Specific photoreceptor cell fate pathways are differentially altered in NR2E3-associated diseases

  1. Izarbe Aísa-Marín
  2. Quirze Rovira
  3. Noelia Díaz
  4. Laura Calvo-López
  5. Juan M. Vaquerizas
  6. Gemma Marfany

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Abstract

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  1. Version published to 10.1016/j.nbd.2024.106463
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    Reply to the reviewers

    FULL REVISION

    The preprint of this article is uploaded in bioRxiv (doi:10.1101/2023.06.03.543550) (June 2023) and has been previously submitted and revised by peers in Review Commons. We attach the full Review, with a detailed answer to the Reviewers and a new revised version of the manuscript following the Reviewer’s comments and suggestions, adding new data, a new Supplementary figure, as well as a revision of the text and discussion. We are thankful to the reviewers for their helpful comments, which have further clarified some of aspects of our work and overall improved the quality of our manuscript.

    All the line’s numbering mentioned in the point by point answer to the reviewers refer to the converted pdf of the revised manuscript, including the main figures.

    Answers to Reviewer #1

    Aísa-Marín et al. present a detailed scRNAseq description of two Nr2e3 mouse models the authors had published in Neurobiology of Disease in 2020. These two mouse models do not mirror known pathogenic variants in human patients, but are useful animal models to understand disease mechanisms in NR2E3-linked retinal degenerations. The present follow-up study has been carefully done, the bioinformatic analysis is sound and selected pathways have been validated at protein level by immunohistochemistry and Western blot. The impact of this data on photoreceptor development and maintenance is somewhat decreased because previous data by the groups of Joseph Corbo, Connie Cepko, Jeremy Nathans, Anand Swaroop and others have already shown several years ago upregulation of cone-specific genes in a spontaneous Nr2e3 mutant mouse, the rd7 mouse. In these papers, hybrid cones both expressing cone- and rod-specific genes have been described and coined 'cods'. The authors are encouraged to use already defined terms in their paper. For instance, the concept of 'half differentiated' photoreceptors is unclear and must be rephrased. In general, existing literature is not always integrated adequately into the submitted work. The detailed remarks are listed below.

    We thank the reviewer for this suggestion to incorporate previous terminology in the field for the sake of clarity. Although it is true that previous authors have described hybrid photoreceptors, they have not quantified them, or compared their contribution to the different subpopulations of photoreceptors in different models of disease. For instance, there may be different types of hybrid photoreceptors, as they can be identified both within the cone and the rod populations, they show different characteristics and might perform different roles or else, be indicative of a pathological phenotype.

    Taking into account the reviewer’s suggestion, we have carefully revised our text and figures and the terms “intermediate” or “half differentiated photoreceptors” have been substituted by the already existing term “hybrid photoreceptors” of “incompletely differentiated”. We have also included some context in previous work regarding ‘cods’.

    See now lines 60, 146, 215, 271, 638-645 in the Discussion, Figure 7 legend and the Graphical Abstract for changes in terminology, and also sentences in lines 518-522, for reference to previous works.

    Reviewer #1

    One important question the authors must also address in their scRNAseq analysis is why the well described 'mixed' S- and M-opsin expressing cones do not seem detectable, are actually not even mentioned?

    We sincerely thank the reviewer for this comment, as it is a relevant piece of information that we missed in our previous analysis. Cones are usually classifed according to the type of opsin they express. In mouse, previous work has described cones expressing solely S- or M-opsins, but also cones that co-express both types of opsins. Indeed, we find these three types of cones, overlapping partially with our cone subpopulations, which are defined by a larger number of signature genes. As previously described, merging the results of the wild-type with the two mutants demonstrate that most cones co-express both opsins (roughly 46%), S- and M-opsins are expressed exclusively in around 13% cone cells each, and somewhat surprisingly around 28% of cones do not express any opsin (Fig. EVF6). However, the dissection of cone results by genotype is more informative, as the percentages vary according to the mutant genotype. In the mutants, the percentage of cones expressing no opsins is higher than in the wild-type at the expense of the cones that should express solely S-opsin (data in EVF5). Again reflecting the impact of Nr2e3 mutations on the differentiation of cones and reinforcing our main message.

    We have introduced several sentences in the text to reflect this results and refer to this new figure EVF6 (see lines 292-307 in the Results as well as 575-580 in the Discussion sections.

    Reviewer 1. Detailed comments:

    1. Graphical abstract: replace 'half differentiated' by incompletely differentiated or similar

    As detailed above, we have followed the suggestion of the reviewer and throroughly revised all the text and all Figures. “Half differentiated” and “intermediate photoreceptors” have been substituted by “incompletely differentiated” and “hybrid photoreceptors”, respectively.

    1. l.86: to my best knowledge, the shorter isoform has only been described at transcript level in humans, no evidence at protein level, please clarifiy. Please also state that the isoform lacking exon 8 is due to retention of intron 7.

