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

鳍再生过程中的离子信号、细胞转变和器官缩放

• 批准号: 10639668
• 负责人: KRYN STANKUNAS
• 金额: $ 41.08万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639668
• 关键词: Adult; Anatomy; Apoptosis; Biological Process; Bone Diseases; Calcineurin; Cell Cycle; Cell Lineage; Cell membrane; Cells; Congenital Abnormality; Development; Developmental Biology; Distal; Ectopic Expression; Exhibits; Fibroblasts; Genes; Genetic; Genetic Epistasis; Genetic Transcription; Growth; Growth Factor; Homeostasis; Human; Image; In Situ; Individual; Injury; Ions; Kinetics; Lead; Link; Malignant Neoplasms; Measurement; Mesenchyme; Modeling; Molecular; Mutate; Mutation; Natural regeneration; Nature; Organ; Organ Size; Output; PPP3CA gene; Pattern; Phase; Phenotype; Positioning Attribute; Potassium Channel; Process; Regenerative Medicine; Reporter; Reporter Genes; Research; Second Messenger Systems; Shapes; Signal Transduction; System; Testing; Transcript; Transgenic Organisms; Transplantation; Voltage-Gated Potassium Channel; Zebrafish; bioelectricity; biophysical properties; blastema; candidate identification; cell behavior; experimental study; gain of function; human disease; loss of function; migration; mutant; novel; organ regeneration; organ repair; pharmacologic; progenitor; regenerative; research study; restoration; restraint; single-cell RNA sequencing; skeletal regeneration; small molecule inhibitor; spatiotemporal; tissue repair; transcriptome sequencing; transcriptomics; tumor; voltage

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.

项目摘要 器官“知道”何时以及如何停止生长以达到正确的尺寸和形状。器官尺寸的破坏 控制机制导致先天性异常，器官稳态和组织修复以及肿瘤。 举例说明这个基本的谜团，成人斑马鱼鳍完美地再生到其原始尺寸和形状 无论受伤程度如何。因此，斑马鱼鳍的再生是一个引人入胜且可拖延的系统 询问“器官缩放”机制。生物电性或离子流跨细胞机制，长期相关 具有器官尺寸控制和再生。但是，离子信号及其效果之间的联系与 确定组织尺寸的特定细胞行为有限。受扰动的离子信号传导，特别是电压升高 - 封闭的K+通道活性并抑制Ca2+依赖性钙调神经蛋白信号传导，导致急剧过度生长 再生斑马鱼鳍。鳍片再生中的“小众”细胞的远端成纤维细胞池池 Blastema维持鳍的产物。利基市场逐渐消耗的生长会减慢，可能是由于净重新降低 与非增长促进状态的区分。我们最近发现了经典的longfint2突变表型 是由成纤维细胞/小裂谱系中KCNH2A钾通道的异位表达引起的。异位 KCNH2A破坏了有序的利基部署，从而延长了生长期。 KCNH2A可能会阻止Ca2+ - 钙调神经蛋白信号传导在再生后期均具有独特的作用。我们做了一个CA2+响应迅速 GCAMP6S转基因报告基因线，发现盘状成纤维细胞 /利基细胞暴露了动态CA2+通量。我们的 单细胞转录组学确定了候选上游电压门控Ca2+通道。我们突变了基因 编码每个通道，生成第一个隐性模型的巨大伸长鳍模型。我们现在 假设由Ca2+通道的干部调制的小众Ca2+信号传导将钙调神经酶激活至 促进利基至间质状态过渡。我们将追求三个特定目标来测试此模型，并 识别将离子信号传导与细胞行为联系起来的机制，以恢复FIN大小：1）表征时空的特征 Wildtype和长期固定斑马鱼的胞质Ca2+动力学和钙调神经酶活性，2）确定如何 电压门控的Ca2+通道调节再生鳍CA2+动力学和鳍片的生长，3）确定 Ca2+动力学和钙调神经酶如何促进鳍生长戒烟的细胞行为。我们提出的研究将 相关电压门控Ca2+通道调节的细胞内Ca2+动力学，下游钙调神经磷酸酶信号， 以及一个小说的“利基”状态过渡，以回答鳍期间稳健器官缩放的经典奥秘 再生。我们的研究的更广泛影响包括识别生物电性的概念和机理联系 针对确定器官大小和形式的特定分子和细胞行为。最后，学习健壮的成人 斑马鱼骨骼再生将为人骨病的再生医学方法提供信息。