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For RT-qPCR experiments, 25 zebrafish embryos were pooled for each condition three biological replicates.

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For heat-shock conditions, controls of each biological replicate were composed of heat-shocked siblings from the same clutch lacking EGFP expression. Cycle threshold Ct values were determined by PikoReal software 2. As an internal reference, we used zebrafish eif1b Renz et al. Control sample values were normalized to 1.

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In the table below, the mean of the fold changes and the corresponding SEM of each biological replicate after normalization is shown. The mean of the ratio indicated in the table below is always between 1 and 0, and this accounts for target downregulation. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "CCM proteins control endocardial mechanosensitivity during zebrafish valvulogenesis" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Ian C Scott Reviewer 1 ; Dimitris Beis Reviewer 3.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. In this manuscript, Donat and colleagues investigate the roles of the CCM-associated genes heg1 and krit1 during atrioventricular valve leaflet formation in the endocardium of the developing zebrafish heart.

Mechanosensitivity of the Heart Mechanosensitivity in Cells and Tissues

The authors first show that heg1 transcripts are positively regulated by blood flow. Heg1 in turn acts to stabilize Krit1, and these genes repress klf2a , a major mechanosensitive gene. As a result, the absence of Heg1 and Krit1 function leads to overexpression of klf2a and mis-activation of Notch signaling throughout the endocardium, which impairs valve leaflet formation.

Cardiac mechanosensitivity and stretch-activated ion channels.

This is an interesting model, particularly in terms of providing a mechanistic link between blood flow and specific features of cardiac morphology, and it would therefore be of interest to the readers of eLife. However, there are a number of caveats inherent in the authors' experimental design and results, and further work is needed to fully support the authors' interpretations and conclusions.

Figure 1A and B , Figure 2A on vascular endothelial cells. This technique, although quantitative, does not provide any information about the localization of the expression of the studied genes. Whole mount in situ hybridizations would greatly help to reveal spatial differences in the expression of these genes, particularly in the heart. Ideally, qPCR should be performed in isolated AV endocardial cells, given the focus of the manuscript. This should be analyzed in the AVC. Thus, it is somewhat challenging to draw a meaningful conclusion about the ability of Heg1 to stabilize Krit1 protein levels from these non-physiologic studies.

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  5. Western analysis would be a better approach in determining Krit1 levels in heg1 morphant and wild-type hearts. In addition, the authors' use of flt1:YFP as a reporter of flow responses is based on the prior studies of Hogan, which were focused on the vascular endothelium. However, the ability of this line to sense hemodynamic flow has not been validated in the heart.

    Is this endocardial or myocardial expression that is observed? Using endocardial and myocardial lines in combination with the flt1:YFP might help resolve these issues. Performing studies to validate the ability of this line to sense hemodynamic flow in the heart would help with interpretation of studies as well. It is possible that the AVC region is expanded in the krit1 mutant. How are the authors defining the AVC?

    Bibliographic Information

    Without atrial, ventricular or AV boundary markers, it is difficult to determine where the endocardial cushions are in the krit1 mutant. Providing this data would help with interpretation of the studies. In addition, it would be useful to examine the ccm2 mutant in addition to the krit1 mutant, to strengthen the demonstration of how loss of ccm genes affects endocardial notch activation. Many assumptions are made in order for the authors to come to their conclusions.

    For example, there is insufficient evidence to show that injected mRNA is no longer present at 55 or 96 hpf. In fact, the phenotype is surprisingly similar to that found in late Notch inhibition and in the klf2a mutant Beis et al.

    The transient rescue of krit1 mutants allows AVC endocardial cells to differentiate properly at 48 hpf. It might be helpful to show a model for the ccm mutant with blood flow and then when there is no blood flow. Based on the authors' current model, it seems that Notch would be activated throughout the endocardium in the ccm mutant with blood flow since ccm is not present to block klf2a expression. If this is the case, this seems to be in conflict with the current ccm mutant and no blood flow model where the lack of blood flow would lead to no klf2a expression and downstream Notch signaling.

    Mechanosensitivity of the Heart

    Additionally, the authors also speculate "Notch-mediated lateral inhibition" in their model; however, there is insufficient data to support this notion. This should be modified in the model. Thank you for resubmitting the revised version of your manuscript entitled "CCM proteins control endocardial mechanosensitivity during zebrafish valvulogenesis" for further consideration at eLife.

    Your revised manuscript has now been favorably evaluated by Marianne Bronner Senior editor , a Reviewing editor, and its original three reviewers. The reviewers appreciate the improvements made in your revised manuscript, but they have also raised several issues that remain to be addressed through further revision, as outlined below.

    As an example: does the abstract make it clear whether heg1 expression is increased or reduced by low blood flow? The last paragraph of the Introduction section is similarly vague and does not refer to the heg1 results. Can the evidence for this be clarified? However, the images in Figure 2B—D are hard to interpret. Although the ratios measured by the authors Figure 2E—F demonstrate the same trend following overexpression of Krit1 and Ccm2, the patterns of expression shown in Figure 2C—D seem markedly different; hard to interpret. This apparent discrepancy should be addressed.

    In addition, in light of recent studies showing myocardial Notch reporter activity in developing zebrafish hearts, it is unclear whether the Notch reporter activity shown here and in Figure 4 is endocardial or myocardial.

    Stretch-activated channels in the heart: contribution to cardiac performance

    Can this be clarified? If they are introducing this marker here, its characterization as such should be clearly described. This marker indicates the edge of the atrium but isn't sufficient to demonstrate whether the AVC is or isn't expanded in the mutant hearts. This is confusing — can it be revised to include these elements of the model? Within the revised version of the manuscript, we now provide expression data of heg1 mRNA Figure 1—figure supplement 1. Consistent with a blood-flow responsive expression, heg1 mRNA has a stronger expression at the AVC and in the ventricle at 48 hpf.

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    We are now also referring to this work in our revised manuscript. The expression of krit1 has been published previously Mably et al. This lends further support to an important role particularly of heg1 in response to biomechanical stimuli. Unfortunately, we have not been able yet to establish a technology for AVC dissection. This finding is in tune with the changes of EGFP-Krit1 levels within the vasculature now shown in Figure 1—figure supplement 2.

    Due to the lack of an antibody that recognizes zebrafish Krit1, it has remained a challenge to quantify the physiological levels of Krit1 upon loss of Heg1. To assess physiological levels of Krit1, we therefore undertook mass spectrometric analyses of the cardiac proteome under WT and heg1 mutant conditions with purified hearts per genetic condition and replicate in an attempt to detect CCM complex proteins. Unfortunately, this approach was not sufficiently sensitive to detect Krit1 protein levels.

    We agree with the reviewers that the ability of the Tg flt1:YFP line to sense the hemodynamic forces of blood flow has not previously been shown. Within the revised manuscript, we have now repeated the above experiments using the well-established blood flow-sensitive Notch reporter line Tg EPV. Odc1 s Ninov et al. Within the revised manuscript, we now show that the forced overexpression of Krit1 or Ccm2 alters the expression of the Tg EPV. Odc1 s reporter within the endocardium which, in WT, is particularly strong within the ventricle and AVC. These expression changes of the Notch reporter line occur while blood flow is not obviously affected as assayed by visual inspection.