A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in <em>Drosophila</em>

<div><p>The GAL4/<em>UAS</em> gene expression system is a precise means of targeted gene expression employed to study biological phenomena in <em>Drosophila</em>. A modified GAL4/<em>UAS</em> system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (<em>LexAop</em>) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/<em>UAS</em> system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in <em>Drosophila</em>.</p> </div

LexPR or XVE as Bridges for Spatial Targeting of Gene Expression.

<p>In a single animal, 3 separate <i>p</i>-element constructs were combined. Two were used for the GAL4/<i>UAS</i> binary gene expression system to specifically express either the LexPR or XVE chimeric proteins in a specific area and one carried the target gene of interest under the control of the <i>LexAop</i> operator sequences (<i>LexAop</i>). (A) A schematic diagram of LexPR under the control of the yeast upstream activating sequence (<i>UAS</i>) with the GAL4 driver. LexPR is a chimeric protein that includes the LexA-DNA binding domain (LexA-BD) fused to the human progesterone receptor ligand-binding domain and the p65 (NFκB) activation domain. Following treatment with RU486, LexPR is activated and binds to <i>LexAop</i> to drive the expression of Your Favorite Gene (<i>YFG</i>) downstream. In the absence of RU486, the target transgene remains silent. (B) A schematic diagram of XVE under the control of the yeast <i>UAS</i> with the GAL4 driver. XVE is a chimeric protein that includes the LexA-BD fused to the estrogen receptor ligand-binding domain and the p65 activation domain. Following treatment with β-estradiol, XVE is activated and binds to <i>LexAop</i> to drive the expression of <i>YFG</i> downstream. In the absence of β-estradiol, the target transgene remains silent.</p

Expression of constitutively active PKA via activated LexPR (or XVE) modulates sleep in <i>Drosophila</i>.

<p>The effect of overexpression of the PKA catalytic activity in MBs on sleep behavior was measured using <i>UAS</i>-mc* that was directly controlled by the <i>MB</i>-GeneSwitch or indirectly controlled by LexPR/<i>LexAop</i>-mc* (or XVE/<i>LexAop</i>-mc*). (A1) Sleep was monitored in the absence or presence of 1.5 mM RU486. Genotypes: +/<i>LexAop</i>-LexPR; +/<i>UAS</i>-mc* (green line without inducer), +/<i>LexAop</i>-LexPR; +/<i>UAS</i>-mc* (gray line with inducer), +/<i>UAS</i>-LexPR; <i>247</i>-Gal4/<i>LexAop</i>-mCD8::GFP (blue line without inducer) and +/<i>UAS</i>-LexPR; <i>247</i>-Gal4/<i>LexAop</i>-mCD8::GFP (cyan line with inducer) (n = 32 for each group). (A2) Sleep was monitored in the absence or presence of 30 mg/ml β-estradiol. Genotypes: +/<i>LexAop</i>-XVE; +/<i>UAS</i>-mc* (green line without inducer), +/<i>LexAop</i>-XVE; +/<i>UAS</i>-mc* (gray line with inducer), +/<i>UAS</i>-XVE; <i>247</i>-Gal4/<i>LexAop</i>-mCD8::GFP (blue line without inducer) and +/<i>UAS</i>-XVE; <i>247</i>-Gal4/<i>LexAop</i>-mCD8::GFP (cyan line with inducer) (n = 32 for each group). After 6 days of induction with (black bars) or without (white bars) a suitable inducer, sleep behaviors were recorded for 2 days: (B) daily total sleep time, (C) sleep bouts in LD binned according to duration, and (D) the number of bouts. Data from each panel were analyzed using Student's <i>t</i> test, and any differences between various concentrations or treatment durations are indicated: n.s. indicates no significant difference; *** indicates p<0.001.</p

Time-course and Dose-response Analysis of the Inducible LexPR Bridge System in Response to RU486.

