An auxin-inducible, GAL4-compatible, gene expression system for Drosophila
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Evaluation Summary:
This work will be of interest to Drosophila geneticists in its development of a new tool for temporal control of gene induction by the widely-used bipartite Gal4/UAS system. By transferring protein modules from plants that mediate auxin-dependent protein degradation to control the stability of a Gal4-inhibitor (Gal80), the authors successfully demonstrate the ability to control Gal4 activity in flies through the provision of auxin in the food substrate and provide evidence for the sensitivity, specificity and non-toxicity of this tool.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila , spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila .
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Author Response:
Reviewer #1 (Public Review):
The authors aimed to develop a new, non-toxic tool for temporal regulation of Gal4-dependent gene expression in Drosophila, by creating a version of the Gal4 inhibitor, Gal80, bearing an auxin-degron sequence, rendering this protein susceptible to degradation upon provision of the plant hormone auxin by feeding. This technology (Auxin-inducible Gene Expression System, AGES) builds upon previous use of this system in other animals, including one study in Drosophila in which a different protein was targeted for auxin-dependent degradation (Trost, Fly 2016).
Strengths:
The authors have identified a need for a better tool for temporal control of transgene expression that is compatible with the vast libraries of Gal4 drivers, that doesn't rely on temperature shifts (as for Gal80ts), and …
Author Response:
Reviewer #1 (Public Review):
The authors aimed to develop a new, non-toxic tool for temporal regulation of Gal4-dependent gene expression in Drosophila, by creating a version of the Gal4 inhibitor, Gal80, bearing an auxin-degron sequence, rendering this protein susceptible to degradation upon provision of the plant hormone auxin by feeding. This technology (Auxin-inducible Gene Expression System, AGES) builds upon previous use of this system in other animals, including one study in Drosophila in which a different protein was targeted for auxin-dependent degradation (Trost, Fly 2016).
Strengths:
The authors have identified a need for a better tool for temporal control of transgene expression that is compatible with the vast libraries of Gal4 drivers, that doesn't rely on temperature shifts (as for Gal80ts), and that is non-toxic. As presented, they have been successful in developing such a tool and providing an initial characterization revealing its functionality, and key technical information for future exploitation (e.g. auxin dose, lag time of gene induction after auxin provision, the ability of auxin to cross the blood-brain-barrier).
Weaknesses:
- The authors fail to give much credit to the previous work (Trost, Fly 2016), which provided the first demonstration of the utility, temporal dynamics, and non-toxicity of the auxin-degron system in Drosophila. While the current study applies the auxin-degron to generate a much more generally useful genetic tool, it is a bit ungenerous to only mention the early work in passing in the Introduction.
We agree that we should have introduced/emphasised the Trost paper more in our manuscript. We have now included two sentences in the introduction highlighting it, plus stated it as an inspiration for our study (86-89).
- The technical testing of the system feels rather light for a tool-development manuscript, using AGES with two broadly expressed (and presumably quite strong) Gal4 drivers and a UAS-GFP effector transgene as a read-out of gene expression. Several simple extensions to this work would have been desirable in this first study.
For example:
- quantitative read-out of auxin-dependent GFP expression is only shown in Figure 2. Figures 3 and 4 show only images of a single animal in a given test condition. Such experiments could be quantified to give a sense of the animal-to-animal variation.
We have quantified GFP expression levels in larvae on different concentrations of auxin, and over a time course using 5 mM (new Figure 3). We have also performed new experiments (with full quantification) switching on GFP expression in a subset of larval brain cells (grh-GAL4) and a subset of adult brain cells (Or85a-GAL4) (new Figure 4).
- the temporal dynamics of the system are only superficially described, despite the importance of this property for researchers to be aware of. The authors write (line 115-116): ""shorter exposure times of adults to auxin containing food were tested (data are not shown), however, 24 hours is the minimal amount of time required for proper induction of GAL4 activity.", but this is exactly the sort of information that should be shown and rigorously explored when presenting a new tool. Ideally, one could compare such properties side-by-side with Gal80ts. In addition, there is no mention of the reversibility of AGES (as is possible with Gal80ts), raising the question of how long auxin remains in the fly after ingestion.
We have now quantified the on and off temporal dynamics in adult flies using 10 mM auxin (Figure 2 – Figure Supplement 2A).
- the authors argue the auxin provision is non-toxic, but the main read-out is survival/lifespan. While these are not affected by continuous exposure to auxin, the developmental time to pupal stages is clearly affected by high doses of auxin, so there is some pharmacological effect of this hormone. As such, more subtle effects of auxin (e.g., on locomotor activity, sleep, fertility etc.) cannot be fully excluded.
We have examined the effect of working concentration (5 mM for larvae and 10 mM for adults) on larval crawling and adult climbing (Figure 6 – Figure Supplement 1). Here we observe no impact of auxin on these behaviours. The circadian experiment data also showed that auxin did not affect locomotor activity (Figure 6 - supplement 2, A-D). However, in males, 2 mM auxin did affect the circadian rhythm of one of the control lines but not the other (Figure 6C). Currently, we do not have an explanation for this but does emphasise the requirement to always perform the appropriate controls.
- the authors also write (line 205-6): "In our experience, auxin-containing food can be stored 4C for up to 4 weeks where the hormone's potency still persists", but, again, such observations would be much more useful to carefully document in this technical study.
