Voltage-gated calcium channels generate blastema Ca 2+ fluxes restraining zebrafish fin regenerative outgrowth.

Abstract

Adult zebrafish fins regenerate to their original size regardless of damage extent, providing a tractable model of organ size and scale control. Gain-of-function of voltage-gated K ⁺ channels expressed in fibroblast-lineage blastema cells promotes excessive fin outgrowth, leading to a long-finned phenotype. Similarly, inhibition of the Ca ²⁺ -dependent phosphatase calcineurin during regeneration causes dramatic fin overgrowth. However, Ca ²⁺ fluxes and their potential origins from dynamic membrane voltages have not been explored or linked to fin size restoration. We used fibroblast-lineage GCaMP imaging of regenerating adult fins to identify widespread, heterogeneous Ca ²⁺ transients in distal blastema cells. Membrane depolarization of isolated regenerating fin fibroblasts triggered Ca ²⁺ spikes dependent on voltage-gated Ca ²⁺ channel activity. Single cell transcriptomics identified the voltage-gated Ca ²⁺ channels cacna1c (L-type channel), cacna1ba (N-type), and cacna1g (T-type) as candidate mediators of fibroblast-lineage Ca ²⁺ signaling. Small molecule inhibition revealed L- and/or N-type voltage-gated Ca ²⁺ channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, cacna1g homozygous mutant zebrafish regenerated extraordinarily long fins due to prolonged outgrowth. The regenerated fins far exceeded their original length but with otherwise normal ray skeletons. Therefore, cacna1g mutants uniquely provide a genetic loss-of-function long-finned model that decouples developmental and regenerative fin outgrowth. Live GCaMP imaging of regenerating fins showed T-type Cacna1g channels enable Ca ²⁺ dynamics in distal fibroblast-lineage blastemal mesenchyme during the outgrowth phase. We conclude "bioelectricity" for fin size control likely entirely reflects voltage-modulated Ca ²⁺ dynamics in fibroblast-lineage blastemal cells that specifically and steadily decelerates outgrowth at a rate tuned to restore the original fin size.