Tools for synchronous temporal control of independent transgenes in discrete Drosophila tissues

  1. Evelyne Ruchti
  2. Brian D. McCabe

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Abstract

Exogenous binary gene regulatory expression systems are a linchpin technology critical for many model organism genetic manipulations including of Drosophila melanogaster. Subsequent to the initial establishment of the Gal4/UAS binary expression system, LexA/LexOp and QF/QUAS binary gene expression systems have been adopted to supplement and expand the tissue specific control of genes expression in Drosophila. Here, we have developed a compendium of modular vectors that enable robust, reproducible, and high throughput parallel construction of transgenes that produce either Gal4, LexA or QF2 transcription factors under specific gene enhancers and compatible partner vectors that allow transgenes to be regulated by these factors under UAS, LexOp, or QUAS control. Expanding upon this foundation, we have generated a novel hybrid binary gene regulatory system derived from QF and Gal4 - QFG4, that enables simultaneous coordinate regulation of UAS and QUAS transgenes by Gal80, allowing synchronous temporal control of independent transgene expression in distinct cells or tissues. We envision these new tools will facilitate novel and increasingly sophisticated interrogation of cell and tissue interactions particularly in contexts where temporal control is paramount.

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    We thank the reviewers for their careful evaluation of our manuscript and their constructive comments. Overall, the reviewers recognize the technical value and potential impact of the pBID2 platform as a unified framework for generating transgenic tools across multiple binary expression systems in Drosophila.

    In response to the reviewer’s suggestions, we will strengthen the manuscript in two main directions. First, we will perform additional experiments to further support key claims regarding the QFG4 system, including (i) assessing temporal dynamics of transgene expression across multiple time points, (ii) extending validation to additional tissues, (iii) generating new driver lines. (iv) In addition we will confirm co-expression of pBID2-VGMN-GAL4 and pBID2-VGMN-QF2 in the same neurons. These experiments are currently underway and will directly address concerns regarding synchronicity and reporducibility. Second, we will revise the manuscript to improve clarity, accuracy, and context within the existing literature. This includes justifying claims where appropriate, refining terminology, expanding discussion of prior work, and improving data presentation.

    Reviewer # 1 __Major Comments __

    1 - Synchronous temporal control: multiple time points in Fig. 7

    Synchronous temporal control has not been rigorously demonstrated in Fig. 7. Only a single time point (7 days after temperature shift) was examined. The synchronicity of expression between the two systems remains unclear, and a temporal delay between them is possible. I suggest examining multiple time points to assess true synchronicity.

    We thank Reviewer 1 for the positive assessment of the study and for the constructive suggestions. We agree with this concern. Our current data examine only a single time point after temperature shift, which is insufficient to support claims of synchronous regulation between the GAL4 and QFG4 systems. We plan to repeat the Fig. 7 experiment and collect imaging data at multiple time points spanning approximately one week after temperature shift. The resulting data will be incorporated into a revised Fig. 7, and the text will be updated accordingly.

    2 - Coordinated control of Gal4 and QFG4 activity in additional tissues

    Expression in distinct cells or tissues is demonstrated only in VGMN and muscle. If the claim is intended to be broadly applicable, additional examples would strengthen it. Including other tissues or cell types would provide stronger support.

    We agree that demonstrating QFG4 in additional tissues is important to substantiate the generality of the approach. We will test the coordinated response of a nSyb-QFG4 driver line (in neurons) together with a Repo-GAL4 line (in glia), in the adult brain. This experiment directly addresses the reviewer's request for a second tissue context and provides a biologically meaningful example of intersectional control across two distinct cell types within the same organ.

    3 - New QFG4 driver lines: demonstrating pipeline scalability

    Demonstrating additional QFG4 lines targeting other tissues would highlight the versatility and scalability of the approach and would represent valuable community resources. Given that this is a methodological paper focused on pipeline development, such experiments would directly test the ease and efficiency of the system.

    We plan to use the pBID2 pipeline to generate at least one additional QFG4 driver line targeting another cell type, which we will image to confirm expression patterns. These lines will serve as further proof-of-concept for the scalability of the platform and will be deposited as community resources.

    __Minor Comments __

    All minor comments from Reviewer 1 will be addressed through text and figure revisions:

    • The typographical error "Kusubira" (line 111) will be corrected to "Kusabira".
    • Figure resolution will be improved: Fig. 3B and 3C panels will be enlarged to better demonstrate nuclear localisation of mKO2, and Fig. 7B will include higher magnification images to illustrate differential localisation between VGMN and muscle.

