An auxin-inducible, GAL4-compatible, gene expression system for Drosophila
- Department of Life Sciences, Imperial College London, United Kingdom
- Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey, United States
Figures
Figure 1 with 1 supplement
Design and action of auxin-inducible gene expression system (AGES).
A tubulin promoter ubiquitously expresses the bicistronic TIR1 and AGES cassette. Auxin-inducible degron (AID)-tagged GAL80 is cleaved from the TIR1 due to the presence of the T2A sequence. In the absence of auxin, GAL80 can inhibit GAL4 activity, whilst the presence of auxin induces degradation of GAL80, allowing GAL4 to drive the expression of UAS-transgene(s).
Figure 1—figure supplement 1
Plasmid map of the auxin-inducible gene expression system (AGES) plasmid.
Map of the pattB-tubP-AtTIR1-P2A-miniAID-Gal80-miniAID-SV40 plasmid (generated using Snapgene). The full sequence is in Figure 1—figure supplement 1—source data 1.
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Figure 1—figure supplement 1—source data 1
Genbank (.gb) sequence file for pattB-tubP-AtTIR1-P2A-miniAID-Gal80-miniAID-SV40.
- https://cdn.elifesciences.org/articles/67598/elife-67598-fig1-figsupp1-data1-v1.zip
Figure 2 with 2 supplements
Auxin-inducible gene expression system (AGES) effectively induces GAL4 activity in Drosophila adults.
(A) Ventral images of live females that express GAL4 in fat body tissue. Ingestion of food containing auxin (≥5 mM for 24 hr) induces GAL4 activity and the expression of GFP. (B) Quantification of GFP levels. Pixel intensity thresholding was performed to isolate abdomens as regions of interest. The average pixel intensities from six replicates were quantified and analysed using Kruskal-Wallis test with Dunn pair-wise comparison (***, p < 0.001 and **, p < 0.01). (C) Quantitative PCR (qPCR) data for GFP mRNA levels using different concentrations of auxin (three biological replicates). Values were normalised to housekeeping gene RpL4 (Ribosomal Protein L4) and relative expression levels (compared to the negative control) were calculated using the ΔΔCt method. Y-axis displaying ΔΔCt values and statistics done using Kruskal-Wallis test with Dunn pair-wise comparison (*, p < 0.05). See Figure 2—source data 1 for raw data.
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Figure 2—source data 1
GFP intensity and qPCR values for Figure 2B, C.
- https://cdn.elifesciences.org/articles/67598/elife-67598-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Auxin-inducible gene expression system (AGES) effectively induces GAL4 activity in Drosophila adult males.
(A) Ventral images of live males that express GAL4 in fat body tissue. Ingestion of food containing auxin (24 hr) induces GAL4 activity and the expression of GFP. (B) Quantification of GFP levels (from six male abdomens). Pixel intensity thresholding was performed to isolate abdomens as regions of interest. The average pixel intensities were quantified and analysed using ordinary one-way ANOVA (**, p = 0.002, ****, p < 0.0001). (C) Quantitative PCR (qPCR) data for GFP mRNA levels using different concentrations of auxin (three biological replicates). Values were normalised to housekeeping gene RpL4 (Ribosomal Protein L4) and relative expression levels were calculated using the ΔΔCt method. Y-axis displaying ΔΔCt values and statistics done using ordinary one-way ANOVA (**, p < 0.005).
Figure 2—figure supplement 2
On-off dynamics of auxin-inducible gene expression system (AGES) in adult flies and stability of auxin fly food.
(A) Quantification of GFP levels in female abdomens (expressed in the fat body). Pixel intensity thresholding was performed to isolate abdomens as regions of interest. The average pixel intensities were quantified and analysed using ordinary one-way ANOVA (*, p < 0.05, ****, p < 0.0001). (B) Quantification of GFP levels in female abdomens with freshly made auxin food and 15-week-old food.
Figure 3
Auxin-inducible gene expression system (AGES) allows induction of GAL4 activity in Drosophila larvae.
(A) GFP fluorescence quantification in fat body tissue after induction (for 24 hr) on food with different concentrations of auxin. The average pixel intensities from six larvae were quantified and analysed using Kruskal-Wallis test with Dunn pair-wise comparison (***, p < 0.001 and **, p < 0.01). (B) Time course of GFP expression in fat body tissue when using 5 mM auxin. The average pixel intensities from six larvae were quantified and analysed using Kruskal-Wallis test with Dunn pair-wise comparison (**, p < 0.01). (C) Representative images of larvae fed on different concentrations of auxin. (D) Representative images of larvae imaged at each time interval since induction. See Figure 3—source data 1 for raw data.
