(a) FOFO construct design: the actin5C promoter is blocked from inducing transcript expression by two efficient transcriptional terminator (STOP) cassettes. Each of these is flanked by FRT or mFRT71, specifically recognized by FLP and mFLP5, respectively. Therefore, miRs and EGFP will only be expressed in cells containing the two flippases. Spatial and temporal control is achieved by providing a spatially restricted FLP and hs-induced mFLP5. SD, splice donor; SA, splice acceptor. Following excision of the fushi tarazu (ftz) intron, miRs are processed without detriment to reporter expression. Gypsy insulators minimize position effects whilst enhancing expression levels; attB sites allow site-specific insertion into attP-containing host strains. (b) Schematic of FOFO application. With the insertion sites chosen for this study, flies of the following genotype can be generated: enhancer-FLP; hs-mFLP5, FOFO-EGFP; enhancer2-GAL4 (exemplifying with the GAL4 transgene on the third chromosome, though it could be placed elsewhere). Expression of deleterious sequences (either knock-down by miRs or overexpression alongside the reporter by means of T2A) can be induced (by heat-shock) in a single fly stock (without need to cross) carrying FOFO, a lineage-specific enhancer1-FLP and hs-mFLP5. The point is then to add in the same flies (F0 generation) a GAL4 transgene (enhancer2-GAL4) and cross to UAS responders. The FOFO containing stock expresses FLP in the spatially restricted domain defined by enhancer1 (yellow) in a tissue represented by the grey shape. FLP expression will constitutively excise the first STOP cassette but the presence of a second STOP cassette precludes expression of anything downstream unless flies are subject to hs. The F1 progeny expresses a transgene (purple) in the GAL4-expressing domain defined by enhancer2 (black). Following hs, mFLP5 expression leads to excision of the second STOP cassette and thus expression of miRs and EGFP in the domain covered by the lineage-specific enhancer. Even if the domain of the latter overlaps with that of enhancer2 as depicted, GAL4 miRs will delete GAL4 expression in the EGFP-expressing domain so that the GAL4 domain never overlaps with that of enhancer1 and only cell nonautonomous effects are assessed. (c) Schematic representation of a FOFO application with the tools designed for this study. EGFP-labeled neural tumors (green) are generated within brain lobes (grey shape) in a stock also carrying a GAL4 expressed in glia (purple). Crossing this stock to any UAS-responder lines (could be genome-wide gain- or loss-of-function) will allow identification of genes whose glial expression affects tumor size.