Cooperation between proximate cell layers drives large-scale wound closure prior to whole-body regeneration

Wound healing is a fundamental property of animal life, but the extent to which large injuries can be endogenously repaired varies across phyla. In vertebrates, minor wounds can re-epithelialize on their own, but major wounds often result in imperfect re-epithelialization and fibrotic scarring. In contrast, more regenerative species are capable of both scarless healing and subsequent replacement of missing structures. Due to limitations in many animals' ability to recover from large, multi-tissue injuries, much of our understanding of wound closure comes from experiments studying gaps in epithelial monolayers. In comparison, the mechanisms by which animals can heal large wounds have been characterized primarily at the histological level, usually in non-model species that undergo substantial regeneration after wound healing. Here, we investigate the cellular dynamics of large-scale wound healing in the acoel Hofstenia miamia, an invertebrate worm capable of whole-body regeneration. H. miamia lack a traditional basement membrane and epidermal cells are not separated from underlying muscle by an organized layer of extracellular matrix - making the system incompatible with canonical mechanisms of crawling-based wound healing and raising the possibility that novel mechanisms may be driving closure. By labeling injured animals with an actin dye, we found that H. miamia epidermal cells extend long, actin-rich cytoplasmic protrusions across the wound edge until they reach the nearest epithelial layer and establish cell-cell contact. This process is dependent on muscle contraction; when animals are anesthetized and immobile, they are unable to form these cellular bridges, and epidermal cells cannot migrate independently. At injury sites that lack underlying muscle connecting the wound edge, wounds heal through the formation of organism-wide contractile purse strings. Together, this work identifies mechanisms by which multiple tissue types work together to close large wounds in vivo, indicating a deep conservation of proximate epithelial-mesenchymal interactions in morphogenetic processes.