    Reviewer # 2 __Major Comments __

    1 - Novelty of this study

    There is very little novelty in this study. The gateway compatible vectors to generate LexA, Gal4 and QF drivers were generated in Janelia years ago and are currently in use. The only observable difference is the use of insulators in this manuscript.

    We thank the reviewer for their critical evaluation of our work. We respectfully disagree with the reviewer’s assessment that the study lacks novelty. While individual components such as Gateway-compatible vectors and binary expression systems have been previously developed, the pBID2 platform provides a unified, modular framework that minimizes transcriptional leakage (through the use of Gypsy insulators and a DSCP promoter) and achieves strong expression (through a p10 UTR terminator, multiple repeats of activator sequences, and a Syn21 element upstream of drivers), integrating the GAL4/UAS, LexA/LexOP, and QF/QUAS systems within a single architecture. This standardization enables the streamlined generation of complex transgenic combinations that would otherwise be fragmented. In addition, the QFG4 system introduces a GAL80-sensitive QF-based activator, enabling coordinated temporal regulation across independent binary systems. We believe this represents a conceptual advance beyond existing implementations. To better reflect this contribution, we will revise the Introduction and Discussion to more clearly position our work relative to existing tools and explicitly acknowledge prior developments, including Janelia-based constructs.

    2 - P2A versus T2A

    T2A is the 2A peptide that has been used in Drosophila research. P2A was shown to work worse than T2A in the Diao and White 2012 paper. This decreases the novelty of this finding.

    We will revise the relevant sections of the Results and Discussion to more accurately reflect the existing literature on 2A peptide performance in Drosophila, including the findings of Diao and White 2012 and their demonstration of T2A efficacy. We will clarify the rationale for our use of P2A in the pBID2 system and discuss this choice in proper context of both the Daniels et al. 2014 and Diao and White 2012 publications, as suggested also by Reviewer 3.

    3 - QFG4 co-regulation and leakiness

    The proof of principle experiment does not show how co-regulation of QF driver and Gal4 driver by Gal80 can be beneficial, and shows that the regulation of QFG4 is not as tight when used in conjunction with a Gal4 driver. The reason for the leakier QFG4 regulation is not clear and not explored.

    The reviewer raises important points regarding the functional advantages and potential limitations of QFG4, including its regulatory tightness and biological utility. To address this, we will (i) expand the Discussion to better articulate the contexts in which coordinated regulation of independent systems is advantageous, (ii) clarify that QFG4 is intended as a flexible tool whose performance may vary depending on experimental context and discuss the observed differences in repression efficiency between QFG4 and GAL4, (iii) moderate our claims where appropriate to reflect the current level of validation. In addition, the new experiments outlined above for Reviewer 1 (multiple time points and additional tissues) will provide further insight into the performance and applicability of the system.

    __Minor Comments __

    All minor comments from Reviewer 2 will be addressed through text revision:

    • The use of "permissive" and "restrictive" temperatures will be corrected throughout to align with conventional usage in the field (restrictive = 29°C, permissive = 18°C).
    • The discussion of the LexA-GAD strategy will be incorporated into the Results section where relevant, rather than appearing only in the Discussion.
    • The Diao and White 2012 reference will be appropriately cited alongside Daniels et al. 2014 in the P2A/T2A discussion.

    Reviewer # 3 Major Comments

    Reviewer 3 raised no major experimental concerns and found the data sufficient to support the main claims of the paper. All comments from Reviewer 3 will be addressed through text and figure revisions. Nevertheless, we are still planning to perform an additional experiment in response to the remarks about the VGMN dual labelling in the same cells.

    New experiment

    1 - Co-expression of VGMN-GAL4 and VGMN-QF2 in the same neurons

    It is not clear if pBID2-GAL4 and pBID2-QF2 constructs express in exactly the same neurons, e.g., with VGMN. Figure 4 shows independent labelling, but it is not clear if these were validated as the exact same expression pattern. Dual labelling experiments in the same animal would clarify this.

    We thank the reviewer for recognising the relevance of the topic and for this valuable suggestion. We plan to perform dual-labelling experiments using the VGMN enhancer to directly compare the expression patterns driven by pBID2-VGMN-GAL4 and pBID2-VGMN-QF2 within the same cells. Fig. 4 currently shows independent labelling in separate animals, which does not allow direct comparison at single-cell resolution. The dual-labelling data will allow us to confirm whether the two constructs drive expression in the same neurons and will directly support the claim that pBID2 produces equivalent and interchangeable driver lines across binary systems.

