Quantitative spatial and temporal assessment of regulatory element activity in zebrafish

  1. Shipra Bhatia
  2. Dirk Jan Kleinjan
  3. Kirsty Uttley
  4. Anita Mann
  5. Nefeli Dellepiane
  6. Wendy A Bickmore

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Abstract

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

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  1. Version published to 10.7554/elife.65601 on eLife
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    Reply to the reviewers

    We thank the reviewers for their enthusiastic support for our work and their insightful comments and suggestions which we believe strengthen the manuscript. Below we detail how we propose to respond to each of the specific points raised by each reviewer.

    __Reviewer #1______

    1). It is convincingly shown that adding insulator elements (cHS4) reduces crosstalk between the two PAX6 CREs tested (Fig. 3). However, it is unclear if this approach will work for other CREs. This point should be discussed, and perhaps the authors could give some troubleshooting advice (e.g. adding more insulators or trying different insulator elements?).

    We will include these suggestions in the discussion and describe some ongoing efforts to characterise another insulator element in our assay.

    2). All CREs used in proof-of-concept experiments in this work have well known activities in zebrafish embryos. A new/uncharacterized CRE has not been tested yet using this system. It is unclear from the workflow (Fig. 1B) what happens if the CRE does not drive detectable levels of EGFP/mCherry. How does one determine whether lack of reporter expression is due to technical problem (with the transgene or phiC31 integration) or that the CRE is not active in zebrafish? Perhaps adding a PCR-based genotyping step could address this potential problem?

    We will include a PCR-based genotyping assay in the description of the assay pipeline and discuss its utility in assessing successful integration events as suggested by the reviewer.

    3). Other limitations of the system should also be discussed. For example, the system appears to be useful for identifying variant CREs that result in a change (either loss or gain) of temporal or spatial activity, but it is not clear how subtle changes in expression level (either slightly increased or decreased) would be identified or quantified. Perhaps other approaches could be used in combination with this system to fully analyze mutant CRE activity. Another limitation is that this approach is only be applicable to CREs that are active in the first few days of zebrafish embryonic development.

    *We will include these suggestions in the discussion and clearly address the limitations of the system *

    **Minor points:**

    i) Although it is discussed in the previous work published in PLoS Genetics, it is probably worth mentioning here why the gata2 minimal promoter was chosen for the reporter system.

    The choice of the gata2 promoter in our constructs was based on previously published work from our group. We will re-iterate and reference these studies in the workflow description.

    ii) It would be helpful if the cSH4 element is briefly described (e.g. "insulator element") in Fig.1 legend. We will modify the figure legend according to the suggestion.

    3). It is not clear from the manuscript whether the new reagents reported here-including dual reporter vectors and transgenic attB landing site zebrafish strains-will be made available to the scientific community, or how these reagents would be distributed.

    We would include a section describing our plans for distribution of reagents and tools described in the manuscript. All the vectors would be deposited in Addgene for distribution and all the zebrafish lines would be openly shared with the scientific community.

    __Reviewer #2: __

    1. The dual reporter system uses EGFP and mCherry to report the activities of two different CREs in the same animal. However, EGFP and mCherry have drastically different fluorescence properties which have not been measured particularly well in vivo and especially not in zebrafish. They have different maturation times (mCherry is much quicker). Both are quite stable in vivo, but mCherry is particularly stable in cell culture and in vivo, even resisting lysosomal degradation (EGFP does not - it is acid and protease sensitive) (Katayama et al., 2008; McWilliams et al., 2016). Often, promoter activity assays in zebrafish employ short lived "destabilized" FPs, such as destabilized GFP and destabilized dsRed. With stable FPs, false positives could be reported due to the fluorescent signal remaining for a long period of time after promoter activity has ceased. Replacing the traditional FPs with destabilized versions could be one way to improve the temporal resolution of this assay. This is probably not necessary to do in the present study but might be a worthy future direction.

    We would discuss these points in the possible limitations of our assay and will also endeavour to incorporate these suggestions in future versions of our assays.

    However, no matter which pair of FPs is chosen, there will be differences in signal intensity/brightness and decay rate. Thus, the FP swap experiments should be employed for any experiment claiming a temporal (Fig. 4) or quantitative (Fig. 5) difference between CRE activation or deactivation. If the EGFP/mCherry swap experiments show the same results, the confidence in the assay will be significantly bolstered.

