SBDSR126T rescues survival of sbds−/− zebrafish in a dose-dependent manner independently of Tp53

We created a transgenic line expressing the disease-associated mutation SBDSR126T in the sbds KO zebrafish background. We demonstrated that amount of SBDSR126T is important for development and tp53 pathway activation. Tp53M214K did not rescue neutropenia or survival in the sbds-null zebrafish.

We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): In this study, the authors set out to determine whether transgenic expression of the human SBDS protein carrying a missense SBDS R126T mutation genetically complements sbds null zebrafish.Based on their studies, the authors claim that the R126T mutation is hypomorphic and that the ability to rescue the development of sbds null fish is dose dependent.The sbds null fish carrying the SBDS R126T transgene exhibit neutropenia and suppression of female sex differentiation, which the authors suggest is a consequence of tp53 activation.Unfortunately, I have some major concerns with the work as it is currently presented.
The novelty of the work is unclear based on previously published work on the SBDS R126T mutant allele in mice/cell lines/in vitro (Zhang et al 2006;Tourlakis et al 2015;Calamita 2017;Finch et al 2011) showing that this is indeed a hypomorphic allele.The current study does not seem to significantly advance understanding of how the R126T missense mutation impairs SBDS function.
I also have concerns that a key control experiment seems to be lacking.I may have misinterpreted it due to lack of clarity in the labeling/annotation of the mutant proteins/genes, but it is essential to test whether the WT human SBDS protein fully complements the sbds null zebrafish mutant to support the claim that the "SBDS R126T mutant is hypomorphic".For me, the conclusion "Our results show that SBDS R126T is hypomorphic" is not currently supported by the data.However, this aside, we already know from previous studies and from the human genetics that the SBDS R126T missense mutation is indeed hypomorphic andimpairs SBDS function compared to WT.
To validate the conclusion that tp53 activation is causing the neutropenia and suppression of sex differentiation in the SBDS R126T fish, it is necessary to remove tp53 genetically.
Additional specific points 1.No page or line numbers makes it difficult to comment on text 2. "Most mutations result in a premature termination codon".This is not true-the donor splice site mutation results in low level expression of the full-length protein.
3. In the discussion: "Most mutations result in a premature termination codon and hypomorphic protein"-the term hypomorphic indicates impaired gene function 4. In the abstract: "However, its higher expression" Higher expression of what?Please clarify 5.In the first paragraph, it is difficult to understand exactly what has been done.Initially WT zebrafish sbds is expressed in the sbds null background, but then I understand that the authors have expressed the human SBDS R126T mutant.Is that correct?This needs to be stated explicitly.This begs the question whether human WT SBDS can fully rescue the sbds null mutant zebrafish?Clarity of the text is hampered by inconsistent use/explanation of abbreviations used in labeling the figures and as written in the text: Eg hSBDS/hSBDSR126T/SBDSR126T/sbds/R126T/sbdsnu132/-132/Mut/WT_020 Please keep consistent and explain the nomenclature.6. ubi promoter: it would be helpful if the text in the discussion is moved to the main results section to provide more explanation for the general reader on the nature of the ubi promoter."via the ubi promoter which drives constitutive transgene expression during all developmental stages and adult organs.(Mosimann et al. 2011) " 7. Figure 1A: what is cmlc2?Please explain 8. Figure 2A: why was the time point of 1 yr chosen?14. "our data show that activation of tp53 pathways involving cdkn1a may account for the neutropenia" Does removal of tp53 rescue the neutropenia phenotype?15.SBDS physically interacts with the GTPase Elongation Factor Like 1 (EFL1) to release the Eukaryotic Initiating Factor 6 (EIF6) from the cytoplasmic pre-60S ribosomal subunit.This release facilitates the assembly of the mature 80S ribosome.Reference should be included 16. "These patients present with severe hematological phenotypes.(Zhang et al. 2006b)" What is the evidence for this statement?It is not provided in the cited reference.
