Intracellular pH dynamics promotes zebrafish larval tail regeneration

Intracellular pH (pHi) dynamics regulates numerous cellular processes, including migration and proliferation. While these functions are well-established in cell lines, the role of pHi changes in vivo is less well understood. We generated a transgenic zebrafish line expressing a fluorescent ratiometric pHi biosensor and identified functional changes in pHi during zebrafish larval tail regeneration. We found that tail amputation led to a transient decrease in pHi, followed by a prolonged increase in pHi above pre-amputation values. Moreover, we showed that pharmacologically inhibiting Na⁺/H⁺ exchanger (NHE) activity or decreasing extracellular pH attenuated the post-amputation increase in pHi, reduced subsequent cell proliferation, and impaired tail regeneration. We further found that inhibiting NHE activity post-amputation led to elevated inflammation, disrupted myeloid cell behavior, decreased reactive oxygen species, and increased glycogen synthase kinase-3 (GSK3) activity. Finally, we showed that the regeneration defects in larvae with disrupted pHi were partially rescued by pharmacological GSK3 inhibitors. Collectively, our data reveal a previously unrecognized role for pHi dynamics in coordinating tissue behaviors in vivo and enabling zebrafish larval tail regeneration.