    We estimate the proposed experiments to take about 4 months to allow for molecular cloning of the FP swapped constructs, injection into the "landing" strain, raising to sexual maturity (2.5 mo), screening for founders, and performing the imaging. These are the only two suggested experiments I would need to feel confident in the results and to recommend publication

    We appreciate the reviewer’s suggestion but would point out that we included dye-swaps for the PAX6-CREs described in Figure 3 in this manuscript. The dye-swap experiment for SBE2WT/SBE2Mut were described in our previous work published in Plos Genetics. However, to increase the confidence of the readers in our current system we would include the other suggested dye swaps in the revised version of our manuscript.

    __Reviewer #3: __

    **Major comments**

    1. First, given the importance of quality landing lines for the methodology, I would like to see more clarity and emphasis on validation of the Shh-SBE2 landing pad in the main text. Based on supplemental tables 1 and 2, this reviewer is somewhat unclear on whether there is one or three lines with Shh-SBE2 based landing pads (one site is mentioned in table 1, but table 3 mentions three F0 lines, and the text is ambiguous). The authors also state that the Shh-SBE2 landing pad is a single copy integration, but the data supporting this conclusion does not appear to be included (linker mediated PCR does not rule out other integrations).

    *We will provide a detailed description of the landing lines addressing all the concerns raised by the reviewer. *

    It would also be useful to have more clear numbers indicating the reproducibility of the expression pattern in F1 animals. Do 100% of F1 progeny from multiple crosses show the integration show the expression pattern in image 2 A? If there is variability how much, and how many fish were examined? This reviewer also wonders whether appropriate expression of Shh-SBE2 in this landing site is enough to call it neutral. For example, perhaps position effects might be observed with a different weaker CRE in this site? Better documentation will allow for more widespread and appropriate use of the landing pad.

    We will expand the description for the part of the pipeline the reviewer is referring to, providing the details of transgene segregation.

    Similar concerns apply to the integration of test constructs. To evaluate the practicality of the approach, it would be useful to have numbers reporting the frequency of recovering F1 individuals with PhiC mediated integration of the reporter into the desired landing site. It is also important to provide better documentation of the degree of reproducibility in expression patterns between F1 progeny. Numbers of embryos imaged and fraction with the indicated expression pattern are needed for all data in the main text. At minimum, gross expression patterns should be examined in at least 10 F1 larvae. If there is variability between individuals, some image documentation of this in supplementary data would be welcome.

    We will include the suggested information in the results and provide the supplementary data as suggested by the reviewer.

    **Minor comments:**

    i) For figure 1, it may be clearer to present generation of the landing pad lines and screening of CRES using these lines in separated figure panels (B) for generation of landing pads, and (C) for CRE analysis.

    We will modify figure 1 as suggested.

    ii) Landing pads that were less effective might also be moved out of figure 2, to the supplemental material to help improve clarity and to allow for focus on the tools with the most utility

    We will modify figure 2 as suggested.

    iii) Scale bars should be included in all images,

    This will be done for all the images

    iv) In some cases, image labeling somewhat obscures the relevant features

    We will rectify this in the revised version

    v) To help evaluate consistency, in all relevant figures (4, 5, sup fig 3 ect) the number of embryos examined should be included in the legend.

    We will modify the figure legends to include this information

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Bhatia addresses a longstanding need for rigorous methods to directly compare the effectiveness of cis-regulatory elements (CREs) during vertebrate embryogenesis. The manuscript describes a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and mutant CREs using live imaging in zebrafish embryos. The approach takes advantage of a predefined neutral docking site, and dual-CRE reporter cassette that can be integrated into this site using PhiC31. Using this method, the authors demonstrate subtle differences in the spatial and temporal dynamics of two shh CREs that have been previously reported to have similar domains of activity, and they demonstrate changes in CRE activity in embryos harboring a disease specific mutation in the SBE2 CRE.

    Major comments

    Overall this manuscript describes a valuable tool and key conclusions regarding its need and utility convincing. However, some additional documentation of methods and key reagents, and numbers would be of value.

    First, given the importance of quality landing lines for the methodology, I would like to see more clarity and emphasis on validation of the Shh-SBE2 landing pad in the main text. Based on supplemental tables 1 and 2, this reviewer is somewhat unclear on whether there is one or three lines with Shh-SBE2 based landing pads (one site is mentioned in table 1, but table 3 mentions three F0 lines, and the text is ambiguous). The authors also state that the Shh-SBE2 landing pad is a single copy integration, but the data supporting this conclusion does not appear to be included (linker mediated PCR does not rule out other integrations). It would also be useful to have more clear numbers indicating the reproducibility of the expression pattern in F1 animals. Do 100% of F1 progeny from multiple crosses show the integration show the expression pattern in image 2 A? If there is variability how much, and how many fish were examined? This reviewer also wonders whether appropriate expression of Shh-SBE2 in this landing site is enough to call it neutral. For example, perhaps position effects might be observed with a different weaker CRE in this site? Better documentation will allow for more widespread and appropriate use of the landing pad.

