Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reports the identification of the genetic basis for a mutation that leads to retinal developmental defects particularly in the ciliary marginal zone.  Overall the manuscript is well-written and the experiments are generally appropriate for detailing the effects of this mutation.  There are a couple of places where the study could be improved a little or additional details reported, but in general the study makes a solid case for the effects of the mutation on the stem cells at the retinal margin.  Unfortunately, the nmz mutation is in the same gene as another published study from a decade ago which comes to nearly the same conclusions, greatly dampening the significance of the findings in this paper.  That said, it does help to validate the impact of this gene on neural progenitors and is worthy of note.

Major comments:

• The Abstract, Introduction and Methods sections are very well written with clear information presented well.
• In the results (for example around Figure 2) it is notable that the RPE is somewhat thicker in this mutant, particularly around the CMZ. This seems similar to many of the CMZ mutants reported in papers like Wehman et al 2005.  While not needing extensive comments, it seems worth noting as it may help with understanding connections between retinal degenerative conditions and RPE.
• The electron micrographs shown in Figure 3 are rather hard to see and interpret. Would it be possible to provide some additional information in the legend and perhaps directly on the micrographs to aid in identification (e.g. I presume the darker spots are pigment granules in the RPE, but it isn’t indicated).  If there is any way to have images with more contrast it would be helpful!  I’m not entirely sure of whether the arrow in the WT images really reflects a cleavage furrow (again there is little guidance about what we are seeing and the images have very low contrast).  Lastly, the images seem to be just 2 magnifications of one section each from a control and a mutant.  Given that this is trying to make a claim about cell death, it would be nice to have a sense of how often such nuclei (again VERY hard to see in the micrographs as presented) were seen and it might be more helpful to see either additional micrographs from other fish or some other sense of consistency of this finding.  (In terms of cell death, while not necessary it would be relatively simple to do a TUNEL or other cell-death label that could provide a more holistic view of death across the retina)
• Line 262 and Figure 4: I think it is a little challenging to say that two cell populations were labeled given this experiment.  If I understand the timeline correctly, BrdU should have labeled all of the CMZ stem/progenitor cells from 3-4dpf and then there was a 1 day chase, where those cells could continue proliferating and/or initiate differentiation.  One of the challenges in interpreting this is that there is just one sample WT micrograph shown and while that micrograph shows a pattern kind of like a CMZ labeling (the labeled zone 2) along with some of the cells that have now exited the CMZ (the labeled zone 1) that pattern does not appear as clearly in the ventral retina.  It would be helpful to have some rough quantification of how often the clear separation was observed versus a single patch, since that is one of the more important claims about what is happening in the CMZ (or perhaps some other metric that would allow a semi-quantitative comparison with the mutant).  In terms of the mutant micrograph, there is additional BrdU labeling within the central retina (looks like perhaps particularly in the RGC layer and around the nerve).  Whether this represents merely a developmental delay in the final differentiating divisions of the eye or a persistent maintenance of some central proliferation (or maybe proliferation of microglia or other phagocytic cells?) seems worth some note.
• The importance of the CPP experiment seems muted since the authors claim that the genetic background of the nmz fish doesn’t give a response leading to the study only happening with the m836 line, and it is only done in heterozygotes (since the homozygotes are lethal), with no finding of an effect.
• Similarly, the OKR results seem to add confusion rather than clarity, as there appears to be more background effects or experimental effects than genotypic effects. It isn’t clear that this adds substantially to the findings of the paper rather than reflecting some other variables.  If a key point is trying to be made about the vision of heterozygotes it might be worth including a little more about heterozygotes in either the morphological or BrdU analysis to show whether there is a potential incomplete dominance or anything (though the mutation is described as recessive so likely not).
• The transcriptomic analysis happens on whole larvae rather than on isolated retina or neural specific tissue. While understandable from a technical perspective, the authors should be careful in the interpretation of the results.  While dlb (as one example) is a CMZ gene, it is also expressed in many other tissues and from a sample of whole larvae the changes in expression in the CMZ itself are going to represent a tiny fraction of the change across the body, so the interpretation needs to be softened a bit.  The authors make claims that imply changes in the CMZ itself (for example Lines 501-503 of the conclusions) but the analysis done in this paper does not show that tp53 or any of the other genes listed are changed in the CMZ itself, rather across the whole fish.  Also there is no validation of the transcriptomic analysis, such as with an in situ of a small number of the genes that might be able to both show up/down regulation of those genes but also provide the spatial analysis that could strengthen the argument (while not an absolute requirement, this would provide a great deal more evidence for a little work).
• The discussion feels a little long, particularly in going through an extensive pathway of tp53 though whether any of those specific genes are actually in CMZ is not clear.
• Although the discussion talks a little about the Hu paper that found similar effects from a separate ddb1 mutation, the discussion feels like it doesn’t fully disclose the full work on the CMZ in that paper. For example in Lines 405-408, the authors talk about the Hu paper finding genomic instability and cell cycle arrest and apoptosis in the dopaminergic system, which is part of that paper, but Hu also examines explicitly the CMZ of the retina (showing ddb1 expression there, loss of proliferation in the CMZ in mutants, increased apoptosis in the CMZ, etc.).  While I know those findings mute some of the impact of this study it does seem important to fully discuss them and connect with the comparable findings in the present study rather than to make claims that the prior study only looked at things separate from this study.