We have tested how long auxin food lasts (Figure 2 – Figure Supplement 2B) and it is still working after 15 weeks (longer than most labs would store fly food).
The AGES system has the potential for use in the Drosophila community as a complementary and very valuable tool for temporal control of Gal4-driven gene expression. As with all tools, only time will tell whether the favorable properties highlighted by the authors' initial tests stand further scrutiny using other Gal4 drivers, other types of phenotypic read-out (gene expression, physiology, behavior etc).
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Evaluation Summary:
This work will be of interest to Drosophila geneticists in its development of a new tool for temporal control of gene induction by the widely-used bipartite Gal4/UAS system. By transferring protein modules from plants that mediate auxin-dependent protein degradation to control the stability of a Gal4-inhibitor (Gal80), the authors successfully demonstrate the ability to control Gal4 activity in flies through the provision of auxin in the food substrate and provide evidence for the sensitivity, specificity and non-toxicity of this tool.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors aimed to develop a new, non-toxic tool for temporal regulation of Gal4-dependent gene expression in Drosophila, by creating a version of the Gal4 inhibitor, Gal80, bearing an auxin-degron sequence, rendering this protein susceptible to degradation upon provision of the plant hormone auxin by feeding. This technology (Auxin-inducible Gene Expression System, AGES) builds upon previous use of this system in other animals, including one study in Drosophila in which a different protein was targeted for auxin-dependent degradation (Trost, Fly 2016).
Strengths:
The authors have identified a need for a better tool for temporal control of transgene expression that is compatible with the vast libraries of Gal4 drivers, that doesn't rely on temperature shifts (as for Gal80ts), and that is non-toxic. As …
Reviewer #1 (Public Review):
The authors aimed to develop a new, non-toxic tool for temporal regulation of Gal4-dependent gene expression in Drosophila, by creating a version of the Gal4 inhibitor, Gal80, bearing an auxin-degron sequence, rendering this protein susceptible to degradation upon provision of the plant hormone auxin by feeding. This technology (Auxin-inducible Gene Expression System, AGES) builds upon previous use of this system in other animals, including one study in Drosophila in which a different protein was targeted for auxin-dependent degradation (Trost, Fly 2016).
Strengths:
The authors have identified a need for a better tool for temporal control of transgene expression that is compatible with the vast libraries of Gal4 drivers, that doesn't rely on temperature shifts (as for Gal80ts), and that is non-toxic. As presented, they have been successful in developing such a tool and providing an initial characterization revealing its functionality, and key technical information for future exploitation (e.g. auxin dose, lag time of gene induction after auxin provision, the ability of auxin to cross the blood-brain-barrier).
Weaknesses:
1. The authors fail to give much credit to the previous work (Trost, Fly 2016), which provided the first demonstration of the utility, temporal dynamics, and non-toxicity of the auxin-degron system in Drosophila. While the current study applies the auxin-degron to generate a much more generally useful genetic tool, it is a bit ungenerous to only mention the early work in passing in the Introduction.
2. The technical testing of the system feels rather light for a tool-development manuscript, using AGES with two broadly expressed (and presumably quite strong) Gal4 drivers and a UAS-GFP effector transgene as a read-out of gene expression. Several simple extensions to this work would have been desirable in this first study.
For example:
- quantitative read-out of auxin-dependent GFP expression is only shown in Figure 2. Figures 3 and 4 show only images of a single animal in a given test condition. Such experiments could be quantified to give a sense of the animal-to-animal variation.
- the temporal dynamics of the system are only superficially described, despite the importance of this property for researchers to be aware of. The authors write (line 115-116): ""shorter exposure times of adults to auxin containing food were tested (data are not shown), however, 24 hours is the minimal amount of time required for proper induction of GAL4 activity.", but this is exactly the sort of information that should be shown and rigorously explored when presenting a new tool. Ideally, one could compare such properties side-by-side with Gal80ts. In addition, there is no mention of the reversibility of AGES (as is possible with Gal80ts), raising the question of how long auxin remains in the fly after ingestion.
- the authors argue the auxin provision is non-toxic, but the main read-out is survival/lifespan. While these are not affected by continuous exposure to auxin, the developmental time to pupal stages is clearly affected by high doses of auxin, so there is some pharmacological effect of this hormone. As such, more subtle effects of auxin (e.g., on locomotor activity, sleep, fertility etc.) cannot be fully excluded.
- the authors also write (line 205-6): "In our experience, auxin-containing food can be stored 4C for up to 4 weeks where the hormone's potency still persists", but, again, such observations would be much more useful to carefully document in this technical study.
The AGES system has the potential for use in the Drosophila community as a complementary and very valuable tool for temporal control of Gal4-driven gene expression. As with all tools, only time will tell whether the favorable properties highlighted by the authors' initial tests stand further scrutiny using other Gal4 drivers, other types of phenotypic read-out (gene expression, physiology, behavior etc).
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Reviewer #2 (Public Review):
This paper's authors have developed a novel system that enables temporal control of Gal4-induced expression in Drosophila melanogaster. There are various drawbacks to existing systems used to control Gal4 expression, including toxicity and the need to shift temperatures, both of which are circumvented with the new AGES system described in this paper. The authors successfully display the efficacy of using this system to induce Gal4-driven reporter gene expression in the adult and larvae while also highlighting that the system is non-toxic and affordable and can be used in combination with existing Gal4 lines and will thus be of broad interest to the Drosophila community.
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