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The revised manuscript is acceptable in the current form.
# PeerJ Staff Note - this decision was reviewed and approved by Keith Crandall, a PeerJ Section Editor covering this Section #
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The authors have significantly improved the clarity of the manuscript. I recommend to consider the manuscript for publication in PeerJ after resolving the concerns raised by the first reviewer.
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As suggested by both reviewers:
Please revise manuscript for clarity, resolve issue related to pooling of samples, results on ocular biometrics and enrichment analysis. Validating core gene expression changes using independent methods and provide rational of the experiments in result section as suggested.
Title: Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/choroid in chick model of form-deprivation myopia (#24645)
Giummarra L, et al., present an interesting manuscript investigating the mechanisms of form-deprivation myopia and recovery from FD myopia using chick model, and identified the involvement of mitochondrial metabolism, neurotransmission, structural pathways, complement cascade, and bile acid and bile salt metabolism pathways in retina/RPE/choroid. This manuscript is well written and structured. Authors employed genome-wide gene expression profiling and various analysis methods to investigate the role of different signaling pathways in myopia development. This study provides interesting insight into the role of retina/RPE/choroid in FD myopia model, but some questions need to be addressed for the consideration of publication.
1. One of the major concerns for this study is the pooling of samples, or pooling n=5 for each conditions to n=1 for Affymetrix chips. Pooling samples together from a couple of individuals in general is not preferred if researcher can obtain enough sample or RNA from each eye. Collecting enough RNA from each eye should not be a problem for this study since combined retina/RPE/choroid tissues are being collected, and more than 1 ug of RNA can be easily collected from each eye for microarray assay. Authors please give the reason of pooling samples together. Please also give explanation that n=1 for each condition works.
In addition, authors emphasized the similarity between retina/RPE/choroid and retina/RPE (McGlinn et al. study) (Line 153-154), as well as pooled and individual samples (Line 190-197). They also discussed the limitation of this study at the end of discussion. However, the difference between these situations should be discussed more. First, every tissue has unique anatomical properties and functions. For example, choroid is a very complex tissue which include large amount of vessels, endothelium cells and other cell types that are very different from retina and RPE. Retina also contains so many different cell types including neurons that may respond to FD or defocus differently with different period of treatment. While RPE in nature is epithelial cell. These tissues are so different and they may act differently in myopia development. Second, experiments using combined tissues are a kind of practice of pooling sample in nature. Third, pooling samples together from different animals can be problematic in many ways, and should be only considered when no enough sample is available under certain conditions (L190-197).
2. Figure 1 is very confusing from Introduction to Materials and Methods, as well as Results:
Line 69: Is Figure 1 the result of current study or from references? I checked references listed here but did not find anything related to Figure 1. Legend of Figure 1 indicated that it is curtesy of Egan, G. If this is true, then no figure is needed in this manuscript (just add reference), and no need to describe MRI in Materials and Methods. Please revise manuscript to make it clear.
If Figure 1 is the result of this study as described in Materials and Methods – MRI imaging section, then please describe MRI methods and results as appropriate.
Please label Figure 1, including retina, choroid, and sclera in both panels if this is your original research. It is very confusing that authors indicated 24 h of recovery after FD treatment in this study, but MRI figure (1B) was taken 72 h of recovery.
3. Results for ocular biometrics and Figure 2:
It is appreciated that authors collected extra data to compliment McGlinn et al. study, but these data should be plotted separately. The reason is that these data were collected from 7 days old chicks which is different from current study. Plot these data together can be misleading (Figure 2A & 2B).
I suggest plotting Figure 2B AL and VCD in separate bars. The reasons are: 1) it is hard to compare ALs when they are stacked on top of VCDs which ends at different levels. 2) Readers may misinterpreted black + gray bar as the axial length of the eye, and adding AL and VCD in one bar is not meaningful anyway.
4. Enrichment analysis for short-term FDM induction and Table 1
Summarize genes identified from McGlinn et al. study to 13 enriched pathways and Table 1 is not informative. It will be better to provide gene list for each identified pathway.
Minor points include
1. Line 80, line 135, line 156, line 163-164, etc.: Please be consistence with form-deprivation and form deprivation used in manuscript, as well as 6hrs, 72hrs, 6 hr, 24 hr; short term, short-term, long term, long-term;
2. Line 129: Is this study using 10 days FD treatment? Typo here for “following 7 days of translucent occlusion”?
3. Line 187: Typo “n=65”
Authors need to make clear sample number for each experiments
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The manuscript entitled “Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/choroid in chick model of formdeprivation myopia” by Giummarra et. al. has profiled transcriptome wide gene expression changes in during the induction and recovery from form-deprivation myopia (FDM) in chick. The GSEA analysis revealed important pathways in the pathophysiological process. Overall the manuscript is well executed with clear background knowledge and experimental approaches. However, the rationale/hypothesis were not well described in the result section for the experiment/analysis performed. The quality of this manuscript could be improved by addressing the following concerns. At the present stage of the manuscript, I suggest considering for publication in PeerJ following a revision.
1. I suggest validating core gene expression changes, at least one gene of each of the enriched pathways by an independent method (for instance, quantitative PCR method).
2. The title of a figure legend should describe the figure succinctly, not the experiment. I suggest using conclusions of the result as title of the result sections as well as Figure legend.
3. I suggest including rational of the experiments, preceding the actual experiment and conclusions in each “Result” sections.
4. Figure1; please move the MRI imaging method to the “Materials and Methods” section.
5. Figure 2 a, b, c, supplementary figure 1; include statistical significance test p and r value on the figures and wherever applicable on other figures.
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