Reviewer #2 (Comments to the Authors (Required)): This study explores the role of co-occurring mutations in disease pathophysiology.This manuscript has established a zebrafish transgenic model system to investigate the effects of a missense mutation SBDSR126T in a ribosomopathy, Shwachman-Diamond syndrome (SDS).This study is conceptually like the one performed by Zhang S et al, 2006a.However, embryonic lethality was a huge limitation in the Zhang S et al study.To overcome this limitation, the authors generated a transgenic line introducing a transgene carrying the SDBSR126T mutation in a sbds null background.This methodology allowed the authors to manipulate the levels of SBDSR126T in the sbds null mutants and establish that higher levels of SBDSR126T are critical for organism survival and development when present in a sbds null background.The authors perform several techniques, such as protein estimation by western blotting, qPCR for mRNA quantification, sudan black staining for neutrophil counts, and polysome profiling, to come to their conclusion.However, consistency across the article, an adequate explanation of results, and discussion could be much better.Please refer to the following points.Using a transgene-independent technique to assess copy numbers will be critical, as transgenes may get silenced after the F1 generation.It is more important for this study as it involved 1x and 2x copy of transgenes to acheive the conclusion of the study.4. Introduction, Paragraph 3, line 8 -Please rewrite "SbdsR126T/R126 littermates" to "SbdsR126T/R126T littermates".5. Results, Paragraph 3, line 2 -Please rewrite "Tg(ubi:SBDSR126T:pA) sbds+/-" to "Tg(ubi:SBDSR126T:pA);sbds+/-". 6. 11. Figure 2I -The authors state that they observed a decrease in rpl11 and rpl5 protein levels and accumulation of Eif6 protein in 10dpf sbds-/-larvae from their previous study.In this study, the authors evaluated the levels of these proteins by western blotting of fin lysates from the transgenic adult fish (1 yr-old) and found the ribosomal proteins to be similar in WT, Tg(hSBDSR126T)sbds+/+ and Tg(hSBDSR126T)sbds-/-.However, the authors can detect the accumulation of Eif6 in the fin lysates of Tg(hSBDSR126T)sbds+/+ and Tg(hSBDSR126T)sbds-/-.Have the authors tested the expression of these proteins from 1-year-old liver lysates of WT, Tg(hSBDSR126T)sbds+/+, and Tg(hSBDSR126T)sbds-/-?Since the liver is more metabolically active than the fin, it would be interesting to investigate the ribosomal pathway protein levels from liver lysates.12. Results, Paragraph 6, line 8 -Please rewrite "showed a decreased" to "showed a decrease".13. Figure 4A -The authors indicate healthy embryo (black star) from Tg(ubi:SBDSR126T:pA);sbds-/.However, this embryo visually looks developmentally delayed.In several instances, the authors demonstrate the activation of p53 and stress response pathways.It will be critical to analyze the apoptosis and cell proliferation in the various transgenic animals in sbds-/-null background to validate the physiological impact of activating the aforesaid pathways.14. Figure 4C -The authors write in the text that they "checked the mRNA levels of the three critical glycolysis enzymes (gck, pfkmb and pyrk)"; however, the expression levels of only pfkmb are displayed in the graphs.15. Figure 4G -Please use X-axis split for the standard length (SL) bar plot.This will help to visualize and appreciate the data in a better way.16. Figure 5A -How do the authors distinguish between Tg(ubi:SBDSR126T:pA);sbds-/-embryos (arising from the cross male Tg(ubi:SBDSR126T:pA);sbds-/-and female Tg(ubi:SBDSR126T:pA);sbds-/-) having 1 transgene and 2 transgenes?17. Figure 5C -Does the graph presented for the percentage of deformed embryos correspond to the sbds+/+ and sbds-/-only?Do these embryos have the transgene?If these embryos have transgene, then according to the breeding cross, 25% of the embryos must have 2 copies of transgene in the sbds-/-MZ background and would be able to survive.In that case, the statement by the authors "Interestingly, embryonic development was defective in 25% of the sbds-/-embryos after 1dpf, and the mortality was 100% after 3 dpf (Figure 5B-C), presence of transgene did not ameliorate the defects" does not make sense.18. Figure 5D -qPCR analysis corresponding to sbds gene is confusing.The bar graphs suggest an increase in sbds mRNA levels in TgR126T;sbds+/+ and TgR126T;sbds-/-as compared to sbds+/+ 2 dpf embryos.Does this mean the TgR126T transgene triggers endogenous sbds expression?19. Figure 5D -qPCR analysis corresponding to hSBDS mRNA seems incorrect.There are data points visible for sbds-/-.How can you quantitate an mRNA derived from the transgene which is not present in the sample?Similarly, why are the mRNA levels corresponding to hSBDS lower in TgR126T;sbds-/-as compared to TgR126T;sbds+/+? 20. Figure 6: The authors demonstrate that high expression (2 transgene) of SBDSR126T rescues neutropenia at 5dpf.However, at 10dpf, the high expression of SBDSR126T is unable to rescue neutropenia.The authors need to talk about this in the discussion section.21.Results, Paragraph 9, line 6 -Please rewrite "cas9" to "casp9".22. Discussion, Paragraph 6, line 3 -Please rewrite "SBDS protein is important for zebrafish developmented" to "SBDS protein is important for zebrafish development".23.Does the SBDSR126T allele in the SDS patients display any sex specificity (prevalent in males)?24.The authors need to emphasize the use of maternal zygotic sbds null mutants in their results.25.Does suppression of cdkn1a in the sdbs null background result in reversed neutropenia?26.Performing qRT-PCR for all the transgenics (sbds+/+, sbds-/-, TgR126T;sbds+/+, TgR126T;sbds-/-1copy and 2 copy) at Day1 , Day2 , Day5 and Day10 of development would be informative.Thank you for giving us the opportunity to submit a revised draft of our manuscript titled "SBDS R126T rescues survival of sbds -/-zebrafish in a dose dependent manner independently of Tp53" to Life Science Alliance.We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript.We are grateful to the reviewers for their insightful comments on our paper.We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers.All the changes within the manuscript are denoted in red font.
