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    Reply to the reviewers

    Point-by-point response:

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    **SUMMARY**

    This MS tackles a largely unknown topic of vessel formation: how vessels anastomose and lumenise. The authors demonstrate that a matrix protein svep1 produced by neural tube during zebrafish embryogenesis plays a key role with blood flow to orchestrate anastomose formation. Actually in absence of this protein concomitantly with blood flow reduction results in significant decrease of lumenised DLAV segments.

    In absence of svep1 they observed an expansion of apelin positive endothelial cells connected with a defect in tip/stalk cell specification. Interestingly the phenotype is amplified by blocking the kinase activity of VEGFR2

    **MAJOR COMMENTS**

    The most solid evidence on the role of blood flow in cooperating with svep1 relies on the use of tricaine, which reduces heart contractility. Interestingly the authors report some data by using embryo lacking cardiac troponin T2. In my opinion I suggest the author to better analyze the phenotype obtained by the deletion of svep1 together a dose-dependent reduction of tnnt2. This approach is more elegant and physiologic than the use of a chemical compound. Furthermore this approach will allow to better analyze the relations ship between blood flow and the expression of svep1 in neural tube. It should be relevant to establish a sort of flow threshold required to dampen lumenisation. *

    Response: We appreciate the comment and have previously attempted to titrate the tnnt2 morpholino as published to have a graded reduction in blood flow. In our hands, this has not proved to be a robust approach, but we are willing to give it another try. In addition, we propose use alternative compounds to tricaine for blood flow reduction without affecting neural physiology. Alternatively we will use a-bungarotoxin mRNA injection to selectively affect neural activity to immobilize the embryos without effects on heart rate and blood flow (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526548/)

    To further improve the findings here reported I suggest to analyze the expression of klf2, which is a well known mechano-sensor of blood flow in several animal species including zebrafish.

    Response: We will perform klf2 expression analysis

    *It's likely that apelin is relevant in the observed phenotype. Which is the phenotype of a double mutant lacking both apl and svep1? Is there a direct influence of blood flow on apl expression? *

    Response: We will investigate the double loss of function. However, double mutants would take some time, and a combination of morpholino and mutant would likely be the first and best option to answer this question in a reasonable time frame. The effect of flow on apl expression can be tested.

    Is there any suggestion that this mechanism is oprative in mammalian?

    Response: This is an interesting question and certainly relevant for follow up studies. At present, we can only speculate on a possible connection with flow, given that Svep1 mutations have recently been associated with artherosclerosis. However, whether the anastomosis defect we identify is conserved remains to be seen.

    *Reviewer #1 (Significance (Required)):

    The data here reported might represent a step forward in the field because a new mechanism is suggested.

    The interest is sufficiently broad.

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

    **Summary:**

    The authors demonstrated that loss of svep1 in zebrafish contributed to defective anastomosis of intersegmental vessels, in addition, such Svep1 acted synergistically with blood flow to modulate vascular network formation in the zebrafish trunk.

    **Major comments:**

    The expression of svep1 is localized in neurons of neural tube, dorsal epithelial cells (as indicated by transgenic zebrafish) and ventral somite boundary (as indicated by in situ) but is excluded from endothelial cells nor the vasculature. It remains puzzling and the authors have not addressed this very reason of how a gene that is expressed in non-vascular tissue play a crucial role in vessel anastomosis, ie DLAV, ISV lumenization, during angiogenesis. As the entire story of this svep1 is related to its function in angiogenic sprout and lumen formation of vascular tissues, it will be helpful for reader to be able to put the pieces together of how such gene may be functionally involved in such angiogenic process. Previous publication of this gene involved in lymphoangiogenesis, as in this manuscript the authors could provide more evidence of how such gene and its localized expression contribute to different tissue in the vascular system, ie DLAV, instead of the neural tube, dorsal epidermis or ventral somite boundary.*

    Response: We appreciate the wish to understand exactly how non-endothelial expression of Svep1 causes an endothelial phenotype selectively under reduced flow conditions. The very nature of this new phenotype requires analysis in vivo, and can not easily be transferred to an ex vivo assay. Therefore, selective loss of function in different cell populations is not easily available. More importantly, the interpretation of such efforts, when mosaic, are marred with issues. At this point, we feel that full molecular characterization of how Svep1 affects endothelial cells during anastomosis will require entirely new approaches and lies beyond what can be achieved in this manuscript.

    We will however attempt to clarify the findings and the potential mechanisms in the discussion.

    *Another puzzling point is that tricaine is the center of the subject in this study. As the authors claim that tricaine-dependent blood flow reduction synergistically augmented the effect of svep1 deficiency. However, tricaine is known acting on neural voltage-gated sodium channels, whether svep1 function was affected by tricaine in the neural tissues and possibly its expression, the authors could provide more explanation and argument in the discussion. *

    Response: As mentioned in our response to reviewer 1, we will perform additional experiments to try to clarify whether an effect of tricaine on neuronal sodium channels contributes to the phenotype.