    Following the suggestion of the reviewer, we have clarified that the short NR2E3 transcript isoform is due to retention of intron 7, both in human and mouse. We have also clarified that in human, the protein has been computationally predicted, whereas there are experimental evidences in mouse. (lines 94-104).

    1. l.89: lacks repressor and dimerization domains. Exon 8 is not coding all repressor and dimerization domains. The authors do not mention neither the D-box in the DBD that also contributes to dimerization, in addition to the LBD (von Alpen et al., Hum Mut, 2015). Furthermore, repressor domain should be presented in the context of the auto-repressed structure of NR2E3 (Zhu et al., Genes Dev, 2015).

    We agree with the reviewer that the original text was simplified. For the sake of clarity and following his/her suggestion we now include more context about the structure of NR2E3 and included the suggested references (lines 87-93).

    See also below the answer to the points 6, 9 and 13, which are also related.

    1. l.90: typo NR2E3

    The corresponding line is now on line 105 and the typo has been corrected.

    1. l.93-106: incorrect, please rewrite whole paragraph. There is only one single pathogenic variant leading to NR2E3-Gly56Arg-linked autosomal dominant retinitis pigmentosa, all other pathogenic variants are recessive and cause ESCS!

    Now these paragraph starts in line 105. We agree with the reviewer that so far only mutation Gly56Arg in *NR2E3 *is associated to autosomal dominant retinitis pigmentosa. however there are also some (even if few) *NR2E3 *mutations associated to autosomal recessive forms of RP, in addition to the most well known autosomal recessive mutations that cause ESCS. Here we provide a selection of reports supporting NR2E3 as causative of arRP (that are some more included in HMGD).

    In order to avoid further confusion to some readers, we also include these references in the new version (see lines 109-112).

    • Gerber, S. et al. The photoreceptor cell-specific nuclear receptor gene (PNR) accounts for retinitis pigmentosa in the Crypto-Jews from Portugal (Marranos), survivors from the Spanish Inquisition. Hum Genet 107, 276–284 (2000). https://doi.org/10.1007/s004390000350
    • Kannabiran, et al. Mutations in TULP1, NR2E3, and MFRP genes in Indian families with autosomal recessive retinitis pigmentosa. Mol. Vis. 2012; 18:1165-74..
    • Bocquet, B. et al. Homozygosity mapping in autosomal recessive retinitis pigmentosa families detects novel mutations. Mol Vis 19:2487-500.
    1. l.110: see comment above about dimerization.

    We have modified the sentence in the text accordingly, see now line 125.

    1. l.162: ok, but the main reason for restricting the analysis to photoreceptors should be the photoreceptor-specific expression of* Nr2e3* though...

    We have specified that Nr2e3 is solely expressed in photoreceptor cells (see line 176).

    1. l.165: please specify what is the percentage of rods with respect to all retinal cells

    The percentage of rods is around 77.7% of all cells (now on line 180). We value the suggestion, as it adds information to the reader. Therefore, the percentage of each main cell type is now included in Figure 1B.

    1. l.210: idem l.89

    We have modified the sentence in the text accordingly (now on lines 226-227).

    1. l.310: replace 'halfway'

    As specified in the answer to the first main point, we have throroughly revised the text and amended the references towards hybrid and incompletely differentiated photoreceptors.

    1. l.337: as expected? please detail

    We agree that the sentece was not clear. We have now clarified that our results agree with previous studies (see lines 370-371).

    1. l.388: discuss also crystallins in other RD models, v.g. Rpe65 ko mice

    We thank the reviewer for this suggestion and have included a brief description of the expression of crystallins in response to retinal stress in other RD models, and include the appropriate references (now on lines 422-428).

    1. l.469: idem l.89

    We have modified the sentence in the text accordingly (see lines 506-509).

    1. l.526: Please discuss increase in non-apoptotic cell death markers with respect to published data in the rd7 mouse (Venturini et al., Sci Rep, 2021)

    We have included published data in the *rd7 *mouse and discussed that multiple non-apoptotic cell death markers might be activated in response to NR2E3 disfunction (see lines 587-593).

    1. l.580: the proposed dominant negative effect is overtly speculative and not supported by any presented data, please remove.

    We have rewritten the sentence removing the dominant negative effect and referring exclusively to our results.

    Answers to Reviewer #2

    Aísa-Marín et al. present a detailed scRNAseq description of two Nr2e3 mouse models the authors had published in Neurobiology of Disease in 2020. These two mouse models do not mirror known pathogenic variants in human patients, but are useful animal models to understand disease mechanisms in NR2E3-linked retinal degenerations. The present follow-up study has been carefully done, the bioinformatic analysis is sound and selected pathways have been validated at protein level by immunohistochemistry and Western blot.