    Similar concerns apply to the integration of test constructs. To evaluate the practicality of the approach, it would be useful to have numbers reporting the frequency of recovering F1 individuals with PhiC mediated integration of the reporter into the desired landing site. It is also important to provide better documentation of the degree of reproducibility in expression patterns between F1 progeny. Numbers of embryos imaged and fraction with the indicated expression pattern are needed for all data in the main text. At minimum, gross expression patterns should be examined in at least 10 F1 larvae. If there is variability between individuals, some image documentation of this in supplementary data would be welcome.

    Presumably nearly all of this data has already been collected during validation of the tools and just isn't reported clearly, so these updates would not require significant time or cost.

    Minor comments:

    With respect to clarity, while the authors do an excellent job of explaining the rational for their system, the details of execution in the manuscript can be difficult to follow at times, below are minor suggestions to help the reader follow more easily.

    For figure 1, it may be clearer to present generation of the landing pad lines and screening of CRES using these lines in separated figure panels (B) for generation of landing pads, and (C) for CRE analysis.

    Landing pads that were less effective might also be moved out of figure 2, to the supplemental material to help improve clarity and to allow for focus on the tools with the most utility

    Scale bars should be included in all images,

    In some cases, image labeling somewhat obscures the relevant features

    To help evaluate consistency, in all relevant figures (4, 5, sup fig 3 ect) the number of embryos examined should be included in the legend.

    Significance

    This manuscript is significant as if provides useful tools for direct comparison of CRE activity in stable transgenic embryos, where two CREs are integrated into a single genomic location. The method offers an advance in efficiency and rigor compared to past approaches. As a zebrafish researcher, it is easy to recognize the value of having a transgenic line with a validated neutral landing site for transgene analysis, and having a well-designed construct for detailed in vivo comparison of CRE activity.

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    Referee #2

    Evidence, reproducibility and clarity

    This study presents a dual fluorescent protein (FP) reporter system to determine differential activities of Cis regulator elements (CREs) on transcription factor behavior in an in vivo setting. The strategy uses the PhiC31 system to ensure single copy insertion into a consistent genomic locus and is an important improvement over the authors' previous work using a similar system with random genomic integration and separated FP constructs. Because different genomic loci are more accessible than others, comparing the activities of randomly inserted CREs cannot be quantitative and requires generation and comparison of multiple lines for each CRE to validate. The bulk of this study is validation of the new specifically inserted, dual FP system including showing that including insulator sequences between the CREs of interest is necessary to prevent crosstalk. The last two figures demonstrate the utility of the system to interrogate spatial and temporal regulation of CRE variants and the quantitative expression levels of a mutant and WT CRE pair. This is an exciting tool with clear potential to uniquely compare CRE activities in vivo, and the results are clearly presented. However, given that the impact of this study is as a technical improvement over previous methods and that it is aimed to demonstrate the robustness and utility of the reporter system, additional controls are necessary to demonstrate that FP choice does not influence the temporal or quantitative readouts.

    The dual reporter system uses EGFP and mCherry to report the activities of two different CREs in the same animal. However, EGFP and mCherry have drastically different fluorescence properties which have not been measured particularly well in vivo and especially not in zebrafish. They have different maturation times (mCherry is much quicker). Both are quite stable in vivo, but mCherry is particularly stable in cell culture and in vivo, even resisting lysosomal degradation (EGFP does not - it is acid and protease sensitive) (Katayama et al., 2008; McWilliams et al., 2016). Often, promoter activity assays in zebrafish employ short lived "destabilized" FPs, such as destabilized GFP and destabilized dsRed. With stable FPs, false positives could be reported due to the fluorescent signal remaining for a long period of time after promoter activity has ceased. Replacing the traditional FPs with destabilized versions could be one way to improve the temporal resolution of this assay. This is probably not necessary to do in the present study but might be a worthy future direction. However, no matter which pair of FPs is chosen, there will be differences in signal intensity/brightness and decay rate. Thus, the FP swap experiments should be employed for any experiment claiming a temporal (Fig. 4) or quantitative (Fig. 5) difference between CRE activation or deactivation. If the EGFP/mCherry swap experiments show the same results, the confidence in the assay will be significantly bolstered.

