Plasticity of salamander skeletal muscle cells

Sammanfattning: Regeneration is unevenly spread throughout the animal kingdom. Some of the invertebrates have a very high regenerative capacity but the capacity to replace lost or damaged tissues in mammals is limited. Among the vertebrates, the aquatic salamanders are the champions of regeneration, being able to regenerate body parts such as their limbs, tail, and jaw. Understanding the cell- and molecular machinery underlying these events can in the end lead to improvements in the field of regenerative medicine. When a salamander limb is amputated, a so called blastema is formed at the tip of the stump. This is a mesenchymal growth zone, from where the cells for the new limb originate. The skeletal muscle is believed to contribute to the blastema and the newly formed limb during regeneration in salamanders. There are two possible mechanisms by which this could occur. First, the muscle reserve cells, satellite cells, become activated, and after subsequent expansion they incorporate in to the blastema. Second, the multinucleated muscle cell dedifferentiates in a cellularization process, forming several new progenitor cells that contribute to the regenerate. These two mechanisms are the subjects of study in this thesis. In the first paper, satellite cells were monitored during the regeneration of the salamander limb. The satellite cell population was found to be restored after several rounds of amputation/regeneration, demonstrating a robust mechanism to replenish this cell population. By genetically labeling satellite cell progeny with GFP, cells were traced during limb regeneration. The labeled cells were found to contribute to cartilage tissue as well as to muscle, suggesting that they have lineage switching potential. The satellite cell marker Pax7 was rapidly down regulated in the blastema, indicating that the blastema has a reprogramming activity. In the second paper, the link between injury and dedifferentiation of muscle was explored. Previously it was shown that the skeletal muscle fiber must suffer a direct cellular injury in order to dedifferentiate in vivo. This led us to hypothesize that a programmed cell death response initiated by the injury, is linked to the dedifferentiation of this cell type. In order to investigate this, cellular events were examined after treating the cells with a known cellularization inducer called myoseverin, and pro-apoptotic drugs. The responses after these treatments were found to be similar, they both evoked cellularization of the muscle cells, and this was preceded by features of an activated apoptotic machinery. By inhibiting the intrinsic pathway of the apoptotic machinery, cellularization was inhibited. Finally, dedifferentiated progeny generated by pro-apoptotic stimuli was found to be capable of reentering the cell cycle and proliferate. Together these results indicate that the injury is directly linked to dedifferentiation through the apoptotic machinery.