<p>(A) The trans-activation of LexPR was monitored in flies (<i>Or22a</i>-Gal4/<i>UAS</i>-LexPR-attP40; +/<i>LexAop</i>-mCD8::GFP-attP2) fed various concentrations of RU486 (0, 0.5, 1, 1.5, 2, and 3 mM) for 5 days. <i>LexAop</i>-mCD8::GFP expression was observed in one of the antennal lobes of adult brains. (B) <i>LexAop</i>-mCD8::GFP expression in flies fed 1.5 mM RU486 for 1–6 days (d1–d6). (C) The inducer was removed by replacing the food with fresh food for 2–24 days ((−) d2–d24). Using 3D projections, (D) the green fluorescence intensity of single glomeruli was analyzed in 5 samples from each group of induction by different concentrations of RU486 (0, 0.5, 1, 1.5, 2, and 3 mM) for 5 days, and (E) the green fluorescence intensity of single glomeruli was analyzed from 5 samples for each group of induction for 1–6 days. Each bar represents the mean, and the error bars represent the standard error (± s.e.). Data from each panel were analyzed using Student's <i>t</i> test, and any differences between various concentrations or treatment durations are indicated: n.s. indicates no significant difference; *** indicates p<0.001; ** indicates p<0.01; and * indicates p<0.05. Scale bar, 20 µm.</p

Induction and Activation of LexPR and XVE in the Adult Fly Central Nervous System.

<p>The trans-activation of LexPR and XVE was visualized in adult brains carrying the respective drivers and <i>LexAop</i>-mCD8::GFP. Confocal images are displayed of the <i>UAS</i>-mCD8::GFP expressed directly by several Gal4 drivers (A1–E1); <i>UAS</i>-LexPR acting as a bridge in the absence of RU486 (−) (A2–E2); <i>UAS</i>-LexPR acting as a bridge in the presence of 1.5 mM RU486 (+) for 5 days (A3–E3 are whole-brain images and A4–C4 are focused on 1 antennal lobe); <i>UAS/</i>XVE in the absence of β-estradiol (−) (D4–E4 and A5–C5); <i>UAS/</i>XVE in the presence of 30 mg/mL β-estradiol (+) for 5 days (A6–C6 and D5–E5 are whole-brain images and A7–C7 are focused on 1 antennal lobe). All induction methods activated <i>LexAop</i>-mCD8::GFP expression. Genotypes: (A1) <i>Or22a</i>-Gal4><i>UAS</i>-mCD8::GFP-attP2, (A2–4) <i>Or22a</i>-Gal4><i>UAS</i>-LexPR-attP40; <i>LexAop</i>-mCD8::GFP-attP2, (A5–7) <i>Or22a</i>-Gal4><i>UAS</i>-XVE-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (B1) <i>Or47b</i>-Gal4><i>UAS</i>- mCD8::GFP, (B2–4) <i>Or47b</i>-Gal4><i>UAS</i>-LexPR-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (B4–7) <i>Or47b</i>-Gal4><i>UAS</i>-LexPR-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (C5–7) <i>Or47b</i>-Gal4><i>UAS</i>-XVE-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (C1) <i>Or67a</i>-Gal4><i>UAS</i>- mCD8::GFP, (C2–4) <i>Or67a</i>-Gal4><i>UAS</i>-LexPR-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (C4–7) <i>Or67a</i>-Gal4><i>UAS</i>- XVE-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (D1) <i>247</i>-Gal4><i>UAS</i>- mCD8::GFP-attP2, (D2–3) <i>247</i>-Gal4><i>UAS</i>- LexPR-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (D4–5) <i>247</i>-Gal4><i>UAS</i>- XVE-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (E1) <i>GH146</i>-Gal4>UAS- mCD8::GFP-attP2, (E2–3) <i>GH146</i>-Gal4><i>UAS</i>-LexPR-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (E4–5) <i>GH146</i>-Gal4><i>UAS</i>-XVE-attP40; <i>LexAop</i>- mCD8::GFP-attP2, (F1–2) <i>MB</i>-GeneSwitch^12-1><i>UAS</i>- mCD8::GFP-attP2. Scale bar, 20 µm.</p

Analysis of <i>UAS-</i>Luciferase Lines to Identify Loci with Low Basal Luciferase Activity After Different Temperature Treatments.

<p>Luciferase activity was measured in 16 tests of 3 adults each. Activity was normalized to total protein levels and is shown as arbitrary units (a.u.). Each bar represents the mean (n = 16 for each group), and the error bars represent the standard error (± s.e.). Luciferase activity was measured in heterozygous <i>UAS</i>-luciferase transgenic flies without a Gal4 driver. (A) Gray bars indicate the luciferase activity of 6-day-old flies reared at 18°C. (B) Black bars indicate the luciferase activity of 2-day-old flies reared at 18°C that were exposed to 25°C for 2 days and then to 30°C for 2 days. White bars indicate the luciferase activity of 2-day-old flies reared at 18°C that were exposed to 30°C for 4 days.</p