Zebrafish Posterior Lateral Line primordium migration requires interactions between a superficial sheath of motile cells and the skin

Dear Editors and Reviewers,

Thank you for your thorough consideration and review of our paper. To address the concerns of the reviewers (listed under the “essential revisions”), we have made the following modifications. In some cases, we have performed additional experiments, however due to the restricted work schedule we are under because of COVID-19, we could not experimentally address all of the reviewers' criticisms. However, we hope the revised manuscript satisfies the majority of the reviewers’ concerns.

Additional experiments and the role of superficial cells In addition to the experiment described above to examine confinement of the PLLp, we have also performed the experiment suggested by reviewer #2 looking at basal protrusions in skinned but matrigel embedded embryos. We report that, unlike superficial protrusions, the directionality of basal protrusions is rescued when embryos are embedded in matrigel. This surprising result further argues against a purely mechanical confinement mechanism (as recovery of polarization is not observed after agarose embedding), and so we have, as discussed above, softened the relevant conclusions. However, despite recovery of polarized basal protrusions, directed primordium migration is not recovered in matrigel embedded embryos. We feel that this failure to migrate, when basal protrusions are polarized but superficial protrusions are not, provides stronger support for the necessity of polarized superficial protrusions in migration. We have discussed this experiment in the relevant portions of the results and discussion. In addition, we would like to address the following comments by the reviewers directly .

Reviewer #1

Actin flow rates: We have added some discussion of actin flow rates in a variety of other contexts to better situate our measurements. As we note, measurement of Actin retrograde flow rates in vivo are very rare, and often significantly different from those observed in in vitro studies, and so we refrain from interpreting too much from our measurement, other than situating it in context of other measured rates (both in vivo and in vitro).

Reviewer #2

As discussed above, we have made significant changes to the way we discuss “confinement” in the manuscript, including softening conclusions about the role of confinement, given the new results reported in Figure 7. We agree with the reviewer that we could have done a more thorough job in describing the nature of this population. To this end, we have added a section in the results describing external contacts of each cell population, which is further explained and quantified in a new Figure S2. In addition, we have provided two examples of the morphology of leading, protoneuromast, and superficial cells in a new Figure 2 – figure supplement 1. We feel that these new figures and results text will do a better job giving context to the reader. We have rewritten parts of the results and discussion to increase clarity, and included a supplementary table showing the number of embryos and movies used for each quantification. In addition, we have added a new supplementary figure with examples of each type of quantification used (actin rods in a variety of contexts, retrograde flow kymographs). We have also added a discussion of these measurements to the main text.

Reviewer #3

We have made all of the modifications in the text and figures suggested by this reviewer. We hope that the additional experiments described help to alleviate their major concerns, however we regret that we were unable to perform some of the experiments suggested. We have, in these cases, toned down the relevant conclusions. Some experiments (for example modulating the elastic modulus of matrigel) we plan to perform in upcoming studies. We agree with the reviewer that figure S1 showing a track of 5 nuclei is underwhelming. We have replaced Figure S1 with a more robust figure and movie (Figure 2 – figure supplement 2, Movie S3). In this figure, we segment, 3D reconstruct and track every nucleus in a primordium over two hours. This data is used to construct a new figure showing the averaged nuclear position of all nuclei in a 120 micron cross section of the PLLp (centered around a neuromast). We have also included a reconstruction movie of all nuclei in the PLLp over this time course, color coded by cell type, and showing the cross section and position heatmaps as movie S3, and the area used to generate the position heatmap is indicated in this movie. We also agree that we do not have complete enough data to make firm statements about the ultimate fate of these cells. Although we strongly suspect that they become interneuromast cells, we have not conclusively demonstrated this, and so have removed this claim from the paper.

Essential revisions:

1) Confinement of the primordium. The reviewers agree that the height of the primordium cells with and without skin should be measured to see if the primordium cells are higher along the medial-lateral axis after skin removal. If the cells are higher, this would be in agreement with the mechanical confinement idea.

While we could extract some values from our existing data, we felt the better approach was to perform an additional experiment to quantify this potential change. We have therefore performed additional paired measurements of the height of the PLLp in primordia before and after skin removal. These data are discussed in the Results section, and presented in Figure 4. Removal of the skin results in an almost 20% increase in average primordium height (p = 6e^-7, paired t-test). Note that for this measurement, we could not robustly measure individual cell height paired between skin intact and skin removed, and so we have devised an average measure of primordium height, measured at 4 equally spaced points along the length of the primordium, as detailed in the Materials and methods.

However, all reviewers feel that the skin removal experiments do not show that confinement is essential for primordium migration and this claim should be softened and other possibilities should be discussed (changes in the environment, wounding response etc).

Despite the results described above suggesting that the primordium is in fact under mechanical confinement by the skin, we agree with the reviewers critique that our experiments do not adequately demonstrate that mechanical confinement, per se, is essential for migration. We have toned down the relevant conclusions and expanded discussion of additional ways confinement by the skin could contribute to migration.