    *It is unclear on p12 "These results suggest that while svep1 loss-of-function produces a cardiac defect that enhances the effect of tricaine on reducing blood flow, svep1 has an additive effect in modulating blood vessels anastomosis" that svep1 deficiency enhances the effect of tricaine leading to reduced blood flow, however, it is not accurate to state that svep1 loss-of-function produces a cardiac defect. It is not sure if the effect of svep1 was actually neural rather than cardiovascular tissue, for example, tricaine acts on neural voltage-gated sodium channel that slowing down heart beat. Whether the authors can explore the possibility that svep1 function in neural rather than cardiovascular tissues, may be discuss why the authors think svep1 enhances the blood flow defect (tnnt2a knockdown or tricaine) on angiogenesis such as DLAV phenotype. *

    Response: We will attempt to dissect potential contributions by neural effects from cardiac and flow related effects as stated above. Tnnt2 MO and alternative drugs to reduce heart function selectively will be used. We will also clarify the discussion.

    *On p13, the authors stated that svep1 expression was inhibited by reduced blood flow, however, is it really the effect of reduced blood flow or caused by the chemical tricaine? If tnnt2a knockdown showed a similar phenotype, then it may be more convincing. *

    Response: see above

    ***Minor comments:**

    The work on "svep1 loss-of-function and knockdown are rescued by flt1 knockdown" was beautifully done and it is very clear and convincing.

    The last two sections, "Vegfa/Vegfr signalling is necessary for ISV lumenisation maintenance and DLAV formation" and "Vegfa/Vegfr signalling inhibition exacerbates svep1 loss-of-function DLAV phenotype in reduced flow conditions" are more related to the flt1 knockdown phenotype. These 3 different sections are actually related in the sense that the rescue phenotype should be explained in the vegf signaling pathway. They are better off to discuss more cohesively about this vegf pathway that will help readers to appreciate more their work in svep1. *

    Answer: We agree and will do so.

    *Reviewer #2 (Significance (Required)):

    This manuscript of svep1 in zebrafish provides new insight in angiogenesis, particularly in development of vessel anastomosis in zebrafish embryo, is very significant in the field and readers who are interested in angiogenesis and zebrafish development, including myself.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This manuscript reports that the secreted extra-cellular matrix protein Svep1 plays a role in vascular anastomosis during developmental angiogenesis in zebrafish. Further, the study demonstrates that flow and Svep1 modulate the vascular network in a synergistic fashion. This is a high quality manuscript presenting novel data which compellingly support the conclusions that are made. I have no suggestions for further experimentation but list minor points below.

    1. The final paragraph of Discussion is underdeveloped in that it claims regulation of phenotypic robustness in angiogenesis and its failure promises crucial insights into the mechanisms causing breakdown of vascular homeostasis in human disease. However, this issue is not pursued in any substantial way in Discussion. For example, are there known mutations in humans which lead to anastomosis defects and, if so, do any of them relate to the molecules or signaling pathways which are the subject of this manuscript? *

    Response: We agree with the wish to see more substantial discussion of the issue of phenotypic robustness and potential links to human disease. The question of anastomosis itself is something that has not been addressed in humans, as it is a rather detailed phenotype observable where predictive patterning occurs and can be dynamically studied. As such, there is a lack of literature and knowledge on signalling pathways that drive anastomosis in humans, and also not many that have been identified in experimental systems or animal models. Flt1 and Vegf signalling, junctional molecules and a few other pathways have been shown to be involved, but nothing is known so far about Svep1 and anastomosis in other system. We will attempt to complement the discussion to make this more clear.

    • There are typographical errors in the text so a further proof-read is required. *

    Response: thank you, these will be corrected

    *Reviewer #3 (Significance (Required)):

    This manuscript provides an incremental conceptual advance in our understanding of the molecular mechanisms responsible for vascular anastomosis during developmental angiogenesis. The manuscript will be of interest to developmental biologists and vascular biologists.

    My field of expertise pertains to angiogenesis and lymphangiogenesis in the setting of cancer and other diseases. **I am not a developmental biologist.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript reports that the secreted extra-cellular matrix protein Svep1 plays a role in vascular anastomosis during developmental angiogenesis in zebrafish. Further, the study demonstrates that flow and Svep1 modulate the vascular network in a synergistic fashion. This is a high quality manuscript presenting novel data which compellingly support the conclusions that are made. I have no suggestions for further experimentation but list minor points below.

    1. The final paragraph of Discussion is underdeveloped in that it claims regulation of phenotypic robustness in angiogenesis and its failure promises crucial insights into the mechanisms causing breakdown of vascular homeostasis in human disease. However, this issue is not pursued in any substantial way in Discussion. For example, are there known mutations in humans which lead to anastomosis defects and, if so, do any of them relate to the molecules or signaling pathways which are the subject of this manuscript?
    2. There are typographical errors in the text so a further proof-read is required.

    Significance

    This manuscript provides an incremental conceptual advance in our understanding of the molecular mechanisms responsible for vascular anastomosis during developmental angiogenesis. The manuscript will be of interest to developmental biologists and vascular biologists.

    My field of expertise pertains to angiogenesis and lymphangiogenesis in the setting of cancer and other diseases. I am not a developmental biologist.