    Minor Comments

    All minor comments from Reviewer 3 will be addressed through text revision:

    • Figure 1 will be revised to add arrows indicating that Activator and Responder constructs are inserted at position 0 of pBID2.
    • The name of the MCS variant (pBID2-MCS) will be made explicit in the relevant results section (lines 86-87).
    • The contribution of Diao and White 2012 to the validation of T2A in Drosophila will be more clearly described in the Results section, and Ref #68 will be cited at line 120.
    • The Figure 3 legend labelling errors ("B)" covering panels B and C; "C)" covering panel D) will be corrected.
    • Lines 258-260 will be revised: the discussion of GAL80 binding to the GAL4 activation domain will be clarified to avoid implying a role for the middle domain without supporting experimental data. As correctly noted by Reviewer 3, demonstrating a role for the middle domain would require a QFG4 construct using only the GAL4 activation domain.
    • The typographical error "otor" in the Fig. 7 legend will be corrected to "motor".
    • A comparison of pBID2-UAS constructs with Janelia UAS constructs (e.g., pJFRC7-20XUAS-IVS-mCD8::GFP) will be added to the Discussion, including any direct comparisons we have performed.
    • Dual-labelling experiments to confirm co-expression of pBID2-GAL4 and pBID2-QF2 in the same neurons will be performed (see Major Comment 4 above).
    • The Acknowledgments will be corrected: "Christopher G. Potter" will be corrected to "Christopher J. Potter".
    • The Materials and Methods section will be corrected: "CsChrismson" will be corrected to "CsChrimson".
  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In the manuscript by Ruchti and McCabe, the authors introduce and validate many new constructs for use in generating new transgenic reagents in Drosophila. The authors introduce a number of improvements, including the pBID2 plasmid for generating flies that utilize the binary expression systems GAL4/UAS, LexQF/LexOp, QF2/QUAS. The authors generated two new reporters that introduce a nucleus-membrane marker to simultaneously label both the nucleus of the cell and its processes. The authors also identify a new enhancer derived from the VGlut genomic area that drives specific expression in motor neurons (labeled as VGMN). The authors also introduce a new activator, QFG4, which utilizes the QF DNA binding domain and the Gal4 activation domain. They demonstrate that this new reagent induces robust expression and has the additional benefit of being GAL80 sensitive. Overall, this work represents new and useful additions to the Drosophila toolkit that have the potential to become widely adopted.

    Major Comments:

    I do not have any major concerns regarding the work presented. The authors demonstrate the practical use of their new reagents via a number of experiments with new transgenic flies. As such, the conclusion that these new reagents are an improvement over existing reagents is justified. Additional experiments are not necessary to support the major claims on this paper. The data and methods are presented in a way that allows reproduction as well as utilization of the newly introduced reagents. The figures are well presented and adequately demonstrate the function of the new reagents in transgenic Drosophila.

    Minor Comments:

    In Figure 1, it was not entirely clear that the Activator constructs and the Responder Constructs have been inserted at position 0 of the pBID2 construct. Perhaps adding an arrow onto the lines that point to 0 could make this point clearer.

    Line 86-87. The authors have a variation of pBID2 that uses a MCS. What is the name of these constructs? Please add this to this section so its obvious. I assume it is pBID2-MCS as reflected in Figure S2.

    Regarding T2A (lines ~106-146). T2A was first validated to be useful for transgene expression by Ref#68 (Diao and White 2012.). This paper is why many current Drosophila constructs use T2A. This should be better reflected in the results section when reporting on the use of T2A and P2A experiments. As written, it was not clear that T2A was previously validated as a useful method for expression in Drosophila. As one example that could be updated, Ref #68 should also be cited on line 120 "we used ribosomal skipping sequences (63-65)".

    Figure 3 legend. "B)" should be "B) and C)". "C)" should be "D)".

    Lines 258-260. GAL80 binds directly to the activation domain of GAL4 at its C-terminus (~aa 761- 880). The middle domain likely doesn't play a role in GAL80 binding and might just function for structural stability. To make this statement in the discussion, the authors would need to make a QFG4 that uses just the GAL4 activation domain without its middle domain, similar to what was used to make QF2.

    Figure 7 legend. 3rd to last line. "otor" should be "motor".

    To the discussion section, please comment on how pBID2-UAS constructs might compare to Janelia UAS constructs, eg., pJFRC7-20XUAS-IVS-mCD8::GFP. If the authors have made direct comparisons, it would be helpful to include their observations. The Janelia constructs have similar features, and it would be helpful to include the authors thoughs on why to choose pBID2-20xUAS vs pJFRC7-20xUAS (for example).