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Figure 3—source data 1
GFP intensity values for Figure 3.
- https://cdn.elifesciences.org/articles/67598/elife-67598-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
Auxin-inducible gene expression system (AGES) allows induction of GAL4 activity in the Drosophila adult and larval brain.
(A) GFP fluorescence driven by Or85a-GAL4 in the antennal lobe, with and without auxin. (B) GFP fluorescence quantification in the antennal lobe 24 hr after induction (four to six replicates). Statistics performed using ordinary one-way ANOVA (*, p < 0.05). (C) GFP fluorescence driven by grh-GAL4 in the larval ventral nerve cord, with and without auxin. (D) GFP fluorescence quantification in the ventral nerve cord 24 hr after induction (six replicates). Statistics performed using ordinary one-way ANOVA (*, p < 0.05). See Figure 4—source data 1 for raw data.
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Figure 4—source data 1
GFP intensity values for Figure 4.
- https://cdn.elifesciences.org/articles/67598/elife-67598-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Auxin-inducible gene expression system (AGES) allows induction of pan-neuronal GAL4 activity in the adult brain.
Confocal images of adult brains stained with anti-GFP and anti-Elav. Adults were fed food containing 5 mM auxin for 24 hr. Scale bars represent 20 µm.
Figure 5 with 1 supplement
Impact of auxin on developmental timing and survival.
(A) Time taken from egg-laying to wandering L3 larvae. (B) Time taken from egg-laying to pupation. (C) Survival across developmental stages (larval to pupal and pupal to adult) with 10 mM auxin. (D) Survival of adult females during continuous exposure to auxin. (E) Survival of adult males during continuous exposure to auxin. Logrank test and weighted Gehan-Breslow-Wilcoxon model (ns) were used for the adult survival assays. See Figure 5—source data 1 for raw data.
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Figure 5—source data 1
Raw data for larval crawling and adult climbing assays.
- https://cdn.elifesciences.org/articles/67598/elife-67598-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Working concentrations of auxin do not impact locomotor function of wild-type Drosophila.
(A) Larval crawling speed on 0 mM food (10 larvae) and 5 mM auxin food (10 larvae). (B) Distance climbed in climbing assay for males and females on 0 mM food (five separate vials and a total of 144 flies for males and 130 flies for females) and 10 mM auxin food (five separate vials and a total of 141 flies for males and 141 flies for females). Analysed using a paired t-test.
Figure 6 with 2 supplements
Auxin-inducible gene expression system (AGES) induced expression of Kir2.1 in PDF+ clock neurons ablates circadian locomotor rhythms.
(A, B) Representative double-plotted actograms for 5- to 10-day-old male flies maintained on standard food (A) or food supplemented with 2 mm NAA (B) for 3 days in 12 hr light:12 hr dark (12:12 LD) and 7 days in DD. Left: parental control UAS-Kir2.1, centre: parental control PDF-GAL4; AGES, right: experimental PDF-GAL4/UAS-Kir2.1; AGES/+. Bars indicate LD cycle, grey shaded days indicate constant darkness. (C) Amplitude of circadian rest:activity rhythms on DD days 2–8 represented by fast Fourier transform (FFT) power at 24 hr for male PDF-GAL4;AGES > UAS-Kir2.1 flies and their parental controls on standard food (orange) and food supplemented with 2 mM NAA (blue). Points represent individual flies, box shows 25–75% confidence interval, median line, and outliers. (D) Twenty-four hr FFT power as in (C) for female PDF-GAL4;AGES > UAS-Kir2.1 flies and their parental controls. (E) Twenty-four hr FFT power for as in (C) male PDF-GAL4-Geneswitch > UAS-Kir2.1 flies and their parental controls maintained on vehicle control food (orange) and food supplemented with 466 mM RU-486 (red). (F) Twenty-four hr FFT power as in (E) for female PDF-GAL4-Geneswitch > UAS-Kir2.1 flies and their parental controls. For all panels, means were compared by two-way ANOVA by genotype and food substrate, see Figure 6—source data 1 for raw data, p-values, and key resource data. Means sharing the same letter are not significantly different from one another by Tukey’s post hoc test (p > 0.05).
