Osmolarity-independent electrical cues guide rapid response to injury in zebrafish epidermis

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The ability of epithelial tissues to heal after injury is essential for animal life, yet the mechanisms by which epithelial cells sense tissue damage are incompletely understood. In aquatic organisms such as zebrafish, osmotic shock following injury is believed to be an early and potent activator of a wound response. We find that, in addition to sensing osmolarity, basal skin cells in zebrafish larvae are also sensitive to changes in the particular ionic composition of their surroundings after wounding, specifically the concentration of sodium chloride in the immediate vicinity of the wound. This sodium chloride-specific wound detection mechanism is independent of cell swelling, and instead is suggestive of a mechanism by which cells sense changes in the transepithelial electrical potential generated by the transport of sodium and chloride ions across the skin. Consistent with this hypothesis, we show that electric fields directly applied within the skin are sufficient to initiate actin polarization and migration of basal cells in their native epithelial context in vivo, even overriding endogenous wound signaling. This suggests that, in order to mount a robust wound response, skin cells respond to both osmotic and electrical perturbations arising from tissue injury.

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  1. ###Reviewer #3:

    This is an interesting paper examining the role of electric fields as a tissue damage signal for epithelial cells in vivo. Previous work had indicated the presence of electric fields in wounded tissues. But whether these phenomena play a role in early wound detection by epithelial cells has been unclear. The authors use live imaging in zebrafish to track the behaviour of epithelial cells in response to wounds. Imaging of actin dynamics was used as a readout for directional sensing in these cells. The authors show that directional sensing depends on the local concentration of specific electrolytes and that application of external electric fields can stimulate directional migration. These major conclusions are interesting and well supported. Although this is not the first time that electric fields are suggested to play a role, the study offers valuable direct evidence, in vivo evidence, and introduces a new system in which the mechanisms can be studied further.

    Main comment:

    The study is focused on establishing whether electric fields play a role in wound sensing and does not touch on how these effects are mediated. The experiments were designed to distinguish osmotic from electric effects, establish whether the effects are global or local and assess the direct effects of electric fields on epithelial cell motion. These are significant and do not appear trivial. Nevertheless, some insight, even in the form of discussion, into how these effects might be sensed by epithelial cells seemed to be lacking. At the minimum, the authors could provide ideas based on the literature. Ideally, the study would include an analysis of cytoskeletal rearrangements and calcium dynamics in response to electric fields or alterations of electrolytes for completion. The authors introduce these key readouts of epithelial signalling, but they did not make full use of these in their functional assays. Depending on whether electric fields influence the calcium wave, different mechanistic hypotheses can be made for future studies.

  2. ###Reviewer #2:

    I enjoyed the manuscript. Driving cell movement and even overriding wound migrational cues with an electric field is very interesting. My principal concern is that it appears the manuscript has been written in a way to downplay the previous findings in this field. I am no expert on the effects of electric fields on wound healing and chemotaxis, but a cursory look at the literature shows that that lot has been published in this arena. It appears that most if not all of the findings in this manuscript have been seen before in other contexts.

    The zebrafish offers a great set of tools to interrogate electric fields on chemotaxis and wound healing. I am simply asking for a bit of clarity with respect to the history of electrical fields, cell chemotaxis and wound healing. The authors need to provide more context for their work in the introduction with respect to electrical fields and more clearly describe what has been done before. In addition, the authors need to make additions to the conclusion that clearly define what is novel in their findings and how it relates to previous studies of electric fields and cell chemotaxis.

  3. ###Reviewer #1:

    This manuscript by Kennard and Theriot reports that electrical cues guide skin cells directional migration in response to injury. The authors bring molecular tools and analysis to study environmental cues, like osmolarity and electric fields in vivo. The effects of electrical cues are most studied in vitro. The in vivo model, the vivo approaches with molecular and imaging techniques bring bioelectricity research closer to mainstream techniques. Demonstrating the direct effect of electrical effects independent of osmolarity represent a significant step in this field. The results demonstrating the effects of NaCl, but not quite a few osmolarity control are impressive.

    I have the following questions and suggestions, which I do not expect the authors to address with new experiments, because as with other pioneering research, this manuscript suggests more research questions/directions on the basis that it answers some very important questions. I believe perhaps the authors already have some results to some of those questions.

    1. Good reason for choosing laceration over transection is given. I am a bit puzzled if the EFs and osmolarity are the mechanisms, why were there such differences? The endogenous EFs and osmolarity would be expected to be the same in both the laceration and transection models. Could the laceration stretch the tissue during injury procedure, so the marked increased migration was present in the laceration model? The stretch could activate stretch activated channels, stimulate cells, and realign matrix.

    2. It is not clear what relationship can be established between GCaMP6f response and migration speed (Fig.1E, G, H). inhibition of the calcium response may help to test the relationship.

    3. The local concentration of NaCl showed remarkable inhibitory effects on cell migration, and cell volume. As we know injury may activate channels and pumps, which then facilitate the ionic fluxes, thus generate persistent ionic currents. Channel and pump inhibition experiments could quickly point to some molecular basis of the involvement of NaCl.

    4. I consider using Iso KCl is very interesting, because high K+ would significantly modulate cell membrane potential, however the effect on cell migration is very similar to those of Iso Choline Cl, iso NaGlunate, Iso Sorbitol. This would provide another side evidence for the role of wound electric fields in cell migration.

    5. 200V DC is much higher than endogenous EFs expected in such a model. Caution should be given when interpreting the results. I also wonder whether the authors attempted experiments (Fig. 4B, C) using wounded animals, perhaps the tissues after injury are not technically plausible (too fragmented) for such experiments.

    6. One assumption in the paper is the TEP and wound EFs in vivo. Glass microelectrodes may be able to verify those in space and time. If this works (the TEP and wound EFs can be mapped), the effects of various treatments can be tested and exclude other possibilities.

  4. ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.


    This paper examines the role of electric fields as a damage signal for epithelial cell wounding using a zebrafish tail laceration in vivo model. While electrical fields had been previously noted in vitro, whether they played a role in early wound detection by epithelial cells has been unclear. They tracked the ability of epithelial cells to sense direction by imaging actin dynamics in zebrafish epidermis. From these studies, they find that directional sensing depends on the local concentration of specific electrolytes. Additionally, external electric fields can independently stimulate directional migration.