    Read the original source
    Was this evaluation helpful?
  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors demonstrated that loss of svep1 in zebrafish contributed to defective anastomosis of intersegmental vessels, in addition, such Svep1 acted synergistically with blood flow to modulate vascular network formation in the zebrafish trunk.

    Major comments:

    The expression of svep1 is localized in neurons of neural tube, dorsal epithelial cells (as indicated by transgenic zebrafish) and ventral somite boundary (as indicated by in situ) but is excluded from endothelial cells nor the vasculature. It remains puzzling and the authors have not addressed this very reason of how a gene that is expressed in non-vascular tissue play a crucial role in vessel anastomosis, ie DLAV, ISV lumenization, during angiogenesis. As the entire story of this svep1 is related to its function in angiogenic sprout and lumen formation of vascular tissues, it will be helpful for reader to be able to put the pieces together of how such gene may be functionally involved in such angiogenic process. Previous publication of this gene involved in lymphoangiogenesis, as in this manuscript the authors could provide more evidence of how such gene and its localized expression contribute to different tissue in the vascular system, ie DLAV, instead of the neural tube, dorsal epidermis or ventral somite boundary.

    Another puzzling point is that tricaine is the center of the subject in this study. As the authors claim that tricaine-dependent blood flow reduction synergistically augmented the effect of svep1 deficiency. However, tricaine is known acting on neural voltage-gated sodium channels, whether svep1 function was affected by tricaine in the neural tissues and possibly its expression, the authors could provide more explanation and argument in the discussion.

    It is unclear on p12 "These results suggest that while svep1 loss-of-function produces a cardiac defect that enhances the effect of tricaine on reducing blood flow, svep1 has an additive effect in modulating blood vessels anastomosis" that svep1 deficiency enhances the effect of tricaine leading to reduced blood flow, however, it is not accurate to state that svep1 loss-of-function produces a cardiac defect. It is not sure if the effect of svep1 was actually neural rather than cardiovascular tissue, for example, tricaine acts on neural voltage-gated sodium channel that slowing down heart beat. Whether the authors can explore the possibility that svep1 function in neural rather than cardiovascular tissues, may be discuss why the authors think svep1 enhances the blood flow defect (tnnt2a knockdown or tricaine) on angiogenesis such as DLAV phenotype.

    On p13, the authors stated that svep1 expression was inhibited by reduced blood flow, however, is it really the effect of reduced blood flow or caused by the chemical tricaine? If tnnt2a knockdown showed a similar phenotype, then it may be more convincing.

    Minor comments:

    The work on "svep1 loss-of-function and knockdown are rescued by flt1 knockdown" was beautifully done and it is very clear and convincing.

    The last two sections, "Vegfa/Vegfr signalling is necessary for ISV lumenisation maintenance and DLAV formation" and "Vegfa/Vegfr signalling inhibition exacerbates svep1 loss-of-function DLAV phenotype in reduced flow conditions" are more related to the flt1 knockdown phenotype. These 3 different sections are actually related in the sense that the rescue phenotype should be explained in the vegf signaling pathway. They are better off to discuss more cohesively about this vegf pathway that will help readers to appreciate more their work in svep1.

    Significance

    This manuscript of svep1 in zebrafish provides new insight in angiogenesis, particularly in development of vessel anastomosis in zebrafish embryo, is very significant in the field and readers who are interested in angiogenesis and zebrafish development, including myself.

    Read the original source
    Was this evaluation helpful?
  4. 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

    This MS tackles a largely unknown topic of vessel formation: how vessels anastomose and lumenise. The authors demonstrate that a matrix protein svep1 produced by neural tube during zebrafish embryogenesis plays a key role with blood flow to orchestrate anastomose formation. Actually in absence of this protein concomitantly with blood flow reduction results in significant decrease of lumenised DLAV segments.

    In absence of svep1 they observed an expansion of apelin positive endothelial cells connected with a defect in tip/stalk cell specification. Interestingly the phenotype is amplified by blocking the kinase activity of VEGFR2

    MAJOR COMMENTS

    The most solid evidence on the role of blood flow in cooperating with svep1 relies on the use of tricaine, which reduces heart contractility. Interestingly the authors report some data by using embryo lacking cardiac troponin T2. In my opinion I suggest the author to better analyze the phenotype obtained by the deletion of svep1 together a dose-dependent reduction of tnnt2. This approach is more elegant and physiologic than the use of a chemical compound. Furthermore this approach will allow to better analyze the relations ship between blood flow and the expression of svep1 in neural tube. It should be relevant to establish a sort of flow threshold required to dampen lumenisation.

    To further improve the findings here reported I suggest to analyze the expression of klf2, which is a well known mechano-sensor of blood flow in several animal species including zebrafish.

    It's likely that apelin is relevant in the observed phenotype. Which is the phenotype of a double mutant lacking both apl and svep1? Is there a direct influence of blood flow on apl expression?

    Is there any suggestion that this mechanism is oprative in mammalian?

    Significance

    The data here reported might represent a step forward in the field because a new mechanism is suggested.

    The interest is sufficiently broad.

    Read the original source
    Was this evaluation helpful?