Epithelial wound healing in Clytia hemisphaerica provides insights into extracellular ATP signaling mechanisms and P2XR evolution

Epithelial wound healing involves the collective responses of many cells, including those at the wound margin (marginal cells) and those that lack direct contact with the wound (submarginal cells). How these responses are induced and coordinated to produce rapid, efficient wound healing remains poorly understood. Extracellular ATP (eATP) is implicated as a signal in epithelial wound healing in vertebrates. However, the role of eATP in wound healing in vivo and the cellular responses to eATP are unclear. Almost nothing is known about eATP signaling in non-bilaterian metazoans ( Cnidaria, Ctenophora, Placozoa, and Porifera ). Here, we show that eATP promotes closure of epithelial wounds in vivo in the cnidarian Clytia hemisphaerica (Clytia) indicating that eATP signaling is an evolutionarily ancient strategy in wound healing. Furthermore, eATP increases F-actin accumulation at the edges of submarginal cells. In Clytia, this indicates eATP is involved in coordinating cellular responses during wound healing, acting in part by promoting actin remodeling in cells at a distance from the wound. We also present evidence that eATP activates a cation channel in Clytia epithelial cells. This implies that the eATP signal is transduced through a P2X receptor (P2XR). Phylogenetic analyses identified four Clytia P2XR homologs and revealed two deeply divergent major branches in P2XR evolution, necessitating revision of current models. Interestingly, simple organisms such as cellular slime mold appear exclusively on one branch, bilaterians are found exclusively on the other, and many non-bilaterian metazoans, including Clytia, have P2XR sequences from both branches. Together, these results re-draw the P2XR evolutionary tree, provide new insights into the origin of eATP signaling in wound healing, and demonstrate that the cytoskeleton of submarginal cells is a target of eATP signaling.