Unique functions of two overlapping PAX6 retinal enhancers

  1. Kirsty Uttley
  2. Andrew S. Papanastasiou
  3. Manuela Lahne
  4. Jennifer M. Brisbane
  5. Ryan B. MacDonald
  6. Wendy A. Bickmore
  7. Shipra Bhatia

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Abstract

Enhancers play a critical role in development by precisely modulating spatial, temporal, and cell type-specific gene expression. Sequence variants in enhancers have been implicated in disease, however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development, and has a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive, redundant, or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers, HS5 and NRE. Using a unique combination of live imaging and single-cell RNA sequencing in dual enhancer-reporter zebrafish embryos, we find significant differences in the spatiotemporal activity of these enhancers, and show that HS5 and NRE are active in distinct cell types of the developing retina. Our results show that although overlapping, these enhancers have distinct activities in different cell types and therefore likely non-redundant functions. This work demonstrates that unique cell type-specific functions can be uncovered for apparently similar enhancers when investigated at high resolution in vivo.

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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 (Evidence, reproducibility and clarity (Required)):

    • Summary:*

    • In the article entitled "Unique functions of two overlapping PAX6 retinal enhancers", Uttley and coworkers characterize in detail the activity of two conserved human enhancers (i.e. NRE and HS5) previously reported to drive Pax6 expression to the neural retina. By integrating these enhancers in a PhiC31 landing site using a dual enhancer-reporter cassette, they generated a zebrafish stable line in which their activity can be followed by the expression of GFP (NRE) and mCherry (HS5). The authors show that although the enhancers have a partially overlapping activity at early stages (24hpf), later on (48 and 72hpf) they activity domains segregate: to stem cells and differentiated amacrine cells for NRE, and to proliferating progenitors and differentiated Müller glia cells for HS5. To this end they used two different approaches: a scRNA-seq analysis of sorted cells from the transgenic line and a immunofluorescent analysis employing cell specific markers. The authors conclude that their analysis allowed the identification of unique cell type-specific functions.*

    • Major comments:*

    • In general terms, the article is technically sound (please, see section B for an assessment of the significance of the findings). The methodology used and the data analysis are accurate. The work is well presented, the figures are clear, and the previous literature properly cited. My main concerns are the following:*

      1. A general concern on the main conclusion of the work "the identification of unique cell type-specific functions for these enhancers". This is in my opinion only partially addressed by the study, as the conclusions are limited due to the absence of genetic experiments: such as deleting the enhancers in their native genomic context (either in human organoids or the homologous sequence in animal models), or at least assessing the effect of mutating their sequence in transgenesis assays in zebrafish. I understand that these functional assays may be out of the scope of the current work, but then the text should be toned down (the word "function" is extensively used) to make clear that the authors mean just expression. I would suggest substituting the word by "activity" in many instances.*

    • The absence of further genetic experiments also limits the significance of the study (see section B).*

    We appreciate and agree with the reviewer’s concern and would substitute the word “function” with “activity” throughout the manuscript.

    2) Whereas the work in general is technically correct (particularly transgenic lines and scRNA-seq data are well described and presented), the co-expression analyses using cell-specific markers (figure 5) need to be improved. There are several issues here. First, the magnification shown is too low to appreciate the colocalization details in the figure. The panels should be replaced by others with higher magnification/resolution (see also minor comment on color-blind compatible images)

    • In addition, the selection of the markers is suboptimal. Although PCNA is a good general marker of the entire CMZ, it would be advisable to repeat the experiments using more specific markers of the stem cell niche (e.g. rx1, vsx2; Raymond et al 2006; BMC dev Biol) to better define the enhancers expression domain. In addition, HuC/D labels both RGCs and amacrines, and the colocalization could also be refined using amacrine specific markers (e.g. ptf1a : Jusuf & Harris 2009, Neural Dev).*

    In the revised version of the manuscript, we would:

    1. Provide higher magnification images as suggested by the reviewer
    2. Provide additional stainings and justification for our choice of markers used in these colocalizaion experiments Minor comments:
    • 1.- The work includes several figures (1, 2, 5, 6 and S1) showing colocalization experiments in which channels are shown in red and green. I would advise replacing the red channel with magenta (or the green with cyan) in order to make the figures accessible to readers with color-blindness. This also applies to the schematic representations in figure 6.*

    We will change the channel colours throughout the manuscript as suggested by the reviewers

    2.- It is unclear in the text/images whether the expression driven by the HS5 enhancer is exclusively restricted to temporal retina throughout development (By the way, this differential nasal vs temporal expression should also be included in the final scheme in Figure 6). Does this mean that the expression of Pax6 in proliferating progenitors and Müller glia cells in the nasal retina is not controlled by this enhancer? To which extent is Pax6 needed to maintain the identity of these cell types?

