Ion signaling, cell transitions, and organ scaling during fin regeneration

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PROJECT SUMMARY Organs “know” when and how to stop growing to arrive at the correct size and shape. Disruption of organ size control mechanisms leads to congenital abnormalities, poor organ homeostasis and tissue repair, and tumors. Exemplifying this fundamental mystery, adult zebrafish fins perfectly regenerate to their original size and shape regardless of injury extent. Therefore, zebrafish fin regeneration is a compelling and tractable system to interrogate “organ scaling” mechanisms. Bioelectricity, or ion flows across cell membranes, is long-associated with both organ size control and regeneration. However, links between ion signaling and their effectors to specific cell behaviors determining organ size are limited. Perturbed ion signaling, notably by elevated voltage- gated K+ channel activity and inhibited Ca2+-dependent calcineurin signaling, leads to dramatic overgrowth of regenerating zebrafish fins. A distal fibroblast-lineage pool of "niche" cells within the fin's regenerative blastema sustains fin outgrowth. The niche progressively depletes as outgrowth slows, likely by net re- differentiation to a non-growth promoting state. We recently discovered the classic longfint2 mutant phenotype is caused by ectopic expression of the Kcnh2a potassium channel within the fibroblast/niche lineage. Ectopic Kcnh2a disrupts orderly niche depletion, thereby prolonging the outgrowth period. Kcnh2a likely blocks Ca2+- calcineurin signaling with both acting uniquely during late stages of regeneration. We made a Ca2+ responsive GCaMP6s transgenic reporter line and found distal fibroblast / niche cells exhibit dynamic Ca2+ fluxes. Our single cell transcriptomics identified candidate upstream voltage-gated Ca2+ channels. We mutated the genes encoding each channel, generating the first recessive model of dramatically elongated fins. We now hypothesize niche-specific Ca2+ signaling, modulated by a cadre of Ca2+ channels, activates calcineurin to promote niche-to-mesenchyme state transitions. We will pursue three Specific Aims to test this model and identify mechanisms linking ion signaling to cell behaviors restoring fin size: 1) Characterize spatiotemporal cytosolic Ca2+ dynamics and calcineurin activity in wildtype and long-finned zebrafish, 2) Determine how voltage-gated Ca2+ channels modulate regenerating fin Ca2+ dynamics and fin outgrowth, and 3) Determine how Ca2+ dynamics and calcineurin promote cell behaviors for fin growth cessation. Our proposed research will associate voltage-gated Ca2+ channel-modulated intracellular Ca2+ dynamics, downstream calcineurin signaling, and a novel “niche” state transition towards answering the classic mystery of robust organ scaling during fin regeneration. Our study's broader impacts include identifying conceptual and mechanistic links of bioelectricity to specific molecules and cell behaviors that determine organ size and form. Finally, studying robust adult zebrafish skeletal regeneration will inform regenerative medicine approaches for human bone disease.