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

The author team shared

Author Response

Thank you for your thoughtful summary of our work! Here are our thoughts on your questions:

• This work shows that trans-epithelial potential (TEP) is important and disruption of TEP can induce cell polarization. It has been suggested that TEP is maintained by ion channels and sodium-potassium pumps. With wounding, electrical flows are generated locally eliciting cellular responses. Could the authors suggest how are these flows maintained over time?

There are two main factors contributing to long-lived electrical currents. First the resistance to ion flow has to be fairly high, so that the flow of ions out of the tissue will not occur all at once. A hydraulic analogy is appropriate: the electrical potential driving ion flow out of the epidermis is analogous to a lake that is being drained by a river. The lake will drain slower through a small river that permits less water flow than through a large river. In our system we believe this could be accomplished if ion flow were restricted to the narrow spaces between cells within the epidermis (Robinson and Messerli, 2003).

The second contributing factor is the gradual replenishment of ions by compensatory inward ion flow distant from the wound. In the hydraulic analogy, rain can fill up the lake and prevent it from running dry. In the epidermis, small ion flows occur throughout the epidermis (see Reid et al., 2007 Fig 5d for a mapping of these currents across an unwounded zebrafish larva). While the magnitude of these flows is small, integrated across the rest of the unwounded skin, they can be substantial. Furthermore, it is possible that wounding could induce changes to ion flows elsewhere in the skin, by opening channels or increasing the activity of the pumps that maintain the TEP in homeostasis. For example, in the corneal epithelium of the eye, which is a well-studied model system for wound-induced electric currents, chloride has been identified as a major contributor to the wound-induced current, and there is evidence that chloride channels become upregulated throughout corneal tissue following wounding (Vieira et al. 2011). Building on our work, it would be very informative to map the current flows around the zebrafish larvae during wound healing to see how they are affected by injury. Combined with pharmacological inhibition of ion channels, this readout could be very useful for pinning down the molecular basis of electrical activity in the zebrafish epidermis, particularly during wound healing.

• It has also been shown that cell polarization is evident several hundred microns away from the wound. How is this response elicited when the changes in the electrical flows are so local? Or can small lacerations cause large scale electrical flows?

We favor a model in which lacerations induce electrical flows over at least 200-300 microns, the same range of tissue that becomes polarized to a wound following injury. Indeed, the fact that alterations to electrical potentials can rapidly induce changes in electrical activity across long distances is one of the conceptual advantages of electric fields as a physical coordinating cue. Admittedly, we have not directly measured the electric fields at different locations relative to the wound. However, when this has been done in wounded Xenopus tadpoles, electrical gradients can be measured at least a hundred microns away from the wound (Ferreira et al. 2016).

As we followed the science of the remarkably fast and coordinated wound response in the zebrafish skin, this project led us into the exciting yet technically challenging field of bioelectricity. We were impressed with the deep body of work that laid the foundations for the concept of electric fields in wound healing. We believe a major contribution of this paper is to connect the bioelectric measurements that others have made to specific cytoskeletal responses at the cellular level in vivo. We hope that professional electrophysiologists will be inspired by our work to consider zebrafish an ideal model system for integrating electrical and cell biological analyses for a fuller understanding of wound healing.

References

Ferreira F, Luxarxi G, Reid B, Zhao M.  Early bioelectric activities mediate redox-modulated regeneration. Development 2016; 143(24): 4582-4594. doi:10.1242/dev.142034

Robinson KR and Messerli MA. Left/right, up/down: the role of endogenous electrical fields as directional signals in development, repair and invasion. BioEssays 2003; 25(8): 759-766. doi: 10.1002/bies.10307

Reid B, Nuccitelli R, Zhao M. Non-invasive measurement of bioelectric currents with a vibrating probe. Nat Protoc 2007; 2(3): 661-669. doi:10.1038/nprot.2007.91

Vieira AC, Reid B, Cao L, Mannis MJ, Schwab IR, Zhao M. Ionic components of electric current at rat corneal wounds. PLoS ONE 2011; 6(2): e17411. doi: 10.1371/journal.pone.0017411