Dear Dr Nakajima,

Thank you for your patience while we considered your revised manuscript "Distinct stem-like cell populations facilitate functional regeneration of the Cladonema medusa tentacle" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewer 1.

Reviewer 1 has commented that the manuscript has been strengthened in this revision, but s/he feels that the model of tentacle regeneration is not 'definitive'. After discussing this point with our Academic Editor, we think there is consilience around the current interpretations and we would not require further experimentation to address this point. However, in light of this reviewer comment, we think that some of the conclusions should be toned down and that you should add a section to the discussion detailing limitations, alternative interpretations, and future directions. As an example of a toned down conclusion, the Academic Editor has suggested that on page 19: "Combined, these results demonstrate that distinct proliferative cell populations appear during tentacle regeneration and that most blastema cells are not derived from RHSCs" could be changed to address this point by replacing 'demonstrate' with 'suggest'.

Based on the reviews and on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points. Please also make sure to address the following data and other policy-related requests.

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Sincerely,

Luke

Lucas Smith, Ph.D.

Senior Editor,

lsmith@plos.org,

PLOS Biology

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Reviewer remarks:

Reviewer #1: The authors have responded to my and the other reviewers' points from the first review cycle and revised the manuscript by performing new experiments, resulting in new or improved figures. They have also edited the text at places.

The new data, in particular the 24 h EdU treatment that was followed by amputation and BrdU labeling, may indeed suggest that not all proliferative cells in the blastema directly derive from resident tentacle bulb i-cells. Therefore, it is possible that a distinct population (or populations) of cells also contributes to regeneration alongside i-cells and their progeny. This, however, isn't conclusive since the cell cycle length of Cladonema i-cells is yet unknown. A long i-cell cell cycle could still leave many i-cells unlabeled following a 24 h EdU treatment. These cells could increase their proliferation activity following amputation. Some of the other new experiments in the revised manuscript also make a better case for the authors' preferred conclusion; however, the work overall does not provide a definitive model re the cellular composition of the blastema.

Assuming that there are indeed repair-specific cells in the blastema that are distinct from i-cells, the main question arising from this work remains their origin. The authors suggest two alternatives, one being a population of quiescent stem cells (would they call them i-cells too?), and the other is dedifferentiation of existing somatic cells.

Minor comments:

The authors should avoid the term "lineage trace". This term refers to tracking single cells or at least a defined population that, e.g., expresses a marker gene (i.e., a lineage). What the authors performed was S-phase trace, looking at a broad group of cells that are in S-phase, which could encompass multiple lineages.

What is the evidence that cytoplasmic b-catenin marks all i-cells? Identifying i-cells by morphology may overestimate their numbers because they include committed i-cell progeny that still cycle (e.g., nematoblasts).

Related to the above point, the authors should use the term i-cell consistently. First, they have to define them in Cladonema. As it stands now, it is not always clear to which cells the authors refer. Reading the manuscript, one gets the impression that the authors themselves aren't sure. Are i-cells merely small, proliferative cells with undifferentiated morphology, expressing Nanos/Piwi/Vasa, and being able to differentiate into some (which ones?) cell types? Alternatively, if they are unsure of how to define i-cells, they should clearly state it. This issue is always hard to resolve in a new animal model.

Dear Dr Nakajima,

Thank you for the submission of your revised Research Article "Distinct stem-like cell populations facilitate functional regeneration of the Cladonema medusa tentacle" for publication in PLOS Biology, and apologies again for our delay in sending you a decision. As mentioned over email, your revised study was assessed by Reviewer 1, who is fully satisfied by the changes made. Therefore, on behalf of my colleagues and the Academic Editor, Kimberly L Cooper, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

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Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org