A metabolic shift to glycolysis promotes zebrafish tail regeneration through TGF-β dependent dedifferentiation of notochord cells to form the blastema

Mammals are generally poor at tissue regeneration, often resulting in permanent damage (scarring) or complete loss of tissues, organs, and extremities following injury or as a natural consequence of ageing. In contrast, throughout their lifetime fish maintain a high capacity for regenerating complex tissues after injury. Studying these processes should provide insights into the pathways necessary to trigger therapeutic regeneration in humans. We utilize the zebrafish Danio rerio, which are able to regenerate many of their tissues and organs such as: the fin, retina, spinal cord, inner ear, and heart. In particular, the embryonic zebrafish tail serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Importantly, regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, which is a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to glycolysis in cells comprising and surrounding the notochord during the repositioning of these cells near the amputation site. This metabolic switch is similar to the Warburg effect observed in tumor forming cells. Inhibition of glycolysis does not affect the overall health of the embryo but completely blocks the fin from regenerating after amputation due to the failure to form a normal, pluripotent blastema. To gain a better understanding of the molecular pathways that are regulated by metabolic signaling and guide blastema formation, we performed a time series of single cell RNA-sequencing on regenerating tails under normal conditions or in the absence of glycolysis. Strikingly, we detected a transient cell population in the single cell analysis that represents notochord sheath cells undergoing a TGF-β dependent dedifferentiation and epithelium-to-mesenchyme transition (EMT) to become pluripotent blastema cells. We further demonstrated that the metabolic switch to glycolysis is required for TGF-β signaling and blocking either glycolysis or TGF-β receptors results in aberrant blastema formation through the suppression of essential EMT mediators such as snai1. These studies not only provide new insights into tissue regeneration, but also cancer biology by demonstrating that the shift to glycolysis in the Warburg effect is not necessarily only utilized for quick denergy production by rapidly proliferating cells, but acts a cell signaling trigger that induces EMT prior to regeneration.