Velloso, Cristiana Paranhos; (2000) Cell cycle re-entry and the plasticity of myotubes in newt limb regeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Urodele amphibians regenerate various structures including the limb, tail, lens, and heart. Underlying this regenerative ability may be the plasticity of cells at the site of injury. Muscle is a key tissue to study these issues because myogenesis proceeds by fusion of mononucleate precursor cells into a multinucleate syncytium which is in a state of stable post-mitotic arrest. Following implantation into a regenerating limb, newt myotubes re-enter the cell cycle and give rise to proliferating mononucleate progeny. In culture, generation of mononucleate cells has never been observed, but the myotube nuclei re-enter the cell cycle and arrest in G2. This is in contrast to their mammalian counterparts, which are refractory to growth factor stimulation, and suggests that cell cycle re-entry may be one aspect of myotube plasticity. In initial experiments mouse and newt myoblasts were fused to create hybrid myotubes. In these hybrids, DNA synthesis was observed in both mouse and newt nuclei demonstrating that the post-mitotic arrest of mammalian nuclei can be destabilised. Other experiments addressed the hypothesis that mononucleate cells are generated from multinucleate myotubes by fragmentation of the syncytium, as the nuclei progress through mitosis and cytokinesis. I pursued several strategies to overcome the G2 arrest of cultured myotubes. Transfection with SV-40 large T antigen induced endoreplication of DNA in myotubes, but did not lead to mitosis. Treatment with caffeine resulted in the appearance of fragmented nuclei, which are indicative of aberrant mitosis. This response was dependent on traversal of S-phase by the myotube nuclei. These observations suggested that the block to mitosis is stable in cultured myotubes. A critical question is whether cell cycle re-entry is required for generation of mononucleate cells following implantation of myotubes into regenerating limbs. Cell-cycle re-entry was blocked in myotubes by X-irradiation or expression of the cdk4/6 inhibitor pl6. These myotubes were fluorescently labelled and implanted. Both arrested and control myotubes gave rise to mononucleate progeny, demonstrating that cell cycle re-entry is not required for generation of mononucleate cells.

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