2) Sheath cells.

a) The reviewers agree that it is unclear (and unlikely) that the sheath cells are a separate cell population. The authors should rename this cell population using a more descriptive term such as apical cells.

We agree with the reviewers that this population need not represent a separate cell population with a distinct fate. It could, for example, represent the persistence of mesenchymal-like cells that are present in the leading domain. We have rewritten the relevant sections to provide better context for this population, and now refer to these cells as “superficial primordium cells”, rather than “sheath cells”, which inadvertently suggested a distinct cell type. We have, in a small number of places, retained the use of the word “sheath” to describe the overall structure as we feel this is a good intuitive analogy to their position wrapping around the top and sides of the primordium.

b) The claim that the sheath cells are supporting collective migration should be toned down since the authors do not direct evidence for this.

c) The claim that the sheath cells are mesenchymal should be softened since they are labeled by a epithelial promoter.

With regards to the mesenchymal/epithelial nature of these cells, we agree and have changed the text to reflect the ambiguity about the nature of these cells, and included a caveat that they are labeled by an epithelial promoter. We also note that the leading cells of the primordium, commonly referred to in the literature of “mesenchymal” or “mesenchymal-like” are also labelled by the cldnB promoter. We have changed text referring to these types of cells as “mesenchymal-like” rather than “mesenchymal” and discuss them now in the context of polarized epithelial cells incorporated into rosettes as opposed to cells in the leading domain and superficial cells that are not incorporated into a neuromast.

3) Discussion. The Discussion should be more focused and concise as laid out in the individual reviewer's comments.

We have significantly rewritten the discussion and hope that is more precise and clearer than the initially submitted manuscript. In addition, to address the reviewers concerns about the legibility of the figures, we have made several changes to font sizes etc to enhance readability. To this end, we have split Figure 5 (which had panels A-Zz) into a new Figure 5 and Figure 6 to allow for larger panels and a re-designed figure.

Reviewer #1:

Overall, I think this paper is a strong candidate for publication in eLife. Although the study is largely descriptive in nature, the discovery of this new cell population is important, as it fundamentally changes the way we think about how motive forces are generated in this collective. Moreover, the skin healing experiments are elegant and quite revealing of the underlying biology.

1) I am unconvinced that the newly described cell population is mesenchymal, as proposed by the authors. Since a claudin promoter is used to label the cells, it seems more likely that they are epithelial.

We have changed the use of “mesenchymal” in the paper, and have added a section noting that these cells are labelled with the claudinB promoter

2) Results – The authors point out that the rate of retrograde actin flow correlates with traction forces in migrating cells. Please provide some context for the flow rate measured in this paper. How does it compare to other cell types and how can this information help us better understand the underlying biology?

We have added some discussion of other flow rates measured in a variety of contexts to this part of the Results section and attempted to do a better job contextualizing this flow rate

3) Figure 6C – Wild-type control data should be added to this figure.

We have added wild-type (skin intact) data, from embryos embedded both in agarose and matrigel, to this figure.

Reviewer #2:

[…] We recommend concluding further explanation that the skin's role may be mediated by confinement, adhesion, tissue integrity signaling, or a combination thereof (see comment #1). Our other suggestions could strengthen the paper but are not required.

1) The skin removal experiments require a more nuanced interpretation of the variety of mechanism(s) by which skin may contribute to PLLp migration. We ask that the authors address the following points in the Discussion and Abstract:

a) The term "confinement" needs to be used more precisely. In each condition of the skin removal experiments, the primordium is in contact with a different material with unique mechanical and chemical properties. We caution the authors from referring to embryos embedded in agarose as "unconfined", as agarose often acts as a form of confinement for cultured cells. Perhaps the PLLp experiences weaker forces or geometric constraints while embedded in agarose versus contained within the skin, but the authors should more clearly acknowledge these unknown mechanical parameters that are inherent to the experiment (Results and Discussion).

We agree with the reviewer and have modified the text to more precisely use the word “confinement”.

The authors could additionally choose to strengthen the mechanical comparison by measuring the height of the PLLp under each condition--one would expect that taller cells are less confined. This measurement should be possible by further analysis of image data that the authors have already collected.

We have performed experiments to measure this, as discussed earlier, and now show that the primordium is ~15-20% taller on average when the skin is removed than when it is in intact. As we feel these data add more to the narrative than the current Figure 4H (which showed data to support Figure 4G but didn’t add anything new), we have made these height measurements Figure 4H and removed the old Figure 4H.

b) The possible role of adhesions should be elevated. We find this model interesting, and the slow migration under Matrigel does not technically prove that adhesion is insufficient for PLLp migration, as the authors correctly note in the Discussion (eighth paragraph). It is possible that growth factors in Matrigel alter chemotaxis, counteracting the pro-migratory effect of lamellipodial formation.

We have added discussion of the potential role of adhesions and other potential matrigel-related affects to the Discussion.