    We will modify the figures as suggested and also include more details of expression overlap with PAX6 expression in the text of the revised manuscript.

    3.- The following sentence in the Discussion "To the best of our knowledge, ours is the first report where the activities of developmental enhancers have been mapped in vivo at single-cell resolution to reveal distinct patterns of activity" should be removed/rephrased. I would argue that the activity of cis-regulatory regions associated to any developmental gene are genome-wide mapped at single cell resolution in each scATACseq experiment.

    We agree that scATAC-seq gives information about potentially active enhancers but it does not define the precise cell-types unless overlapped with expression data. Our method is aimed at ‘defining’ the precise cell-types where the enhancer is active and has the potential to be used to build high resolution maps of cell-type specific enhancer usage for loci with multiple enhancers driving a single gene. We will discuss this in detail in the revised version of the manuscript.

    4.- In the methods section:

    • (a) FACS experiments: Please provide a supplementary Figure to graphically account for all gating/sorting strategies.*
    • (b) ScRNA-seq analysis: Please provide the values of mean reads per cell and median genes per cell as obtained from Cell Ranger. This would be informative for others performing similar experiments*

    This will be included in the revised version of the manuscript.

    **Referees cross-commenting**

    • I agree with the comments by reviewer #2 on the FACsorting experiments, the description of the landing sites, and the limited significance of the results.*

    Reviewer #1 (Significance (Required)):

    • As described in the previous section, the technical quality of this work is high in general terms. The experiments presented are clear and the conclusions straightforward. In that sense, the study will be a useful reference for those interested in the regulatory logic of Pax6 during eye development, including mainly developmental biologists and human geneticists. This may be particularly the case if new variants can be associated with these enhancers in microphthalmic patients.*

    • The significance and novelty of the findings is however limited by several factors:*

    • a) First, although the level of detail described in this article was not achieved previously, the human enhancers NRE and HS5 (or their conserved homologous in other vertebrates) were previously reported to drive Pax6 expression to the neural retina in transgenesis assays.(Kammandel et al 1999; Marquardt et al 2001; McBride et al 2011; Ravi et al 2013; Kim et al 2017).*

    We agree that the enhancers we describe in this study have been studied before. However, we would like to argue that ours is the first study where we define precise cell-types for the activity of these enhancers. We will revise the discussion to strengthen this argument.

    b) As mentioned in the previous section, the transgenesis assays are not complemented with genetic experiments. The function of the enhancers on retina differentiation and cell fate determination could have been investigated either by deleting them (or their homologous in different species) in their native context, or by exploring their regulatory grammar introducing point mutations or micro-deletions in transgenesis assays.

    We agree that the suggested experiments would be useful for unambiguously establishing the functions of these enhancers and we will discuss these prospects in the revised version of the manuscript.

    c) For reasons not explained in the text, the analysis focuses only in two of the many cis-regulatory regions controlling Pax6 expression in the retina (Lima Cunha et al 2019, Genes). In the absence of a more comprehensive analysis is difficult assessing the relevance of the findings here described.

    We agree that other enhancers for the PAX6 locus should be investigated using similar analysis pipeline to build a complete picture of the enhancer mediated regulation of PAX6. We will discuss this in the revised version of the manuscript.

    d) Finally, from a very general methodological point of view, the approach of using scRNA-seq to investigate enhance activity at a single-cell level is valid and original. However, it is unclear to which extent will be a useful method for many studies, particularly if the activity of endogenous elements is being assessed. In such cases, available scATAC-seq data will provide genome-wide information on the activity of any cis-regulatory element with cell resolution with no need for transgenesis assays and sorting experiments.

    • We thank the reviewer for recognising the novelty of the approach we describe in this manuscript. We will discuss the merits and demerits of our method with scATAC-seq experiments in the revised version of the manuscript.*

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this work, Uttley et al fine characterize two previously described Pax6 retinal enhancers (NRE and HS5) by combining QSTARZ transgénesis method in zebrafish (allowing to produce site-specific integrations of a dual enhancer reporter cassette), scRNAseq and co-immunostaining with specific markers for different retinal cell populations.

    • The work is experimentally very well performed and well presented and only minor considerations are raised below:*
      • Authors observe that a large fraction Of FACs sorted cells do not display expression of mCherry or EGFP RNAm in their scRNAseq analysis and attribute this to read dropout in the scRNAseq data and/ or to false-positive FAC cell selection. However, a third possibility exists: n fact due to the high stability of the EGFP and mCherry reporters cells or their progeny could maintain relatively high levels of these reporters even after transcriptional downregulation. Accordingly, the two reporters are strongly expressed in retinal precursor at early stages (24hpf). Thus, in my opinion, it is possible that some cells expressing these reporters retained significant EGFP/mCherry protein levels at 48hpf. Could the authors comment on this? Besides, authors could provide the FACsorting data to give an idea of whether only highly EGFP/ mCherry expressing cells were selected or whether also the low or mild expressing ones were included in the scRNAseq analysis. Finally, a combination of HCR/FSH and GFP//mCherry immunostaining could be used to assess whether a discrepancy in the protein vs mRNA distribution of the reporters exists.*
      • The authors could provide the information on the landing site used for the QSTARZ transgene integration. While from their previous publication (Bhatia et al 2021) I assume it is the chr6 landing site, it would be worth having this information in the manuscript, as well as a genotyping validation of the correct integration.*

    We will address these points and provide relevant additional data where needed in the revised version of the manuscript.

