PMC

In vivo expression driven by tubulin, actin and GMR promoters. (a) Ubiquitous expression of mtdTomato or GFP reporters in third instar larvae (left column, representative of n=4–6) and in the adult flies (right column, representative of n=5). Larvae in the top row of each subpanel carry a tubulin or actin driver line and an mtdt or mCD8-GFP reporter. Larvae in the bottom row of each subpanel (marked by “+tub-QS”) also carry a tub-QS transgene. Adult flies (right column, middle) are imaged next to the controls (right column, left) that bear only the TA or only effector transgenes (dashed white outline). The rightmost subpanels (marked by “+tub-QS”) show flies that, in addition to the indicated driver and reporter transgenes, also carry a tub-QS. Expression of act-LexAQF driver is visualised with a mCD8-GFP reporter. Imaging settings were identical for all images, apart from the duration of exposure, which is indicated for each image in milliseconds. Scale bars, 1 mm. (b) Scanning electron micrograph images of the adult female eyes (left column, representative of n=10) and GFP expression in the eye-antennal imaginal disc (right column, representative of n=5) for w¹¹¹⁸, pGMR-GAL4/+ and pGMR-QF2^w/+ flies. Scale bars, 50 μm.

Olfactory, phototaxic, and activity/sleep behavioral analyses of nsyb-GAL4, nsyb-QF2 and nsyb-QF2^w transgenic flies. (a) Flies were tested in groups of 50 in a 4-field olfactometer assay. Control scores are attraction index (AI), calculated for the odor quadrant over 5 mins preceding presentation of an odorant. Odorant scores are AI’s calculated for the last 5 mins of the 10 mins odor presentation. We examined a highly attractive odor, 5% apple cider vinegar in water (left); a mildly attractive stimulus, pure water (middle) and a repulsive stimulus, 5% CO₂ gas (right). Numbers in bars indicate number of independent experiments for each condition. Error bars are SEM. (b) Visually-induced responses of two wild-type strains (w⁻ and w⁺), nsyb-Gal4/+, nsyb-QF2/+ and nsyb-QF2^w/+ were investigated in a 10-step countercurrent phototaxis assay (see Online Methods for details). Phototactic index (PI) is the average number of approaches to the light that each fly made. The PI scores of the nsyb-QF2 and nsyb-QF2^w flies were comparable with those of nsyb-Gal4/+ and w⁺ flies. Numbers in bars indicate number of independent experiments for each condition. Error bars are SEM. (c) Daily Activity, (d) daily sleep time, (e) waking activity, and (f) sleep latency for w⁻ (n=29 flies), nsyb-Gal4/+ (n=28), nsyb-QF2/+ (n=20), and nsyb-QF2^w/+ (n=21) flies, assayed as outlined in Online Methods. Error bars represent SEM. “*”, “**”, “***”, and “n.s.” denote P<0.05, P<0.01, P<0.001, and not significant, respectively, for all panels, as determined by the non-parametric Kolmogorov-Smirnov test.

Activity of modified QF transcriptional activators in vitro and in vivo in neuronal tissue. (a) Schematics of GAL4, original QF and four new transcriptional activators. DBD, DNA binding domain; MD, middle domain; AD, activation domain. Vertical hatching indicate Zn2-Cys6 zinc finger motifs, diagonal hatchings mark dimerization domains. Numbers indicate amino acid position. Constructs are drawn to scale. (b) The transcriptional activity (black bars) of QF transcriptional activators (TA) was investigated by transfecting S2 cells with actin-QF_X plasmid, firefly luciferase reporter plasmid (pLexAop-luc2, pQUAS-luc2 or pUAS-luc2), Rhenilla luciferase plasmid (pAC-hRluc) for normalization, and pBluescript (pBS-KS) plasmid for equalizing DNA amount. For QS repression assays (grey bars), pBluescript plasmid was replaced by pAC-QS plasmid in co-transfections. Numbers in bars indicate the number of independent repeats. Error bars are SEM. Luciferase activity is shown on a log scale. (c–e) Pan-neuronal in vivo expression of constructs driven by neuronal synaptobrevin (nsyb) promoter at 25°C. Left column shows mCD8-GFP expression in third-instar larvae (representative of n=4–6; scale bar, 100 μm), middle column shows nuclear lacZ expression in adult brain (representative of n=4–6; scale bar, 50 μm), and the right column shows the Gal80- or QS-induced suppression of lacZ expression in adult brains (representative of n=5–7). Brains were immunostained for elav (red), lacZ (green) and nc82 (blue). (e) Larval and adult expression of mCD8:GFP driven by nsyb-LexAQF construct (representative of n=5). Right panel, tubP-QS suppression of LexAQF activity (representative of n=5). (f) LacZ expression, quantified as described in Online Methods. Numbers in bars indicate the number of brains for each condition. Error bars are SEM.