Development of transgenic knockdown methods in Ambystoma mexicanum to study satellite cell activation during limb regeneration

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Salamander appendage regeneration induces adult tissue to produce progenitor cells with embryonic properties called the blastema. This may occur by dedifferentiation of mature cell types or activation of resident stem cells. The relative importance of dedifferentiation versus satellite stem cell activation to regeneration is completely unknown. Here we will determine the role of muscle dedifferentiation versus satellite cell activation in appendage regeneration using the axolotl as a model system. We will separately track the fate of dedifferentiated muscle and satellite cells using cre/loxP mediated recombination in transgenic animals. We have previously developed transgenesis in the axolotl and demonstrated that the cre/loxP system functions efficiently in mature tissues. We will further test the role of dedifferentiation versus stem cells by inhibiting their occurrence. To do this efficiently we will first develop site-specific integration into the axolotl genome so that we do not have to pass trangenes through the germline before performing experiments. We will then integrate expression cassettes to permanently express the muscle differentiation factor, myogenin, and the cell cycle inhibitor, p16INK4 in muscle or satellite cells to block production of progenitors from these sources. Finally our goal is to dissect the molecular pathways controlling processes like muscle dedifferentiation that are involved in early blastema formation using these integration strategies. Our first target will be the wnt pathway.