Ion Signaling for Organ Size and Scaling During Zebrafish Fin Regeneration
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Date
2024-08-07
Authors
Le Bleu, Heather
Journal Title
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Publisher
University of Oregon
Abstract
Organs “know” how to develop to a specific size and shape conferring optimal function. In humans, very few organs and appendages show a natural ability to repair. Exemplifying this fundamental mystery, adult zebrafish fins regenerate to their original size and shape regardless of injury extent. Therefore, zebrafish fin regeneration provides a tractable system to investigate “organ scaling” mechanisms. Bioelectricity, or ion flow across cell membranes, is long-associated with both organ size control and regeneration. However, the 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 unique distal population of mesenchymal cells within the fin’s regenerative blastema sustains fin outgrowth. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show ectopic expression of the kcnh2a K+ channel in fin ray fibroblast-lineage cells enhances fin outgrowth in late regeneration rather than at early blastema establishment. Epistasis experiments suggest that Kcnh2a likely blocks Ca2+-dependent calcineurin signaling to end fin outgrowth. Mechanisms of putative Ca2+ signaling during fin size acquisition has not been explored. Using a new Ca2+ responsive GCaMP6s transgenic reporter line, we show fibroblast-lineage cells are the nexus of dynamic voltage-gated Ca2+ channel activity and Ca2+ signaling events during zebrafish fin regeneration. Single cell transcriptomics identifies upstream voltage-gated Ca2+ channels cacna1c (CaV1.2, L-type), cacna1ba (CaV2.2, N-type), and cacna1g (CaV3.1, T-type) as candidate mediators of fibroblast-lineage Ca2+ signaling in vivo. Dual chemical inhibition reveal that L/N-type voltage-gated Ca2+ channels are actively required for fin outgrowth during regeneration. Genetic analysis demonstrates cacna1g mutants regenerate extraordinarily long fins, indicating Cacna1g has a key in fin cessation and scaling. Accordingly, live imaging of regenerating animals suggests Cacna1g channel activity in distalmost mesenchymal cells is essential for Ca2+ flux. We conclude that a cadre of ion channels act within fibroblast-lineage cells to fine-tune Ca2+ signaling events and restore fin size and shape.This dissertation includes previously published and unpublished co-authored material.