 

 

 

Minor comments:

• Line 236: “mutants have much fewer” should either be “many fewer” or just “fewer”.
• Line 260: The description of the timeline for the BrdU experiment leaves out how long the chase period was (I think it was 24 hours after the washout?).
• In Table 1, I’m not sure that Stat is a helpful label for the third column. Is that fold-change?  It would be good to know what that number represents.
• In line 363 a reference is made to “genes reported by others to be expressed in the CMZ” but there isn’t a reference there or in the table for those reports and some sort of reference (either in general or for each specific gene) should be included.
• Line 441: I’m not sure the resolution of the micrographs shown in Figure 4 really allow the claim that it is the peripheral-most part of the CMZ.  Several studies have worked to characterize the compartments of the CMZ (e.g. Raymond et al. 2006) and without markers and improved resolution I’m not sure that this data supports a fine distinction about subcompartments of the CMZ.
• Similarly in 448-452, I’m not sure the second compartment is “undoubtably” the amplifying cluster (as some of the BrdU label would include transit amplifying cells that then differentiate and are pushed out). Without markers this is a difficult argument to make.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Darland et al. describes phenotypic changes in zebrafish mutants with a partial deletion of the ddb1 gene, specifically reporting reduced eye size despite unchanged body length, along with decreased cell numbers in the ciliary marginal zone (CMZ) and a potential link to cell death. However, similar findings regarding ddb1-deficient zebrafish mutants were previously reported in a 2015 publication, and there appears to be substantial overlap between that study and the current manuscript.
Therefore, the novelty of the present work is limited, and the authors are encouraged to clearly delineate how their findings differ from or extend prior research.

 

1. It is recommended that the Results section be organized with subheadings according to the type of experiment. This would improve clarity and allow readers to more easily follow the progression of the findings.

 

2. Figure 1　 The authors may consider including the graph depicting body length and eye size alongside the zebrafish images in the main text. This would provide a more comprehensive and quantitative representation of the phenotype.

 

3. Figure 2.　 The clarity of the magnified images could be improved, as they appear somewhat blurred. Providing sharper or higher-resolution images would enhance the visual quality and support more precise interpretation of the data. All figures should be clearly labeled with appropriate figure numbers to ensure ease of reference. Additionally, the unit “μm” in the scale bars should be correctly not “μM”

 

 

4. Figure 4　 In the BrdU incorporation experiment, the authors classify BrdU-positive cells in wild-type (WT) samples into two groups (label 1 and label 2). However, in the ventral region of the retina in WT specimens, cells corresponding to label 1 do not appear to be present. While the manuscript states that at least two distinct populations of labeled cells were observed, it is unclear whether this result was consistently obtained across all samples. Clarification on the reproducibility and consistency of this observation would be helpful.

 

5. Figure 6A

While the photographed individual effectively highlights the phenotypic difference from the wild-type, the inclusion of a graph showing average body length and eye size across genetic groups would provide stronger quantitative support and improve the clarity of the comparison.

 

6. The unit of length is incorrectly written as “µM” in several places and should be corrected to “µm” to accurately represent micrometers.
   
   
7. on page 3, the temperature is listed as 82°F; this should be converted to degrees Celsius (°C) for consistency with standard scientific reporting.

 

8. The term “Brdu” in Figure 4 (page 8, line 1) should be revised to “BrdU” to ensure accurate and standardized nomenclature.

                        

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made several important modifications to the manuscript and also provided clarity about why certain types of experiments are beyond their capacity to add to the manuscript.  The softening of some conclusions, the movement of some of the behavioral data to the supplemental section and the addition of a few other examples in the supplement I think make this appropriate for publication.  (And I completely understand about limited resources!)  Just a few minor comments to be worked out between the authors and the editor:

• On lines 352 and 354 it would be helpful to write out the acronyms for OKR and CPP (though they appear in the methods few people read that and it would be helpful to have in the body).
• In the added figures for Supplemental Figure 2, are both images for the high and low all from wild-type larvae as the legend states? The authors note an apoptotic cell in S2D (which the legend says is wild-type), while the text of the paper (line 266) indicates it is mutant.  I suspect the legend for Supp Fig 2 needs to be corrected to indicate it is examples from both wild-type and nmz
• The formatting of the large data table in the supplemental section is a little awkward as put in as several of the minus signs seem to have become detatched from the numbers (appearing above them?). Rather than including as a text table (which extending across multiple pages makes it hard to recall which column is which) I wonder if it would be better to include as a separate spreadsheet with the image supplemental figures as a separate supplemental file.
• The discussion still feels longer than necessary with the first page (Lines 419-468) entirely background with no direct connections to the literature. This is more succinctly already handled in the last paragraph of the introduction (Line 71-84).  I’m not going to insist on a change here, but there seems the most important pieces could be better integrated with results and things that are more peripheral summarized more succinctly.
• Details of transcriptomics data is not my specialty, but I have never seen a paper that focuses on the Wald statistic over the fold-change. My understanding is that the Wald statistic is a transformation used in doing the statistical test, but that fold change is still the key relevant number that should be presented for each gene.  Again, I’m not going to insist on this, but papers I can find on the math (e.g. Chen et al 2011) suggest that the key measure is still LogFC and most of the plots people do are based on log2 fold change, so I would strongly recommend changing the table in the text to focus on L2FC rather than the Stat column.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Regarding the numbering of figures, in the case of Figure 1, I believe it may be acceptable to label the panels simply as A, B, etc., rather than using 1A, 1B, and so on. The number could be omitted.

In Figure 3, the panels are arranged from left to right as B → A → C. However, it may be more appropriate to reorder them as A → B → C for better logical flow.

In the upper part of Figure 8, the three panels are not labeled with figure letters. To ensure clarity, all panels should be clearly marked. Additionally, the scale bar in the top-left panel should be repositioned so that it does not overlap with the cellular structures.

Author Response

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Author Response File: Author Response.docx