    Major comments:

    1. Overall, the conclusions of the study are well supported by the results. The findings provide valuable insights into retinal development and the pathogenesis of NR2E3-associated retinal dystrophies in an animal model, which need to be validated in humans. This limitation should be noted in the manuscript.

    Indeed, we agree that our work has used animal models, but further work in human-derived models (e.g. retinal organoids) should be performed to confirm these results. We have clarified this point in the Discussion section (see lines 649-650).

    Include an explanation in the text about how the specificity of the different signals detected by immunohistochemistry was assessed.

    The specificity of each antibody was assessed by introducing a negative control into the immunostaining procedure, wherein only the secondary antibody was used, as well as by comparison to the staining of the protein of interest in wild type or basal conditions reported in previous work from ours or other groups. This has been now specified in the corresponding Methods section (see lines 912-916).

    Explain the observed high variability in the percentage of cones, particularly between the two deltaE8 mutants (Figure 3C).

    We believe that the main characteristic of the Nr2e3-mutant retinas is that photoreceptors are not adequately differentiated and thus, affected photoreceptor subpopulations, in this case cones, degnerate and die. The pathogenic phenotype might be somewhat variable between animals from the same genotype (as it also happens in siblings carrying the same mutation). The deltaE8 mutants show a very different cone subpopulation pattern compared to wild-types and they clearly cluster with the other mutant retinas. For instance, cones of subpopulation cone4 might have all died.

    We take note of the question posed by the reviewer, and thus have included a graph of the absolute number of cones in each subcluster per retina and genotype, which may help the reader (see new Fig 3C, panel on the right).

    Explain why PARP-1 signals in Figure 6E are so thick and intense. Why this thick pattern is also present in the wild type retina?

    The IHC of mouse tissue sections using antibodies of mouse origin can result in background by secondary antibody binding to the endogenous Igs (as reported in Eng et al, 2016, https://doi.org/10.1093/protein/gzv054). The PARP-1 antibody is a mouse monoclonal antibody (Abcam, ab14459). The strong signal that we observe in the INL, IPL, and GL of the retina is typically found when using primary mouse antibodies in mouse tissues and corresponds to the reaction of the secondary anti-mouse antibodies binding to the endogenous IgGs in the blood vessels of the retina (we obtain the same result using the secondary antibody as a negative control, see answer to point 2).

    For the sake of clarity, we have clarified this background staining in the corresponding figure legend (lines 831-834).

    Reviewer #2

    Minor comments:

    1. Describe the abbreviations used in the text, such as PARP-1, MLKL, IRD, and VADC.

    A list of abbreviations has been included at the end of the main text (see lines 663-672).

    1. Improve the visibility of number 4 in Figure 3A and describe the meaning of the insert.

    Figure 3A has been amended accordingly

    1. Label the X-axis (cone subclusters) in Figure 3E.

    The X-axis is now correctly labelled in Figure 3E.

    1. Describe the meaning of the insert in Figure 4A.

    Figure 4A legend now contains the meaning of the insert.

    1. Indicate the relationship among inserts in Figure 4F.

    Figure 4F and legend have been modified to clarify the meaning of the insert.

    1. Use an arrow to indicate the higher expression of CSTB in the cone-rich invaginations in the mutant retinas (Figure 6A).

    The Figure 6A has been modified to include white arrowheads indicating the high expression of CSTB in the invaginations of the mutant retinas. This is also indicated in the corresponding Figure 6 Legend (lines 819-820.

    1. Revise the Y-axis values in Figure 6B, as they do not correspond to a percentage. Please, provide an explanation for the number of symbols displayed in this panel.

    The Y-axis was previously expressed on a per-unit basis. For the sake of clarity and following the reviewer’s suggestion, it has now been appropriately modified to percentage of CSTB colocalizing with opsins.

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    Referee #2

    Evidence, reproducibility and clarity

    Aísa-Marin et al. present a study on alterations in photoreceptor cell fate in NR2E3-associated diseases. The results convincingly reveal the presence of heterogeneous rod and cone populations in the mouse retina, as well as the existence of a novel cone pathway that results in hybrid cones characterized by the expression of genes associated with both cones and rods. Furthermore, the authors show that functional alteration of NR2E3 affects the expression of rod and cone signature genes, providing an interesting animal model to unveil the molecular mechanism underlying these retinal disorders.

    Major comments:

    1. Overall, the conclusions of the study are well supported by the results. The findings provide valuable insights into retinal development and the pathogenesis of NR2E3-associated retinal dystrophies in an animal model, which need to be validated in humans. This limitation should be noted in the manuscript.
    2. Include an explanation in the text about how the specificity of the different signals detected by immunohistochemistry was assessed.
    3. Explain the observed high variability in the percentage of cones, particularly between the two deltaE8 mutants (Figure 3C).
    4. Explain why PARP-1 signals in Figure 6E are so thick and intense. Why this thick pattern is also present in the wild type retina?

