Le Bleu Heather K, Kioussi Rea G, Henner Astra L, Lewis Victor M, Stewart Scott, Stankunas Kryn
bioRxiv. 2025 Feb 13:2024.08.21.608903. doi: 10.1101/2024.08.21.608903.
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 -dependent phosphatase calcineurin during regeneration causes dramatic fin overgrowth. However, Ca 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 transients in distal blastema cells. Membrane depolarization of isolated regenerating fin fibroblasts triggered Ca spikes dependent on voltage-gated Ca channel activity. Single cell transcriptomics identified the voltage-gated Ca channels (L-type channel), (N-type), and (T-type) as candidate mediators of fibroblast-lineage Ca signaling. Small molecule inhibition revealed L- and/or N-type voltage-gated Ca channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, 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, 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 dynamics in distal fibroblast-lineage blastemal mesenchyme during the outgrowth phase. We conclude "bioelectricity" for fin size control likely entirely reflects voltage-modulated Ca dynamics in fibroblast-lineage blastemal cells that specifically and steadily decelerates outgrowth at a rate tuned to restore the original fin size.
成年斑马鱼的鳍无论损伤程度如何，都能再生到原来的大小，这为器官大小和尺度控制提供了一个易于处理的模型。在成纤维细胞谱系的芽基细胞中表达的电压门控钾通道功能获得会促进鳍过度生长，导致长鳍表型。同样，再生过程中对钙依赖性磷酸酶钙调神经磷酸酶的抑制会导致鳍显著过度生长。然而，钙通量及其潜在的动态膜电压来源尚未被探索，也未与鳍大小恢复联系起来。我们利用成年再生鳍的成纤维细胞谱系GCaMP成像，在远端芽基细胞中识别出广泛的、异质性的钙瞬变。分离的再生鳍成纤维细胞的膜去极化触发了依赖电压门控钙通道活性的钙尖峰。单细胞转录组学确定电压门控钙通道（L型通道）、（N型）和（T型）是成纤维细胞谱系钙信号传导的候选介质。小分子抑制显示L型和/或N型电压门控钙通道在再生生长过程中起作用，使鳍恢复到原来的尺度。引人注目的是，纯合突变斑马鱼由于生长延长而再生出异常长的鳍。再生的鳍远远超过其原始长度，但鳍条骨骼在其他方面正常。因此，突变体独特地提供了一个功能丧失的遗传长鳍模型，该模型将发育性和再生性鳍生长解耦。再生鳍的实时GCaMP成像显示，T型Cacna1g通道在生长阶段使远端成纤维细胞谱系芽基间充质中的钙动态变化。我们得出结论，鳍大小控制的“生物电”可能完全反映了成纤维细胞谱系芽基细胞中电压调制的钙动态变化，这种变化以特定且稳定的速率减慢生长，该速率经过调整以恢复鳍的原始大小。