    **Referees cross-commenting**

    • I agree with all the points raised by reviewer 1. Particularly I also find that scATACseq experiments already allow testing, to some extent, enhancer activity at cellular level.*

    • Reviewer #2 (Significance (Required)):*

    • From the biological point of view the work provides only an incremental advance in our understanding of the functions of the HS5 and NRE PAX6 enhancers and of PAX6 regulation in the retina. In fact, unraveling the precise contribution of these enhancers to Pax6 retinal expression and the trans-regulatory code controlling their activity would require complex genetic experiments and would fall out of the scope of this work, requiring an extensive amount of work which could not be addressed in the short term. Thus, this work should be regarded as a methodological resource, with its main strength consisting of the use scRNAseq to fine-characterize enhancer activity.*

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    Referee #2

    Evidence, reproducibility and clarity

    In this work, Uttley et al fine characterize two previously described Pax6 retinal enhancers (NRE and HS5) by combining QSTARZ transgénesis method in zebrafish (allowing to produce site-specific integrations of a dual enhancer reporter cassette), scRNAseq and co-immunostaining with specific markers for different retinal cell populations.

    The work is experimentally very well performed and well presented and only minor considerations are raised below:

    •   Authors observe that a large fraction Of FACs sorted cells do not display expression of mCherry or EGFP RNAm in their scRNAseq analysis and attribute this to read dropout in the scRNAseq data and/ or to false-positive FAC cell selection. However, a third possibility exists: n fact due to the high stability of the EGFP and mCherry reporters cells or their progeny could maintain relatively high levels of these reporters even after transcriptional downregulation. Accordingly, the two reporters are strongly expressed in retinal precursor at early stages (24hpf). Thus, in my opinion, it is possible that some cells expressing these reporters retained significant  EGFP/mCherry protein levels at 48hpf. Could the authors comment on this? Besides, authors could provide the FACsorting data to give an idea of whether only highly EGFP/ mCherry expressing cells were selected or whether also the low or mild expressing ones were included in the scRNAseq analysis. Finally, a combination of HCR/FSH and GFP//mCherry immunostaining could be used to assess whether a discrepancy in the protein vs mRNA distribution of the reporters exists.

    •   The authors could provide the information on the landing site used for the QSTARZ transgene integration. While from their previous publication (Bhatia et al 2021) I assume it is the chr6 landing site, it would be worth having this information in the manuscript, as well as a genotyping validation of the correct integration.

    Referees cross-commenting I agree with all the points raised by reviewer 1. Particularly I also find that scATACseq experiments already allow testing, to some extent, enhancer activity at cellular level.

    Significance

    From the biological point of view the work provides only an incremental advance in our understanding of the functions of the HS5 and NRE PAX6 enhancers and of PAX6 regulation in the retina. In fact, unraveling the precise contribution of these enhancers to Pax6 retinal expression and the trans-regulatory code controlling their activity would require complex genetic experiments and would fall out of the scope of this work, requiring an extensive amount of work which could not be addressed in the short term. Thus, this work should be regarded as a methodological resource, with its main strength consisting of the use scRNAseq to fine-characterize enhancer activity.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In the article entitled "Unique functions of two overlapping PAX6 retinal enhancers", Uttley and coworkers characterize in detail the activity of two conserved human enhancers (i.e. NRE and HS5) previously reported to drive Pax6 expression to the neural retina. By integrating these enhancers in a PhiC31 landing site using a dual enhancer-reporter cassette, they generated a zebrafish stable line in which their activity can be followed by the expression of GFP (NRE) and mCherry (HS5). The authors show that although the enhancers have a partially overlapping activity at early stages (24hpf), later on (48 and 72hpf) they activity domains segregate: to stem cells and differentiated amacrine cells for NRE, and to proliferating progenitors and differentiated Müller glia cells for HS5. To this end they used two different approaches: a scRNA-seq analysis of sorted cells from the transgenic line and a immunofluorescent analysis employing cell specific markers. The authors conclude that their analysis allowed the identification of unique cell type-specific functions.

