Characterization of the dedifferentiation process induced by newt regeneration derived extracts

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The newt, Notophthalmus viridescens, has the ability to regenerate structures (eg. limbs, tail, spinal cord, lens) which are lost to injury or amputation. Cells at the site of the injury revert back to an embryonic type of cell that can then proliferate and switch fate to rebuild the lost structure. This process, called dedifferentiation, is one of the keys to the regeneration process, and understanding the molecular basis behind dedifferentiation could help to enhance regenerative potential in humans. Studies have shown that mammalian muscle cells, when treated with newt regeneration extracts, re-enter the cell cycle and break-up from their multinucleated structures to yield proliferating mononuclear cells. This process is not very efficient, however, and many of the cells undergo cell death in the process. The aim of the current research proposal is to study the dedifferentiation process in newt cells in order to better understand how to improve dedifferentiation and regeneration in humans. We will study newt A1 muscle cells in culture that have been induced to dedifferentiate by adding serum to the medium or by treatment with newt-derived extracts. We will use a technique called Representational Difference Analysis (RDA) to isolate and characterize genes with potential regulatory roles during the newt dedifferentiation process. We present a comprehensive strategy for the isolation, prioritization and analysis of candidate genes with regulatory roles in the dedifferentiation process. We will 1) Conduct RDA on cell lines to identify dedifferentiation-initiating candidate genes, 2) Prioritize cDNAs to identify relevant candidates and 3) Perform functional analysis of candidate genes in regeneration. An understanding of the mechanisms involved in dedifferentiation will have tremendous therapeutic applications in degenerative disease and in neuronal injury.