Newts have a remarkable capacity to regenerate several anatomical structures and organs including their limbs, spinal cords, heart, tails, eye lenses, retinas, and upper and lower jaws. These regenerative processes are dependent upon the dedifferentiation of fully-differentiated cells in the vicinity of the amputation site. This degree of cellular plasticity is unique to organisms with marked regenerative capabilities and is not observed in mammals. However, our laboratory has recently demonstrated that terminally- differentiated mouse myotubes can be induced to dedifferentiate when stimulated with a protein extract from regenerating newt limbs. These results indicate that the signaling pathways for cellular dedifferentiation are intact in extracellular signals that initiate dedifferentiation. The goal of this proposal is to identify the newt genes that initiate dedifferentiation and control cellular plasticity in mammalian cells. Candidate cellular plasticity genes will be identified by performing differential display analysis and suppression subtractive cDNA hybridization between early limb regenerates and non regenerating limb tissues. Sequence analysis, degree of induction, and cellular expression patterns will be used as a basis for selecting candidate genes for further study. Genes that exhibit a significant induction and contain sequences suggesting they encode secreted proteins such as growth factors, cytokines or other ligands will be tested for their ability to initiate cellular dedifferentiation by treating cultured mouse myotubes with conditioned medium from cells expressing these candidate genes. Genes expressed in the underlying stump tissue that contain sequences suggesting they encode cellular protein such as receptors, kinases or transcription factors could be genes that respond to the ectopically expressing them in mouse myotubes. Using these methods, we expect to identify newt genes that function in regulating cellular plasticity in mammalian cells. Identifying these genes would have important implication for the future of regenerative medicine.
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