1. Reviewer #3 (Public Review):

    In zebrafish embryo development the surface epithelium, the enveloping layer (EVL), proliferates and migrates along with the yolk sac during epiboly. This process requires the simultaneous proliferation and migration of cells, which must undergo cell shape changes. Co-ordination of these processes is regulated by proliferation, whereby cell number and shape perturb tissue-scale forces necessary for epiboly. This paper investigates explicitly the importance of successful cytokinesis, through abscission of cytokinetic bridges, on regulating these forces and epiboly progression. They show that Rab25, a GTPase belonging to the Rab11 subfamily, regulates abscission through endomembrane trafficking in the EVL. Through their detailed analysis of cellular-level phenotypes, including qualitative and quantitative approaches, this paper presents convincing evidence for this novel role of Rab25. The authors should be congratulated on excellent time-lapse movies of cytokinesis in early zebrafish development.

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  2. Reviewer #2 (Public Review):

    The authors examined the role of Rab25 during cell division within a developing epithelia. Strikingly, they found that the RabGTPase, Rab25, localized to mitotic structures such as centrosomes and cytokinetic midbodies in dividing cells of the developing zebrafish embryo. They went on to create maternal-zygotic Rab25a and Rab25b mutant embryos where they clearly demonstrate that apical cytokinetic bridges fail to undergo abscission leading to anisotropic cell morphologies that likely contribute to a delayed epiboly.

    The major strengths of this study is the clear cell biology defects found in a developing embryo that lead to downstream developmental defects (delayed epiboly). The rab25 localization is beautiful. The examination of the viscoelastic properties is also compelling. The main improvements would be to expand upon the spatio-temporal localization of Rab25a and Rab25b during cell division at different stages of epiboly, present Rab11 localization patterns in the Rab25 mutant embryos, and clearly demonstrate that changes in viscoelasticity are also in their multinucleated cells that occur in Rab25 mutant conditions. These additions will help the authors support their conclusions that Rab25 localization/regulation of endomembranes (potentially recycling endosomes) regulates abscission and subsequently the viscoelastic properties of the developing tissue.

    This study has identified novel roles for Rab25 in cytokinesis/abscission and opens the doors for examining it in regulating mitotic centrosome function. It is paradigm shifting in that it creates a new way to think about Rab25 and potentially its relationship with Rab11 and recycling endosomes during division in the early embryo.

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  3. Reviewer #1 (Public Review):

    In the manuscript by Willoughby et al. the authors examine the role of Rab25 in early embryogenesis in zebrafish. They implicate Rab25 activity in abscission and show various defects including delayed epiboly and altered cell behaviors associated with defective acting dynamics. This is an interesting and well-written paper that uses reverse genetics and microscopy to analyze the function of Rab25, a GTPase previously implicated in membrane recycling, in vivo. Their work illustrates how defects in cytokinesis affect epiboly and establish an interesting link to acto-myosin regulation of the mechanical properties of the EVL. While these pehnotypes are described and demonstrated clearly, the implication of membrane recycling is not fully supported in the present work. It is also unclear whether Rab25 plays a role in oogenesis that may account for some of the observed phenotypes.

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  4. Evaluation Summary:

    Willoughby et al. examine the role of Rab25 in early embryogenesis in zebrafish. They implicate Rab25 activity in abscission and show various defects including delayed epiboly and altered cell behaviors associated with defective acting dynamics. Overall, this is an interesting and well-written paper. However, there are a number of important controls that are missing and some connections such as the implication of membrane recycling that require stronger experimental validation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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