a UAS-eGFP
construct, and its construction and characterization have been recently
described [18 ]. The Geneswitch system drivers Elav-GS, MHC-GS, S₁-32
and S₁-106 were generously provided by T. Osterwalder and R. Davis
[19 (link),20 (link)].


Drosophila culture.
Drosophila culture and life span assays were performed as described previously [16 (link)].
GeneSwitch virgins were used in the crosses with males of other lines, with the
exception of strains in which the target transgene for over-expression was on
the X chromosome. Life span assays consisted of ~25 flies per vial, and a total
5 vials for each cohort. For survival assays performed at 25^oC,
flies were transferred to new vials ever other day. For survival assays
preformed at 29^oC flies were transferred to new vials every other
day during the first 30-40 days, and then every day for the remainder of the
life span. RU486 (Mifepristone, Sigma) was dissolved in ethanol (100%) to make
a stock solution of 3.2mg/ml. For adult feeding, 50ul RU486 stock solution was
added to the surface of each vial to produce a final concentration of
~160ug/ml; 50ul ethanol was added to the control vials. For larval feeding,
0.5ml of 3.2mg/ml RU486 stock solution (or the indicated diluted concentration)
was added to the surface of each bottle to produce a final concentration of
~160ug/ml (or indicated diluted concentration); 0.5ml ethanol was added to
control bottles.


GeneSwitch driver characterization
. Adult
flies were cultured in vials in the presence and absence of drug for two weeks
prior to dissection. Adult male and female flies, head in half, body in half,
midgut and hindgut, ovary and testes, were photographed. Larvae at 1^st instar, 2^nd instar and 3^rd instar, as well as 3^rd instar dissected tissues (brain, midgut and hindgut, salivary gland, imaginal
discs, and fat body) were also photographed. The Leica MZ FLIII fluorescence
stereomicroscope together with the SPOT software were used for photographs: The
GFP pictures were taken under the fluorescent light with exposure time 4 sec
and a gain of 2.


Statistical analysis.
Mean, standard deviation,
median, percent change in mean, percent change in median, and log rank p value were calculated using R 2.6.2 [58 ]. Analysis of mortality rate was
performed with the WinModest statistical package [59 ]. In the
Gompertz-Makeham model, the increaseof mortality (μ_x )
with age (x) is expressed as: μ_x= ae^bx+c,
where the constant a is the initial mortalityrate, b is the rate of exponential increase in mortality, and c is the
age-independent mortality. The age specific mortality rate (μ_x)
was calculated using WinModest by binning the days over which deaths
were counted (since fly deaths were recorded every other day) such that μ_x = (-ln(N_{x + δx} / N_x )) / δ_x(or P_x = N_{x + δx} / N_x and μ_x = -1/δ_x ln(P_x )), where N_xis the number of flies alive at day x and δ_x is the bin size [2 (link)].
Parameters (a, b, c) were also calculated based on a likelihood ratio
test. The full model (ae^bx+c) was plotted, and the
Gompertz-only component (ae^bx) was used to build the
decomposed survival curves, using μ_x: μ_x= ae^bx, P_x= e^-μx.
For the decomposed survival curves, any value below 0.5% survival was
considered to be the final data point.

The expression pattern seen in the late third- instar in stable spilt lines was maintained through metamorphosis using the flip-switch method described in Harris et al., 2015 (link). Using a similar strategy of the gene-switch method (Roman et al., 2001 (link)), flippase was fused to the ligand-binding domain of the human progesterone receptor, rendering it dependent on progesterone or a progesterone mimic to move into the nucleus. Stable split lines were crossed to pJFRC48-13XLexAop2-IVS-myrtdTomato in su(Hw)attP8; Actin5Cp4.6>dsFRT>LexAp65 in su(Hw)attP5; pJFRC108-20XUAS-IVS-hPRFlp-p10 in VK00005/TM6.
We used the progesterone mimic mifepristone (RU486, Sigma-Aldrich; #M8046) to cause translocation of the flippase to the nucleus where it could then flip-out the STOP cassette in the Actin-LexAp65 transgene. We used surface application of RU486 to food vials. Parents were allowed to lay eggs in a food vial for a few days, then transferred to a fresh vial. Approximately 4 days after transfer, 60 μl of an ~10 mM RU486 stock solution (10 mg RU486 dissolved in 2 ml 95% ethanol) was applied to the surface of the food. At 24 hr after treatment, any larvae that had wandered and/or pupariated were discarded to ensure that test animals had fed on RU486 for at least 24 hr. At 48 hr after treatment, the subsequent wandering larvae and pupae (which had all fed on RU486 for 24–48 hr during the L3 stage) were collected and transferred to an untreated food vial. These animals were then dissected in Schneider’s S2 culture medium as adults. This treatment results in constitutive LexA expression in any cells that express GAL4 during the L3 stage, but, because the RU486 persists at least partway through metamorphosis, neurons that start expressing in early to mid-metamorphosis also show up.