Injury-induced Neuregulin-EGFR signaling from muscle mobilizes stem cells for whole-body regeneration in acoels

The activation of progenitor cells near wound sites is a common feature of regeneration across species, but the conserved signaling mechanisms responsible for this step in whole-body regeneration are still incompletely understood. The acoel Hofstenia miamia undergoes whole-body regeneration using Piwi + pluripotent adult stem cells (neoblasts) that accumulate at amputation sites early in regeneration. The EGFR signaling pathway has broad roles in controlling proliferation, migration, differentiation, and cell survival across metazoans. Using candidate RNAi screening, we identify the Hofstenia EGFR egfr-1 and Neuregulin nrg-1 genes as essential for blastema formation. Structure prediction of NRG-1 and EGFR-1 proteins suggests these factors interact directly. After amputation injuries, nrg-1 expression is induced in body-wall muscle cells at the wound site by 6 h and localizes to the tip of the outgrowing blastema by 24 h and sustains for several days, while egfr-1 is broadly expressed, including in muscle and neoblasts. The early phase of nrg-1 expression occurs at incision sites that repair through wound healing while the late phase is specific to blastema formation. Under nrg-1(RNAi) and egfr-1(RNAi) conditions that impair blastema formation, animals still undergo the earliest responses to injury to activate expression of the Early Growth Response transcription factor egr. In addition, RNAi of nrg-1 only after amputation results in regeneration failure, indicating regeneration requires new nrg-1 expression. nrg-1(RNAi) and egfr-1(RNAi) animals possess Piwi+ and H3P + mitotic neoblasts which hyperproliferate normally after amputation, but these cells fail to accumulate at the wound site. Therefore, muscle provides a source for Neuregulin-EGFR signaling after injury and is necessary for the mobilization of proliferative progenitors to enable blastema outgrowth for whole-body regeneration in Hofstenia. These results indicate a shared functional requirement for muscle signaling to enable regeneration between planarians and acoels across 550 million years of evolution.