Throughout their lifetime, fish maintain a high capacity for regenerating complex tissues after injury. We utilized a larval tail regeneration assay in the zebrafish Danio rerio, which serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to promote glucose metabolism during early regeneration similar to the Warburg effect observed in tumor forming cells. Inhibition of glucose metabolism did not affect the overall health of the embryo but completely blocked the tail from regenerating after amputation due to the failure to form a functional blastema. We performed a time series of single-cell RNA sequencing on regenerating tails with and without inhibition of glucose metabolism. We demonstrated that metabolic reprogramming is required for sustained TGF-β signaling and blocking glucose metabolism largely mimicked inhibition of TGF-β receptors, both resulting in an aberrant blastema. Finally, we showed using genetic ablation of three possible metabolic pathways for glucose, that metabolic reprogramming is required to provide glucose specifically to the hexosamine biosynthetic pathway while neither glycolysis nor the pentose phosphate pathway were necessary for regeneration.

Mammals are generally poor at tissue regeneration, in contrast, fish maintain a high capacity for regenerating complex tissues after injury. Using larval zebrafish, we show that tail amputation triggers an metabolic shift to glycolysis in cells surrounding the notochord as they reposition to the amputation site. Blocking glycolysis prevents the fin from regenerating after amputation due to the failure to form a normal, pluripotent blastema. We performed a time series of scRNA-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-b dependent dedifferentiation and epithelium-to-mesenchyme transition to become pluripotent blastema cells. We further demonstrated that the metabolic switch to glycolysis is required for TGF-b signaling and blocking either glycolysis or TGF-b receptors results in aberrant blastema formation through the suppression of essential EMT mediators such as snai1.

Genetic variation in the membrane trafficking adapter protein complex 4 (AP-4) can result in pathogenic neurological phenotypes including microencephaly, spastic paraplegias, epilepsy, and other developmental defects. We lack molecular mechanisms responsible for impaired AP-4 function arising from genetic variation, because AP-4 remains poorly understood structurally. Here, we analyze patterns of AP-4 genetic evolution and conservation to identify regions that are likely important for function and thus more susceptible to pathogenic variation.We map known variants onto an AP-4 homology model and predict the likelihood of pathogenic variation at a given location on the structure of AP-4. We find significant clustering of likely pathogenic variants located at the interface between the β4 and N-μ4 subunits, as well as throughout the C-μ4 subunit. Our work offers an integrated perspective on how genetic and evolutionary forces affect AP-4 structure and function. As more individuals with uncharacterized AP-4 variants are identified, our work provides a foundation upon which their functional effects and disease relevance can be interpreted.

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