Reversibility of the mononucleate-to-multinucleate myogenic transition during amphibian limb regeneration

Muscle differentiation involves the fusion of mononucleate myoblasts to form multinucleate syncytial myotubes. In order to assess reversibility of the mononucleate-to-multinucleate transition in urodele limb regeneration, myotube formation was induced in cultured newt limb blastemal cells. Myotubes were purified, replated at low density and injected with a cytoplasmic lineage tracer. In some cases nuclei of myotubes were labelled by incorporation of tritiated thymidine. Labelled myotubes were stable in culture for 6-8 weeks and no transfer to mononucleate cells was observed. The myotubes were implanted under the wound epidermis of a hindlimb blastema. Labelled mononucleate cells were observed 1 week after implantation and such cells could derive both the cytoplasmic lineage tracer and the nuclear marker. The number of such cells increased by 2-3 weeks after implantation. These results provide strong support for the reversibility of muscle differentiation during urodele limb regeneration, and raise questions about the mechanism of such a reversal.