Summary Regeneration starts with injury. Yet how injuries affect gene expression in different cell types, and how distinct injuries differ in gene expression remains unclear. We defined the transcriptomes of major cell types of planarians – flatworms that regenerate from nearly any injury – and identified 1,214 tissue-specific markers across 13 cell types. RNA sequencing on 619 single cells revealed that wound-induced genes were either expressed in nearly all cell types or specifically in one of three cell types (stem cells, muscle, or epidermis). Time-course experiments following different injuries indicated a generic wound response is activated with any injury regardless of the regenerative outcome. Only one gene, notum, was differentially expressed early between anterior- and posterior-facing wounds. Injury-specific transcriptional responses emerged 30 hours after injury, involving context-dependent patterning and stem-cell-specialization genes. The regenerative requirement of every injury is different; however, our work demonstrates that all injuries start with a common transcriptional response.

The ability to regenerate missing body parts exists throughout the animal kingdom. Positional information is critical for regeneration, but how it is harbored and utilized by differentiated tissues is poorly understood. In planarians, positional information has been identified through RNA interference (RNAi) phenotypes in which the wrong tissues are regenerated. For example, Wnt pathway inhibition leads to regeneration of heads in place of tails1–3. Characterization of such striking adult phenotypes led to identification of genes expressed in a constitutive and regional manner, associated with patterning, called position control genes (PCGs). Most PCGs are expressed within the planarian muscle4. Despite this major positional information role for muscle in planarians, how muscle is specified and how different muscle subsets impact regeneration is unknown. Here we found distinct regulatory roles for different planarian muscle fibers during regeneration. myoD was required for formation of a specific muscle cell subset: the longitudinal fibers, oriented along the anterior-posterior (AP) axis. Loss of longitudinal fibers led to a complete regeneration failure because of defects in regeneration initiation. A different transcription factor (TF)-encoding gene, nkx1-1, was required for formation of circular fibers, oriented along the medial-lateral (ML) axis. Loss of circular fibers led to a bifurcated AP axis with fused heads forming in single anterior blastemas. Our results demonstrate distinct roles for muscle fiber types in orchestrating planarian regeneration. Whereas muscle is often viewed as a strictly contractile tissue, these findings reveal specific regulatory roles for distinct muscle classes in wound signaling and patterning to enable regeneration.