    To the results or discussion section, please comment if the authors have examined if pBID2-GAL4 and pBID2-QF2 constructs express in exactly the same neurons (eg., with VGMN). For example, by conducting dual labeling experiments in the same animal. Figure 4 shows independent labeling, but it is not clear if these were validated as the exact same expression pattern. As the authors correctly pointed out, the promoter can influence expression (hsp70 or DSCP), but so can sequences from the transcription factor (eg., GAL4 or QF). It is possible the gypsy insulators have addressed these issues, but if the authors have data demonstrating that the exact same expression patterns are induced by the different constructs, it would be helpful to include.

    Acknowledgments. Bibliography lists papers by a Christopher J. Potter, not a Christopher G. Potter.

    Materials and Methods, page 13, pBID Gateway responder vector series. It should be "CsChrimson" not "CsChrismson"

    Significance

    This work represents a significant technical advance to the Drosophila toolkit. It introduces and validates many new reagents (both activators and reporters) that will prove useful to the Drosophila community. These new reagents will enable both simple and complex experiments to be more efficiently performed in Drosophila, especially those interested in investigating complex tissues such as the brain.

    This work will be of primary interest to those developing new reagents for studying Drosophila biology, as well as those interested in genetic tool development. The reagents developed here could also be applied to other genetic systems, such as other insect models.

    This reviewer's expertise is in development genetic tools for use in Drosophila and other insects, and in applying these new genetic methods to the field of neuroscience.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript Ruchti and McCabe describe incremental technical developments to create Gateway compatible vectors with increased transgene expression or driver expression, a dual reporter that expresses membrane targeted fluorescent protein together with nuclear targeted fluorescent protein and a QF version that can be repressed by Gal80 (QFG4).

    Major issues:

    • There is very little novelty in this study. The gateway compatible vectors to generate LexA, Gal4 and QF drivers were generated in Janelia years ago and are currently in use. The only observable difference is the use of insulators in this manuscript. Leakiness of driver lines inserted in well characterized landing sites is not a great concern.
    • Except for the initial studies that showed P2A can work in Drosophila cells other Drosophila, T2A is the 2A peptide that has been used in Drosophila research. P2A was shown to work worse and T2A in the Diao and White 2012 paper. This decreases the novelty of this finding.
    • The proof of principle experiment in the paper do not show how having co-regulation of QF driver and Gal4 driver by using Gal80 or Gal80ts can be beneficial and if anything shows that the regulation of QFG4 is not as tight when used in conjunction with a Gal4 driver. The reason for the leakier QFG4 regulation compared to Gal4 regulation is also not clear and not explored.

    Minor issue:

    • The authors use the terms permissive temperature and restrictive temperature in a manner that is against the conventional use. These terms conventionally refer to functionality of Gal80ts in the given temperature, not the activity of Gal4 which is negatively correlated. Hence, in literature restrictive temperature typically refers to 29 C and permissive temperature is 18 C. This is confusing.
    • Although the references are cited, the information about some of the papers are not properly presented. For example, LexA-GAD strategy is only brought up in discussion but in results, the authors make the statement that no Gal80 regulation strategies exist for LexA. When discussing the use of P2A versus T2A the authors mainly refer to Daniels et al. 2014 publication whereas it was also done in Diao and White 2012. The authors do cite both of these papers but discuss the findings in an incomplete manner.

    Significance

    This manuscript has very limited novelty and is better suited for a more specialized journal such as G3.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    Ruchti et al. developed new vectors compatible with the GAL4/UAS, LexA/LexOP, and QF/QUAS systems to enable high throughput construct generation (pBID2). They validated the platform by generating responder constructs (UAS, LexOP, and QUAS lines) and VGMN (glutamatergic motor neuron) driver lines. In addition, they engineered a new hybrid binary system combining QF and GAL4, termed QFG4, and tested its capacity to modulate expression levels. This design permits regulation of QUAS by GAL80, as demonstrated by experiments examining expression of ontogenetic proteins and co expression of transgenes under VGMN and MNC control with GAL80ts. Overall, the experiments are well designed, carefully performed, and quantitatively analyzed. The vectors and fly lines generated will be valuable resources if deposited in Addgene and the Bloomington Drosophila Stock Center. However, several claims appear speculative or overstated, as outlined below.

    Major Comments

    • Are the key conclusions convincing?

