Development and validation of genetic cell ablation systems in axolotl

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Abstract: The axolotl is a classical and robust model of tissue regeneration. Axolotls are well known for studies of limb regeneration but also regenerate their eyes, lungs, heart, kidney, spinal cord, brain and provide a system to study immune regulation of regeneration of all body tissues and organs. Studying mechanisms of regeneration in axolotl provides a roadmap for engineering similar regeneration mechanisms in mammals and ultimately humans. Two key questions in regeneration biology that remain incompletely understood are: (1). What are the sources of replacement cells during injury repair? and (2). How do cells interact during regeneration to create complex tissues that function as well as the original organs? Our work on generating transgenic axolotl has brought this classical system into the modern genomic era where we can now lineage trace cells that contribute to regeneration and start to tease apart specific regeneration mechanisms in different organs. However, to fully understand the contribution of different cell types and lineages to regeneration, cell ablation approaches are required. Cell ablation allows us to ask questions such as: What is the role of a specific cell type in a complex mixture of cells during regeneration? Is a certain cell type necessary for organ/appendage regeneration? Can other cells act as a reserve pool when the cell type that normally participates in the regenerative process is missing? How do different cell types contribute to the overall patterning of the organ or tissue regenerate? To answer these important questions, we propose generating broadly applicable transgenic axolotl tools for lineage specific cell ablation studies. These tools either utilize enzymes that produces toxins or make use of proteins/toxins that activate/induce cell apoptosis. Specifically, we will generate nitroreductase (NTR) or Diphtheria toxin receptor (DTR) transgenic axolotls under LoxP control and cross them with tissue specific inducible Cre lines that are already available in our laboratory to specifically express DTR or NTR in the tissue of interest. This will provide us spatial control over cell ablation. Further by providing metronidazole (drug) or diphtheria (toxin) respectively to NTR and DTR lines, we will be able to achieve temporal control. Such spatiotemporal control of cell ablation can be achieved over any cell type if an inducible Cre line is available for the tissue of interest. Hence, we believe that the development of genetic cell ablation systems in axolotl will open doors for studying a wide variety of organs and appendages for example, limb, tail, eye, lung, heart, kidney, spinal cord, brain and the role of immune cells in axolotl tissue regeneration.