    Minor comments:

    1. Describe the abbreviations used in the text, such as PARP-1, MLKL, IRD, and VADC.
    2. Improve the visibility of number 4 in Figure 3A and describe the meaning of the insert.
    3. Label the X-axis (cone subclusters) in Figure 3E.
    4. Describe the meaning of the insert in Figure 4A.
    5. Indicate the relationship among inserts in Figure 4F.
    6. Use an arrow to indicate the higher expression of CSTB in the cone-rich invaginations in the mutant retinas (Figure 6A).
    7. Revise the Y-axis values in Figure 6B, as they do not correspond to a percentage. Please, provide an explanation for the number of symbols displayed in this panel.

    Significance

    While it is known that NR2E3, an orphan nuclear receptor, plays a role in photoreceptor fate and differentiation during retinal development, its precise biological function remains poorly characterized. Additionally, the mechanisms by which inherited functional alterations of NR2E3 lead to RP or ESCS are not well understood. To address these unresolved questions, the authors employ a comprehensive experimental approach, including scRNAseq, RT-PCR, and immunohistochemistry, using retinas from two NR2E3 mutant mice. The technical procedures are well executed, and the complex scRNAseq data are rigorously analyzed and presented in a clear manner. Overall, this is a careful and meticulous study, that provides the fundamental basis for further verification in humans.

    In my view, this research is highly relevant for the scientific community interested in the study of retinal development and inherited retinal dystrophies, specifically retinitis pigmentosa (RP) and enhanced S-cone syndrome (ESCS).

  7. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Aísa-Marín et al. present a detailed scRNAseq description of two Nr2e3 mouse models the authors had published in Neurobiology of Disease in 2020. These two mouse models do not mirror known pathogenic variants in human patients, but are useful animal models to understand disease mechanisms in NR2E3-linked retinal degenerations. The present follow-up study has been carefully done, the bioinformatic analysis is sound and selected pathways have been validated at protein level by immunohistochemistry and Western blot. The impact of this data on photoreceptor development and maintenance is somewhat decreased because previous data by the groups of Joseph Corbo, Connie Cepko, Jeremy Nathans, Anand Swaroop and others have already shown several years ago upregulation of cone-specific genes in a spontaneous Nr2e3 mutant mouse, the rd7 mouse. In these papers, hybrid cones both expressing cone- and rod-specific genes have been described and coined 'cods'. The authors are encouraged to use already defined terms in their paper. For instance, the concept of 'half differentiated' photoreceptors is unclear and must be rephrased. In general, existing literature is not always integrated adequately into the submitted work. The detailed remarks are listed below.

    One important question the authors must also address in their scRNAseq analysis is why the well described 'mixed' S- and M-opsin expressing cones do not seem detectable, are actually not even mentioned?

    Detailed comments:

    Graphical abstract: replace 'half differentiated' by incompletely differentiated or similar l.86: to my best knowledge, the shorter isoform has only been described at transcript level in humans, no evidence at protein level, please clarifiy. Please also state that the isoform lacking exon 8 is due to retention of intron 7.

    l.89: lacks repressor and dimerization domains. Exon 8 is not coding all repressor and dimerization domains. The authors do not mention neither the D-box in the DBD that also contributes to dimerization, in addition to the LBD (von Alpen et al., Hum Mut, 2015). Furthermore, repressor domain should be presented in the context of the auto-repressed structure of NR2E3 (Zhu et al., Genes Dev, 2015).

    l.90: typo NR2E3

    l.93-106: incorrect, please rewrite whole paragraph. There is only one single pathogenic variant leading to NR2E3-Gly56Arg-linked autosomal dominant retinitis pigmentosa, all other pathogenic variants are recessive and cause ESCS!

    l.110: see comment above about dimerization

    l.162: ok, but the main reason for restricting the analysis to photoreceptors should be the photoreceptor-specific expression of Nr2e3 though...

    l.165: please specify what is the percentage of rods with respect to all retinal cells

    l.210: idem l.89

    l.310: replace 'halfway'

    l.337: as expected ? please detail

    l.388: discuss also crystallins in other RD models, v.g. Rpe65 ko mice

    l.469: idem l.89

    l.526: Please discuss increase in non‑apoptotic cell death markers with respect to published data in the rd7 mouse (Venturini et al., Sci Rep, 2021)

    l.580: the proposed dominant negative effect is overtly speculative and not supported by any presented data, please remove.

    Significance

    Strength: first scRNAseq analysis in Nr2e3 mouse models, validation at protein level

    Limitations: descriptive of gene expression, no mechanims identified, previous literature not adequately incorporated or missing

    Audience: specialized, basic research

  8. Version published to 10.1101/2023.06.03.543550v1 on bioRxiv