A dynamic repertoire of wound closure strategies precedes whole-body regeneration

Abstract

Wound closure is an essential aspect of successful regeneration, often acting as the first morphogenetic event that precedes downstream cellular events. Despite heavy investment in studying whole-body regeneration in many invertebrate systems, the steps by which these organisms heal their wounds remain understudied. Here, we investigate the cellular mechanisms of wound healing in the acoel Hofstenia miamia, an invertebrate worm capable of whole-body regeneration. H. miamia have two distinct epithelial layers, an outer epidermis and the epithelial lining of their pharynx. By labeling regenerating fragments with an actin dye, we found that H. miamia use distinct mechanisms of epithelial wound repair across different injury contexts. In transverse wounds that dont injure the pharyngeal epithelium, the epidermis closes by gradual radial constriction. In contrast, injuries that damage both the epidermis and the pharyngeal epithelium show the formation of long, actin-rich protrusions that cross the wound gap and form heterotypic bridges prior to re-epithelialization. Muscle contraction is required for the formation of heterotypic bridges - when animals are anesthetized and immobile, they are unable to form these cellular bridges, and epidermal cells cannot migrate independently. Global actomyosin contractility also plays a role in repair mechanisms, and pharmacological perturbation of contractility shifts the dynamics of wound closure after amputation. In the presence of blebbistatin, heterotypic bridge formation is inhibited but homotypic re-epithelialization is accelerated through increased cell crawling. Together, this work identifies mechanisms by which epithelial layers close large wounds in vivo, identifying novel heterotypic cellular bridges in wound closure and demonstrating the conservation of actin-mediated processes in an early-diverging phylum.