If experimental follow-ups were feasible, we would propose observing transplanted basal cells under Matrigel confinement, in order to determine whether superficial lamellipodia rescue basal cryptic lamellipodia with this perturbation.

We have performed these experiments, and now show that basal protrusions are in fact recovered after matrigel embedding. Surprisingly, we also show that these basal protrusions (unlike the superficial protrusions) are in fact polarized in matrigel. This may, in part, explain the limited recovery of migratory ability in these primordia when compared to primordia embedded in agarose. The reason for this asymmetric recovery is unclear.

c) Tissue integrity should be discussed as a potential contributor to the skin effect. […] The experiments shown in Figure 4 could shed light on this hypothesis if the authors were to additionally measure cell speed after the skin has entirely closed (and presumably skin integrity restored) and compare that with speeds before/after cell contact with skin (Figure 4G).

2) When defining the sheath of motile cells, we suggest including a more thorough analysis of the entire PLLp population, rather than just the cells above apical constrictions (Figure 2). […] Analogous findings on the significance of differential cell adhesion have been reported in other developmental contexts, for example Tsai et al., bioRxiv 803635.

We have now performed a more thorough analysis of the nature of this cell population from segmented data, and answered several of these questions. We have redesigned Figure 2—figure supplement 1 to include some of these data on cell morphology and external contacts.

3) While there are a lot of interesting issues raised in the Discussion, we found it to be unfocused and confusing. […] In particular, the result that the primordium produced more lamellipodia on Matrigel than on agarose is consistent with the Bergert et al. experiment discussed by the authors, which shows that Walker 256 cells produce more lamellipodia on adhesive regions as compared to non-adhesive regions (Figure 5 in Berget et al.).

We have rewritten the Discussion to attempt to address these points.

In the present version of the manuscript, the Abstract emphasizes the importance of confinement, but the results are also consistent with a more nuanced interpretation where confinement acts along with adhesion and signaling in determining cell behavior.

We agree with the reviewer, and have modified the Results and Discussion to better discuss other interpretations consistent with the data.

Reviewer #3:

1) Stunning time-lapse data reveal that cells on the apical surface of the PLLp display protrusions that are consistent with the idea that they are migrating on the overlying skin layer. However, imaging data alone are insufficient to conclude that this motile 'sheath' of cells supports the collective migration of the PLLp, as stated in the title. The only experimental test of this idea – removal of the overlying skin – results in a very strong defect in protrusions on both sides of the primordium, and thus does not reveal a specific requirement for the newly discovered apical motility.

Recommendation 1: Provide more direct evidence for the proposal that apical cells support PLLp migration by interacting with the overlying skin. Ideally, the authors could use their genetic mosaic technique to target these cells directly (rather than disrupting the skin). Alternatively, it may be possible to apply correlative approaches on pre-existing imaging data to further support the suggestion that the dynamic behavior of sheath cells has an influence on tissue migration. For example, do changes in sheath cell behavior, perhaps when they move from one skin cell to the next, have any effect on the migration of the PLLp (e.g. shearing) as might be expected from a chimneying model?

Comment: It may be argued that the imaging data, which clearly show the motile behavior of apical sheath cells on the skin, are sufficient to demonstrate the existence of circumferential motility mechanism. In that case, the authors should tone down their claim that these cells 'support' collective migration.

We have tried experiments similar to the ones suggested by the reviewer, but have been unable to target these cells with the precision necessary for these experiments, without also damaging surrounding tissues. However, we also now demonstrate that when re-embedded in matrigel basal, but not superficial, protrusion directionality is restored, while migration is still profoundly affected. We argue that this supports our conclusion that polarized superficial protrusive activity is necessary for migration. However, we recognize the limitations of the evidence provided and have we have toned down our claim that these cells support collective migration and added some necessary caveats.

2) The other major claim is that the study 'highlights the broad role confinement plays in the formation of polarized lamellipodia'. While 'confinement' is not clearly defined, the discussion centres around proposed roles for mechanical confinement in regulating protrusion types that have emerged from in vitro model systems. Here, the support for a role for mechanical confinement comes from skin removal experiments. The issue is that this experiment does more than reduce mechanical confinement, it also severely disrupts what is normally a closed and controlled chemical environment. It's hardly surprising that cells that normally migrate in the presence of patterned diffusible cues and organized matrices suddenly arrest when their closed and controlled environment is forced open.

Recommendation 2: In order to support the proposed role for mechanical confinement in the formation of lamellipodia the authors should rule out that the observed changes aren't the result of chemical effects such as osmotic changes. […] Better still, the authors could consider replacing Matrigel with agarose to alter mechanical confinement in a way analogous to the under agarose in vitro experiments.

We have considered these experiments, but felt that a more systematic exploration of the mechanical properties of artificial substrates would be better described in a separate manuscript that focuses on these parameters. We have added a sentence describing the rationale for using 20mg/mL matrigel (this was the undiluted concentration, and we reasoned that we would like to provide as stiff a substrate as possible for primordium migration) in the appropriate place in the Results.