The following fly stocks and transgenes have been used: y w; w
1118; arm-Gal4; Act5C-Gal4; UAS-GFP; UAS-lacZ (all from the Bloomington Drosophila stock center); GMR-Gal4 (a gift of M. Freeman); ppl-Gal4 (a gift of M. Pankratz); UAS-dInR ; Df(3L)AC1 ; tGPH ; GMR>w
+>Gal4 ; UAS-dPTEN ; UAS-dilp2 ; GE24013 (GenExel). All crosses were performed at 25°C unless stated otherwise.
EP screen and isolation of Imp-L2 alleles
The EP screen that led to the identification of Imp-L2 will be described elsewhere (F.W., W.B., H.S., D. Nellen, K. Basler and E.H., unpublished work). A double-headed EP element (containing ten Gal4-binding sites at each end) suppressing the GMR-Gal4, UAS-InR big eye phenotype was identified in the Imp-L2 locus. Plasmid rescue of EP5.66 revealed that it was inserted 6,969 bp upstream of the first exon of the Imp-L2-RB (CG15009-RB) transcript.
To obtain loss-of-function alleles of Imp-L2, we performed an EMS mutagenesis screen in which we selected mutated chromosomes carrying EP5.66 that could no longer suppress the dInR overexpression phenotype in the eye. EP5.66 males were fed with 25 mM EMS and subsequently crossed to GMR-Gal4, UAS-dInR virgins. 39,000 F1 flies were screened for a reversion of the suppressive effect of EP5.66 on the growth phenotype caused by GMR-Gal4, UAS-dInR. Only one of the identified reversion lines, Imp-L2
MG2, could be confirmed. Sequencing the genomic DNA of Imp-L2
MG2 revealed a point mutation that resulted in a truncation (Trp232Stop).
In order to generate additional Imp-L2 mutants, the P-element GE24013 (marked with white
+) inserted 102 bp upstream of the first exon of the Imp-L2-RC transcript was mobilized by supplying Δ2–3 transposase. Jump starter males were mated with balancer females, and single F1 w
- males were recrossed to balancer virgins. Stocks (350) were established and molecularly tested for deletions by single-fly PCR using several primer pairs, leading to the identification of the alleles Imp-L2
Def223 and Imp-L2
Construction of plasmids
In order to generate the UAS-Imp-L2 construct, a BglII/XhoI fragment of Imp-L2 was excised from the Imp-L2-RB containing cDNA clone LP06542 and inserted into pUAST . To obtain UAS-s.Imp-L2, the second and third exons of Imp-L2 were amplified by PCR from genomic DNA. The fragment was subcloned into pCRII-Topo (Invitrogen). The insert was then excised with EcoRI and cloned into pUAST . Because of the lack of the first exon of the Imp-L2-RB transcript (containing three upstream open reading frames), UAS-s.Imp-L2 has a stronger phenotype than UAS-Imp-L2. The EP element contains ten UAS sites, whereas the UAS transgenes contain only five.
For the generation of the genomic rescue construct, the genomic fragment L2G314 (kindly provided by J. Natzle) was used. The fragment (5 kb of genomic sequence upstream of the first exon of the Imp-L2-RB transcript and 1 kb downstream of the third exon) was excised with BamHI and Asp718 and inserted into the pCaSpeR-4 transformation vector .
The Flag-dilp2 construct was created by PCR amplification of the dilp2 coding sequence without the signal peptide sequence from the full-length cDNA clone, EST GH11579 (obtained from Research Genetics). The resulting PCR product was then equipped with the hemagglutinin signal peptide sequence and a Flag tag and inserted into pUAST .
Drosophila embryonic S2 cells were grown at 25°C in Schneider's Drosophila medium (Gibco/Invitrogen) supplemented with 10% heat-inactivated fetal-calf serum (FCS), penicillin and streptomycin.
For the construction of the stably expressing Flag-dilp2 cell line, S2 cells were co-transfected with UAS-Flag-dilp2, Act-Gal4 and a third vector containing a blasticidin-resistance gene, using effectene transfection reagent (Qiagen). Two days after the transfection, the selection medium (Schneider's containing 10% FCS and 25 μg/ml blasticidin) was added to the cells. After 10 days the selection medium was replaced by Schneider's containing 10% FCS and 10 μg/ml blasticidin.