Here is a point-by-point response to the reviewers' comments and concerns: Reviewer 1 Comment 1: The novelty of the work is unclear based on previously published work on the SBDS R126T mutant allele in mice/cell lines/in vitro (Zhang et al 2006;Tourlakis et al 2015;Calamita 2017;Finch et al 2011) showing that this is indeed a hypomorphic allele.The current study does not seem to significantly advance understanding of how the R126T missense mutation impairs SBDS function.Response: Arguably, the most important question in the biology of bone marrow failure disorders is what constitutes the pathogenic mechanism(s).The current dogma, mostly established from another different marrow failure ribosomopathy (Diamond-Blackfan anemia) is the TP53 pathway.Here, we demonstrated that the tp53 pathway is activated in both the zebrafish KO and the transgenic line expressing R126T.But, Tp53 inactivation does not rescue neutropenia or survival -we consider this novel and extremely important to the field.This leads to the hypothesis that there are other pathways that may affect SDS pathophysiology and suggests that not all ribosomopathies behave the same.As importantly, we have generated a novel model organism which did not receive any wild-type mRNA or protein because maternal eggs were derived from parental transgenics.
Comment 2: I also have concerns that a key control experiment seems to be lacking.I may have misinterpreted it due to lack of clarity in the labeling/annotation of the mutant proteins/genes, but it is essential to test whether the WT human SBDS protein fully complements the sbds null zebrafish mutant to support the claim that the "SBDS R126T mutant is hypomorphic".For me, the conclusion "Our results show that SBDS R126T is hypomorphic" is not currently supported by the data.However, this aside, we already know from previous studies and from the human genetics that the SBDS R126T missense mutation is indeed hypomorphic and impairs SBDS function Thank you for submitting your revised manuscript entitled "SBDS R126T rescues survival of sbds-/-zebrafish in a dosedependent manner independently of Tp53".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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9. Figure 2D: what do the labels indicate-132/Mut/WT_020. Difficult to interpret the data if the labeling is unexplained.10. "This extra peak was absent in the WT liver calibrations (data not shown)".What is the interpretation of this additional peak?11.Fig 2G: Eif6 level is increased in 1/3 sbds WT lines expressing human SBDS R126T-please comment on this.12. Fig 2I, 3D: are the expression levels of the cdkn1a protein altered in the different genotypes?13.Fig 3C: "Sudan black staining showed a decreased in the number of neutrophils in sbds KO background fish, independently of the presence of SBDS R126T".Please show primary data.
1. Fig 1A -The Schematic could be prepared better to explain the study design.What do the black rectangles flanking Zf-sbds mean? 2. Fig 1B -Please label the sizes of the proteins in all the western blots.3. Fig 1C -The authors are requested to perform southern blotting to determine the number of copies of the inserted transgene.
Fig 2B -Please rewrite figure legend "(B) Survival rates in one yr-old fish" to "(B) Standard length in one yr-old fish".7. Fig 2D -In the flow cytometry analysis bar plots, samples representing sbds-/-(Green dots) are plotted for erythrocytes, lymphocytes, myeloid, and precursors.According to the authors, the sbds-/-mutants die between 10-21dpf.How did the authors perform these analyses at 1 year for the sbds-/-animals?Is this a case of mislabelling?sbds+/-samples are not plotted in the "no R126T" bars.Please provide clarification.8. Fig 2E -Provide clear and large labeling (inset) for the polysome profiles.What does "+/132" stand for?Please explain the genotypes in the figure legend.9. Results, Paragraph 4, line 3 -The authors refer to 'mutant' in the text.Please refer to sbds+/-mutants for clarity.10.Results, Paragraph 4, line 9 -The authors state, "This extra peak was absent in the WT liver calibrations (data not shown)."In fig 2F, they provide the polysome profile for the WT liver (in red).
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