    We estimate the proposed experiments to take about 4 months to allow for molecular cloning of the FP swapped constructs, injection into the "landing" strain, raising to sexual maturity (2.5 mo), screening for founders, and performing the imaging. These are the only two suggested experiments I would need to feel confident in the results and to recommend publication.

    Significance

    The impact of this study is as a technical improvement over previous methods and is aimed to demonstrate the robustness and utility of the reporter system.

    The manuscript is geared towards zebrafish experts with an interest in the imaging of intracellular and transcriptional processes.

    Our laboratory has expertise in zebrafish developmental genetics and live imaging of reporters.

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    Referee #1

    Evidence, reproducibility and clarity

    This is a technical manuscript that describes a new transgenic reporter system in zebrafish that is designed to simultaneously test the activity of two cis-regulatory elements (CREs) in the same living embryo. This is an extension of previous work from the authors that established methods to compare two CREs in transgenic zebrafish embryos (published in PLoS Genetics; DOI: 10.1371/journal.pgen.1005193). Here, to address the problem of position effects caused by random transgene integration, the authors have created a dual reporter transgene that can be integrated into a specific neutral site (using phiC31 recombination) in the zebrafish genome. Expression of different fluorescent proteins (EGFP and mCherry) are regulated by two CREs of interest in the zebrafish embryo, which allows visualization of the temporal and spatial activity of the CREs in real time during embryonic development. The authors propose this system could be used to directly compare wild-type and mutant CREs, and then provide several lives of evidence that establish proof-of-concept. Overall, the results are clearly presented, and the conclusions are convincing. The description of methods (including supplemental tables) is extensive, which will facilitate reproducibility. The manuscript is succinct, and describes a useful approach to characterize CREs. However, I have a few points for the authors to consider:

    Major points:

    1)It is convincingly shown that adding insulator elements (cHS4) reduces crosstalk between the two PAX6 CREs tested (Fig. 3). However, it is unclear if this approach will work for other CREs. This point should be discussed, and perhaps the authors could give some troubleshooting advice (e.g. adding more insulators or trying different insulator elements?).

    2)All CREs used in proof-of-concept experiments in this work have well known activities in zebrafish embryos. A new/uncharacterized CRE has not been tested yet using this system. It is unclear from the workflow (Fig. 1B) what happens if the CRE does not drive detectable levels of EGFP/mCherry. How does one determine whether lack of reporter expression is due to technical problem (with the transgene or phiC31 integration) or that the CRE is not active in zebrafish? Perhaps adding a PCR-based genotyping step could address this potential problem?

    3)Other limitations of the system should also be discussed. For example, the system appears to be useful for identifying variant CREs that result in a change (either loss or gain) of temporal or spatial activity, but it is not clear how subtle changes in expression level (either slightly increased or decreased) would be identified or quantified. Perhaps other approaches could be used in combination with this system to fully analyze mutant CRE activity. Another limitation is that this approach is only be applicable to CREs that are active in the first few days of zebrafish embryonic development.

    Minor points:

    1)Although it is discussed in the previous work published in PLoS Genetics, it is probably worth mentioning here why the gata2 minimal promoter was chosen for the reporter system.

    2)It would be helpful if the cSH4 element is briefly described (e.g. "insulator element") in Fig.1 legend.

    3)It is not clear from the manuscript whether the new reagents reported here-including dual reporter vectors and transgenic attB landing site zebrafish strains-will be made available to the scientific community, or how these reagents would be distributed.

    Significance

    This work introduces a new method to analyze cis-regulatory element (CRE) activity in vivo. By generating transgenic zebrafish with a neutral phiC31 landing site for reporter transgene integration, this work improves on previous methods by overcoming the problem of position effects caused by random transgene integration. This will be useful approach to characterize CREs during embryonic development, and variant CREs associated with human disease. This paper will be of interest to developmental biologists, and geneticists trying to understand CRE activity. I have expertise in zebrafish genetics, with extensive experience using Tol2 transgenesis, and some experience using phiC31 recombination. The described experimental approach here is straightforward, and will be easy to apply in labs with experience in zebrafish transgenesis, and imaging fluorescent protein expression in embryos.

  6. Version published to 10.1101/2020.09.13.290460v1 on bioRxiv