In vitro pulldown assay
S2 cells expressing Flag-dilp2 were grown to confluence in 175 cm2 culture flasks, washed with ice-cold PBS and extracted in immunopreciptiation (IP) buffer (120 mM NaCl, 50 mM Tris pH 7.5, 20 mM NaF, 1 mM benzamidine, 1 mM EDTA, 6 mM EGTA, 15 mM Na4P2O7, 0.5% Nonidet P-40, 30 mM β-glycerolphosphate, 1× Complete Mini protease inhibitor (Roche)). After incubation for 15 min on an orbital shaker at 4°C, solubilized material was recovered by centrifugation at 13,000 rpm for 15 min and supernatants were collected. Anti-Flag antibody (5 μg, Sigma M2, F3165) was added and incubated over night at 4°C while rotating. Protein G sepharose beads (Amersham Biosciences) were added for 2 h and the beads were washed four times with IP buffer. Cell lysate from native S2 cells was subjected to the same procedure and the resulting beads were used as control. To verify the immunoprecipitation, a fraction of the beads was incubated with SDS loading buffer (62.5 mM Tris-HCl pH 6.8, 20 mM DTT, 2% SDS, 25% glycerol, 0.02% bromophenol blue) for 5 min at 90°C and the proteins were separated by SDS-PAGE. The presence of Flag-Dilp2 was confirmed by immunoblotting.
For the in vitro translation the Imp-L2-RC cDNA (SD23735) was cloned into pCRII.1 (Invitrogen) downstream of the SP6 polymerase promoter. As a control, the point mutation encoding a non-functional, truncated version of Imp-L2 (identified in the EMS reversion mutagenesis) was inserted into Imp-L2-RC (in pCRII.1 see above) using the Quick-Change site-directed mutagenesis protocol (Stratagene). Both the Imp-L2 and the Imp-L2
MG2 constructs were translated in vitro using the TNT Quick coupled transcription/translation system (Promega) according to the manufacturer's protocol. Briefly, 2 μg of DNA was incubated with 20 μCi [35S]methionine and 20 μl TNT Quick Master Mix in a total volume of 25 μl for 90 min at 30°C. The product (2.5 μl) was used in the in vitro pulldown assay together with Flag-Dilp2 bound to beads or with control beads in IP buffer containing 0.05% NP-40. The reaction was rotated overnight at 4°C, the beads were washed six times with IP buffer (0.05% NP-40) and incubated with SDS loading buffer containing 100 mM DTT for 10 min at 80°C. The dissociated proteins were separated using SDS-PAGE and detected by autoradiography.
Freshly eclosed flies were collected, separated according to sex, placed on normal fly food for 3 days and anesthetized for 1 min with ether before weighing. Weight was determined using a Mettler Toledo MX5 microbalance. Wing size was analyzed as described . ImageJ 1.32j software was used to determine the pixels of the wing area. Scanning electron microscope pictures were taken from adult flies that were critical-point dried and coated with gold.
Heat-shock induced overexpression clones (y, w, hs-Flp; GMR>w
+>Gal4) were induced 24–48 h after egg-laying by a 1 h heat shock at 37°C. Tangential sections of adult eyes were generated as described .
For all starvation experiments, eggs were collected for 2 h on apple agar plates supplemented with yeast. After 72 h, larvae were quickly washed in PBS and transferred either to a new apple agar plate with yeast (normal food, called 'yeast' henceforth), a solution containing 20% glucose in PBS, or a filter paper soaked with 1% glucose in PBS or PBS only. After 24 h, dead larvae were counted.
For the tGPH reporter analysis under starvation, the 'PBS' or 'yeast' conditions were used (see above). After 4 h starvation, larvae were dissected in PBS, fixed and stained with Hoechst. Pictures were taken using a Leica SP2 confocal laser scanning microscope.
Immunohistochemistry and in situ hybridization
The antibody against Imp-L2 was described earlier  and kindly provided by J. Natzle (Department of Molecular and Cellular Biology, University of California, Davis, USA). Antibody staining against Imp-L2 was performed using the following dilutions: rat anti-Imp-L2 (1:500), donkey anti-rat-FITC (1:200, Jackson). Other antibodies used were: anti-β-galactosidase (1:2,000, polyclonal, rabbit), an antibody against the carboxyl terminus of dInR (INRcT, 1:10,000) . Nuclei were either stained with 4',6-diamidino-2-phenylindole (DAPI) or Hoechst. Pictures were taken using a Leica SP2 confocal laser scanning microscope.
RNA in situ hybridization using digoxigenin-labeled probes was performed as described . The probes against Imp-L2 were derived from s.Imp-L2 in a pBluescript SK+ vector.