    Major comments:

    In general terms, the article is technically sound (please, see section B for an assessment of the significance of the findings). The methodology used and the data analysis are accurate. The work is well presented, the figures are clear, and the previous literature properly cited. My main concerns are the following:

    1. A general concern on the main conclusion of the work "the identification of unique cell type-specific functions for these enhancers". This is in my opinion only partially addressed by the study, as the conclusions are limited due to the absence of genetic experiments: such as deleting the enhancers in their native genomic context (either in human organoids or the homologous sequence in animal models), or at least assessing the effect of mutating their sequence in transgenesis assays in zebrafish. I understand that these functional assays may be out of the scope of the current work, but then the text should be toned down (the word "function" is extensively used) to make clear that the authors mean just expression. I would suggest substituting the word by "activity" in many instances. The absence of further genetic experiments also limits the significance of the study (see section B).
    2. Whereas the work in general is technically correct (particularly transgenic lines and scRNA-seq data are well described and presented), the co-expression analyses using cell-specific markers (figure 5) need to be improved. There are several issues here. First, the magnification shown is too low to appreciate the colocalization details in the figure. The panels should be replaced by others with higher magnification/resolution (see also minor comment on color-blind compatible images) In addition, the selection of the markers is suboptimal. Although PCNA is a good general marker of the entire CMZ, it would be advisable to repeat the experiments using more specific markers of the stem cell niche (e.g. rx1, vsx2; Raymond et al 2006; BMC dev Biol) to better define the enhancers expression domain. In addition, HuC/D labels both RGCs and amacrines, and the colocalization could also be refined using amacrine specific markers (e.g. ptf1a : Jusuf & Harris 2009, Neural Dev).

    Minor comments:

    1. The work includes several figures (1, 2, 5, 6 and S1) showing colocalization experiments in which channels are shown in red and green. I would advise replacing the red channel with magenta (or the green with cyan) in order to make the figures accessible to readers with color-blindness. This also applies to the schematic representations in figure 6.
    2. It is unclear in the text/images whether the expression driven by the HS5 enhancer is exclusively restricted to temporal retina throughout development (By the way, this differential nasal vs temporal expression should also be included in the final scheme in Figure 6). Does this mean that the expression of Pax6 in proliferating progenitors and Müller glia cells in the nasal retina is not controlled by this enhancer? To which extent is Pax6 needed to maintain the identity of these cell types?
    3. The following sentence in the Discussion "To the best of our knowledge, ours is the first report where the activities of developmental enhancers have been mapped in vivo at single-cell resolution to reveal distinct patterns of activity" should be removed/rephrased. I would argue that the activity of cis-regulatory regions associated to any developmental gene are genome-wide mapped at single cell resolution in each scATACseq experiment.
    4. In the methods section:
      • (a) FACS experiments: Please provide a supplementary Figure to graphically account for all gating/sorting strategies.
      • (b) ScRNA-seq analysis: Please provide the values of mean reads per cell and median genes per cell as obtained from Cell Ranger. This would be informative for others performing similar experiments

    Referees cross-commenting I agree with the comments by reviewer #2 on the FACsorting experiments, the description of the landing sites, and the limited significance of the results.

    Significance

    As described in the previous section, the technical quality of this work is high in general terms. The experiments presented are clear and the conclusions straightforward. In that sense, the study will be a useful reference for those interested in the regulatory logic of Pax6 during eye development, including mainly developmental biologists and human geneticists. This may be particularly the case if new variants can be associated with these enhancers in microphthalmic patients.

    The significance and novelty of the findings is however limited by several factors:

    • a) First, although the level of detail described in this article was not achieved previously, the human enhancers NRE and HS5 (or their conserved homologous in other vertebrates) were previously reported to drive Pax6 expression to the neural retina in transgenesis assays.(Kammandel et al 1999; Marquardt et al 2001; McBride et al 2011; Ravi et al 2013; Kim et al 2017).
    • b) As mentioned in the previous section, the transgenesis assays are not complemented with genetic experiments. The function of the enhancers on retina differentiation and cell fate determination could have been investigated either by deleting them (or their homologous in different species) in their native context, or by exploring their regulatory grammar introducing point mutations or micro-deletions in transgenesis assays.
    • c) For reasons not explained in the text, the analysis focuses only in two of the many cis-regulatory regions controlling Pax6 expression in the retina (Lima Cunha et al 2019, Genes). In the absence of a more comprehensive analysis is difficult assessing the relevance of the findings here described.
    • d) Finally, from a very general methodological point of view, the approach of using scRNA-seq to investigate enhance activity at a single-cell level is valid and original. However, it is unclear to which extent will be a useful method for many studies, particularly if the activity of endogenous elements is being assessed. In such cases, available scATAC-seq data will provide genome-wide information on the activity of any cis-regulatory element with cell resolution with no need for transgenesis assays and sorting experiments.
  4. Version published to 10.1101/2022.11.25.517987v1 on bioRxiv