The right moves: How studying cell movement during embryonicdevelopment may offer new insights into cancer metastasis sloan_kettering elife
. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Ann Sutherland ; Jean-Léon Maître .
Immunostaining is not a quantitative method, even if the staining and imaging are performed in the same conditions. When comparing intensity between WT and Crumbs2 mutants, how do the authors know that they are not measuring a difference in light penetration through the tissue ? Fluorescence intensities need to be normalized within objects within the same confocal plane . It seems that the junction to the medial signal is affected by actin F7B.
I would advise normalizing the junctional signal to the medial one and to show this data for WT and mutants. This will allow for a comparison of relative levels, which is what matters for apical constriction in the end. We have endeavored to address all the reviewers’ comments, with our revisions having included substantial re-analyses as well as additional experiments.
In addition, the findings of the previous publication on Crumbs2 do not support the model proposed here, and in fact, contradict it. The authors should discuss this at the very least and might want to alter their model for the action of Crumbs2 based on it. Data on the difference in ingression behavior between these stages would be really valuable. This may be beyond the scope of this manuscript, but should be considered.
This has been edited throughout the revised manuscript. Myosin Heavy Chain IIB was used when referring to the latest nomenclature . In a few cases, Myosin II was used when referring more generally to Non-muscle Myosin II, in, or when grouping Myosin Heavy Chain IIA and IIB, or when grouping Myosin Heavy Chain IIB and phosphorylated-MLC.
As suggested by the reviewer, we attempted to image with a shorter time interval than 5min on several different microscope systems and modalities available at our institution and were not successful in acquiring usable images with the ZO1-GFP knock-in reporter. We also need to take into account that these single-copy GFP knock-in reporters are often dim, thereby exacerbating the issue.
3. Immunostaining is not a quantitative method, even if the staining and imaging are performed in the same conditions. When comparing intensity between WT and Crumbs2 mutants, how do the authors know that they are not measuring a difference in light penetration through the tissue ? Fluorescence intensities need to be normalized within objects within the same confocal plane . It seems that the junction to the medial signal is affected by actin F7B.
Regarding the colocalization analysis, the authors detect little to no correlation between myosin and crumbs. They then refer to these proteins as being"complementary". Yet in their schematic in F5E, they represent them as mutually exclusive. I think this schematic and the term"complementary" are exaggerated. Complementarity or mutual exclusion, which are synonyms, would require an anti-correlation between the signals.
The authors argue that the noise in the data supports a dynamic localization of the proteins. Currently, I do not think this is supported by the data. Are ZO1-GFP levels dynamic? Could the authors use their ZO1-GFP movies and immunostaing of ZO1 to measure the dynamics of this protein between live and immunostaining?
Could the authors also comment on spatio-temporal variability ? Do authors imply that this is a generalizable mode of epiblast ingression in the mouse embryo? If so, how can dynamic apical constriction be coordinated with other junctional and cell-matrix remodeling necessary for ingression? Overall, I think the authors presented high-quality imaging data, and that the observations reported in this manuscript are intriguing .
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