    Most experiments are of high quality and generally convincing. However, two conclusions would benefit from further clarification: (i) simultaneous regulation by GAL4 and QFG4, and (ii) coordinated expression of two transgenes in distinct tissues.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The summary states that QFG4 "enables simultaneous coordinate regulation of UAS and QUAS transgenes by Gal80, allowing synchronous temporal control of independent transgene expression in distinct cells or tissues." I have several concerns regarding this claim:

    1. Synchronous temporal control has not been rigorously demonstrated in Fig. 7. Only a single time point (7 days after temperature shift) was examined. The synchronicity of expression between the two systems remains unclear, and a temporal delay between them is possible. I suggest examining multiple time points to assess true synchronicity.
    2. Expression in distinct cells or tissues is demonstrated only in VGMN and muscle. The title also refers to "discrete Drosophila tissues." If the claim is intended to be broadly applicable, additional examples would strengthen it. Including other tissues or cell types would provide stronger support.
    3. Related to point (2), the pBID2 system is presented as a pipeline for generating new lines. Demonstrating additional QFG4 lines targeting other tissues would highlight the versatility and scalability of the approach. Such lines would also represent valuable community resources.
    • Would additional experiments be essential to support the claims of the paper? Additional experiments would be necessary unless the authors adopt more conservative language in their claims. Are the suggested experiments realistic in terms of time and resources? The proposed experiments appear realistic.

    For point (1), the authors could repeat the existing experiment and collect images at multiple time points over approximately one week, as the fly lines are already available. For points (2) and (3), generating two to three additional QFG4 lines and imaging their expression in distinct tissues would provide meaningful validation. Given that this is a methodological paper focused on pipeline development, such experiments would directly test the ease and efficiency of the system. Generating new lines may require approximately 2-3 months, followed by ~1 month of imaging and analysis, which is a reasonable investment.

    • Are the data and methods presented in a reproducible manner?

    Yes, the presentation is generally clear and detailed enough to ensure reproducibility.

    • Are the experiments adequately replicated and statistically analyzed?

    Overall, replication and statistical analysis appear appropriate. However, inclusion of additional time points for Fig. 7 would strengthen the conclusions.

    Minor Comments

    • Specific experimental issues that are easily addressable

    No major additional concerns beyond those noted above.

    • Are prior studies referenced appropriately?

    A minor typographical issue: Line 111 lists "Kusubira," which should be corrected to "Kusabira."

    • Are the text and figures clear and accurate?

    As noted above, certain phrases (e.g., "simultaneous coordinate regulation," "distinct cells or tissues," "discrete tissues") appear overstated relative to the current data. Clarifying or moderating this language would improve accuracy. Some figures require higher resolution presentation. In Fig. 3B and 3C, the images are too small to clearly demonstrate nuclear localization of mKO2; larger panels would help. In Fig. 7B, higher magnification images would better illustrate differential localization between VGMN and muscle.

    • Do you have suggestions to improve presentation?

    Overall, the data presentation is strong and well organized. Clarifying the scope of the claims and providing higher resolution images where noted would further improve the manuscript.

    Significance

    • Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field.

    Drosophila research relies heavily on binary expression systems for spatial and temporal control of gene function. The unified vector platform developed here, incorporating Gateway compatibility across GAL4/UAS, LexA/LexOP, and QF/QUAS systems, represents a meaningful technical advance. By streamlining construct generation across multiple systems within a single framework (pBID2), the authors lower the technical barrier for complex genetic manipulations. The previously developed pBID system for UAS/GAL4 has been widely adopted and highly cited, underscoring community demand for standardized and scalable tools. The current expansion to additional binary systems is therefore likely to have broad impact. Inclusion of a few additional validation experiments, as noted above, would further strengthen confidence in the robustness and versatility of the platform.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    This work builds directly upon foundational binary expression systems widely used in the Drosophila field, including GAL4/UAS (Brand and Perrimon, 1993), LexA/LexOP (Lai and Lee, 2006), and QF/QUAS (Potter et al., 2010). By providing a unified and modular cloning strategy compatible with these systems, the authors enhance the practicality and interoperability of established genetic tools rather than introducing an entirely new paradigm. This technical consolidation is valuable for laboratories that routinely combine multiple binary systems for intersectional or parallel manipulations.

    • State what audience might be interested in and influenced by the reported findings.

    The primary audience will be researchers working with Drosophila genetics, particularly those employing complex intersectional strategies, circuit mapping, developmental biology, and functional manipulation of defined cell populations. Laboratories developing new driver or responder lines will especially benefit from the streamlined cloning pipeline.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Field of expertise: Drosophila genetics, neurobiology, binary expression systems, and circuit analysis. I have sufficient expertise to evaluate the genetic and technical aspects of the manuscript.

  5. Version published to 10.64898/2026.02.02.703225 on bioRxiv