Roles of body-wide injury responses in axolotl limb regeneration - ABSTRACT Patients whose lives have been permanently altered by limb loss are currently left with prosthetic options for restoration of function, but these are not perfect. Prostheses are limited in sensation, articulation, and integration, among other concerns. Complete restoration of the lost limb via therapeutic intervention remains a distant goal of regenerative medicine. This goal would be made significantly more tangible if a clear roadmap for how complex tetrapod limbs can be regenerated was elucidated. Such a roadmap would detail possible sources of progenitor cells and the cues that activate them to proliferate as well as instructions for how these substrate cells undergo the changes necessary for them to be competent to build a new limb and to do so with morphological precision. Since humans, mice, and other mammals do not naturally regenerate full limbs, the roadmap is not likely to arise from mammalian studies alone. However, salamanders, such as axolotls, are profoundly regenerative and can completely replace amputated limbs with precision throughout life, and, with experimentation, they thus offer a chance to build this roadmap. How processes that operate at the whole-body, systemic level to regulate progenitor cell activation and blastema formation remain critical unsolved issues in the limb regeneration field. A blastema is the bud-like structure that grows at the tip of the stump following amputation and contains the progenitor cells necessary to build a new limb. The blastema is essential for regeneration, and, except for the distal-most dip of digits, mammals do not form blastemas following limb amputation. Thus, understanding the controls, including the system-wide responses and inputs, that govern progenitor cell activation and blastema formation in salamanders is key to evaluating limitations observed in mammals. We hypothesize systemically-activated cells promote local limb regeneration in axolotl salamanders. We further hypothesize that axolotls use the hypothalamus-pituitary-adrenal (HPA) neuroendocrine system to stimulate systemic activation and that the central nervous system coordinates injury responses to orchestrate limb regeneration. This project uses modern tools, such as cell sorting and transplantation, paired scRNA-seq and ATAC-seq, and genome editing, to understand the biology of systemically-activated cells. This project will also use implanted flexible-mesh sensors and live neuronal activity recording for evaluating how the axolotl brain responds to limb amputation and how its activity changes as limbs regenerate. This work will lay the basis for understanding how the CNS may be required in the limb regeneration process. This project will provide important molecular and systems- level insights into how a highly-regenerative tetrapod responds to amputation and, ultimately, grows a new limb. It is therefore likely to have implications in regenerative medicine and evolutionary biology.
