| The formation of a blastema during regeneration of an axolotl limb involves important changes in the behavior and function of cells at the site of injury. One of the earliest events is the formation of the wound epithelium and subsequently the apical epidermal cap, which involves in vivo dedifferentiation that is controlled by signaling from the nerve. I have investigated the role of epigenetic modifications to the genome as a possible mechanism for regulating changes in gene expression patterns of keratinocytes of the wound and blastema epithelium that are involved in regeneration. I report a modulation of the expression DNMT3a, a de novo DNA methyltransferase, within the first 72 hours post injury that is dependent on nerve signaling. Treatment of skin wounds on the upper forelimb with decitabine, a DNA methyltransferase inhibitor, induced changes in gene expression and cellular behavior associated with a regenerative response. Furthermore, decitabine-treated wounds were able to participate in regeneration while untreated wounds inhibited a regenerative response. Elucidation of the specific epigenetic modifications that mediate cellular dedifferentiation likely will lead to insights for initiating a regenerative response in organisms that lack this ability. To this end, a novel RNA seq experiment has been designed to compare the transcription profiles of tissues that have undergone dedifferentiation to uninjured tissues and tissues that participate in simple wound healing. In addition, tissues treated with a DNA methyltransferase inhibitor will be evaluated for changes in gene expression that are a result of DNA methylation dynamics specifically. As the mechanisms behind in vivo cellular dedifferentiation are elucidated and applied, it will be important to use an appropriate assay in order to determine the success and extent of dedifferentiation. I include here a developed protocol designed to assay for cellular dedifferentiation called the Excisional Repair Model (ERM) model. Briefly, treated cells or tissues are grafted into a cavity in the lower forearm created by the excision of a segment of the radius. The ability of the treated cells or tissue to differentiate and replace the missing bone fragment, and incorporate into the existing bone reveals their regenerative capacity. |