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Figure 6—source data 1
Raw data, p values and key resource data for circadian and activity behaviourial experiments.
- https://cdn.elifesciences.org/articles/67598/elife-67598-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Dose-dependent NAA effects on behaviour of auxin-inducible gene expression system (AGES) parental controls.
Behavioural data for PDF-GAL4; AGES > UAS-Kir2.1 flies and their parental controls on standard food (orange), 2 mM NAA (blue) or 10 mM NAA (purple). (A, B) Twenty-four hr fast Fourier transform (FFT) power on DD days 2–8 for male (A) and female (B) PDF-GAL4; AGES > UAS-Kir2.1 flies. Two mM NAA data is replotted from Figure 6. Means were compared by two-way ANOVA by genotype and food substrate. Means sharing the same letter are not significantly different from one another by Tukey’s post hoc test (p > 0.05). (C, D) Period length for male (C) and female (D) flies. Means were compared by Student’s t-test for flies of the same genotype on different food substrates. (E, F) Average 24 hr activity counts for male (E) and female (F) flies. Means were compared by two-way ANOVA by genotype and food substrate. Means sharing the same letter are not significantly different from one another by Tukey’s post hoc test (p > 0.05). See Figure 6—source data 1 for raw data and p-values.
Figure 6—figure supplement 2
Effects of auxin-inducible gene expression system (AGES) and GeneSwitch induced expression of Kir2.1 in PDF+ clock neurons on circadian period length and average 24 hr locomotor activity.
(A, B) Period length estimated by chi-squared periodogram on days 2–8 of DD for male (A) and female (B) PDF-GAL4; AGES > UAS-Kir2.1 flies and their parental controls on standard food (orange) and food supplemented with 2 mM NAA (blue). Means were compared by Student’s t-test for flies of the same genotype on different food substrates. (C, D) Average 24 hr activity counts on days 2–8 of DD for male (C) and female (D) PDF-GAL4; AGES > UAS-Kir2.1 flies and their parental controls on standard food (orange) and food supplemented with 2 mM NAA (blue). Means were compared by two-way ANOVA by genotype and food substrate, and Tukey’s post hoc test. Only genotype had significant effects on activity (p = 2.86 × 10–7) and there was no significant interaction effect. Means sharing the same letter are not significantly different from one another by Tukey’s post hoc test (p > 0.05). (E, F) Period length for male (E) and female (F) PDF-GAL4-Geneswitch > UAS-Kir2.1 flies and their parental controls maintained on vehicle control food (orange) and food supplemented with 466 mM RU-486 (teal). Statistics as in panels A and B. (G, H) Average 24 hr activity counts for male (G) and female (H) PDF-GAL4-Geneswitch > UAS-Kir2.1 flies and their parental controls maintained on vehicle control food (orange) and food supplemented with 466 mM RU-486 (teal). Statistics as in panels C and D. Genotype had significant effects on activity in both males and females (p = 4.48 × 10–7, p = 3.60 × 10–2, respectively) and there was a significant interaction between the effects of genotype and food substrate in both males and females (p = 0.007, p = 3.76 x 10–9, respectively). See Figure 6—source data 1 for raw data and p-values.
Tables
Table 1
Main advantages and disadvantages of the most common drug-inducible gene expression systems currently available to the Drosophila community.
| Expression system | Reference | Advantages | Disadvantages |
|---|---|---|---|
| GeneSwitch | Osterwalder et al., 2001 | Non-toxic for adult flies | Not compatible with existing GAL4 linesLeaky expression (Poirier et al., 2008; Scialo et al., 2016)Larval exposure impacts on adult sleep patterns (Li and Stavropoulos, 2016)Drug is unsafe to handle for female researchersDrug is expensive |
| QF System | Potter et al., 2010 | Non-toxic for flies | Not compatible with existing GAL4 lines |
| Tet-off GAL80 | Barwell et al., 2017 | GAL4/UAS compatible | Long induction time (>5 days)Requires x2 copies of the GAL80 transgeneAffects microbiota |
| TMPINDUCIBLE | Kogenaru and Isalan, 2018; Sethi and Wang, 2017 | GAL4/UAS compatible | Currently not compatible for general use with existing GAL4 linesTMP drug is only dissolvable in DMSO (affects survival of larvae) or has to be added as a dry powder to food |
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An auxin-inducible, GAL4-compatible, gene expression system for Drosophila
eLife 11:e67598.
https://doi.org/10.7554/eLife.67598
