Planarian microtubules form a network within muscle and regulate injury-induced genes essential for regeneration patterning

Planarian muscle produces key wound signal patterning whole-body regeneration. Within muscle, generic induction of wnt1 promotes tail regeneration, while polarized expression of the Wnt inhibitor notum at anterior-facing wounds drives head regeneration. Classic experiments indicate that microtubules are also involved in blastema fating, but the cell biology of planarian muscle is still poorly understood. We raised an antibody to muscle-expressed TUBA-2 and found that planarian muscle possesses a microtubule network linking contractile fibers with their mononucleated cell bodies. Microtubules were required for muscle fiber regrowth across wound sites at times that correlated with expression of wound-induced genes. Expression profiling found that sublethal colchicine treatment disrupted a subset of muscle-expressed injury-induced genes, with strongest effects on wnt1 and notum. Higher colchicine doses (>200 µg/ml) prevented wnt1 and notum expression, while, surprisingly, lower doses (125 µg/ml) elevated notum at posterior-facing wounds, thereby implicating microtubules in both the activation and polarization of genes expressed from injured muscle. Furthermore, microtubules functionally interact with Wnts to control head/tail determination. Together, planarian microtubules act in specific regulatory pathways to express key muscle-expressed and injury-induced factors used for blastema fating.