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，以达到正确的大小和形状。器官大小破坏 控制机制导致先天性异常、较差的器官稳态和组织修复以及肿瘤。 成年斑马鱼的鳍可以完美地再生到原来的大小和形状， 无论受伤程度如何。因此，斑马鱼鳍再生是一个引人注目的和易处理的系统， 询问“器官缩放”机制。生物电，或离子流穿过细胞膜， 器官大小控制和再生。然而，离子信号传导及其效应器之间的联系， 决定器官大小的特定细胞行为是有限的。干扰离子信号，特别是高电压- 门控K+通道活性和抑制Ca 2+依赖性钙调神经磷酸酶信号传导，导致细胞过度生长， 再生斑马鱼的鳍一个远端成纤维细胞谱系池的“龛”细胞内的鳍的再生 芽基维持鳍的生长。随着增长放缓，利基市场逐渐枯竭，可能是由于净再生产， 分化为非生长促进状态。我们最近发现了经典的longfint 2突变表型， 是由成纤维细胞/小生境谱系内Kcnh 2a钾通道的异位表达引起的。异位 kcnh 2a破坏了有序的生态位耗竭，从而延长了生长期。Kcnh 2a可能阻断Ca 2 +- 钙调神经磷酸酶信号传导，两者在再生的晚期阶段独特地起作用。我们使钙离子反应 GCaMP 6s转基因报告细胞系和发现的远端成纤维细胞/小生境细胞表现出动态Ca 2+通量。我们 单细胞转录组学鉴定了候选的上游电压门控Ca 2+通道。我们突变了基因 对每个通道进行编码，生成显著伸长的鳍的第一隐性模型。我们现在 假设小生境特异性Ca 2+信号传导，由Ca 2+通道的干部调节，激活钙调神经磷酸酶， 促进生态位到间充质状态的转变。我们将追求三个具体目标来测试这个模型， 鉴定将离子信号传导与恢复手指大小细胞行为联系起来的机制：1）表征时空 野生型和长鳍斑马鱼的胞质Ca 2+动力学和钙调神经磷酸酶活性，2）确定如何 电压门控Ca 2+通道调节再生鳍Ca 2+动力学和鳍生长，和3）确定 Ca 2+动力学和钙调神经磷酸酶如何促进生长停止细胞行为。我们的研究计划将 相关的电压门控Ca 2+通道调节的细胞内Ca 2+动力学，下游钙调神经磷酸酶信号， 以及一种新颖的“利基”状态转变，以回答鳍状突起过程中强大的器官缩放的经典之谜。 再生我们的研究更广泛的影响包括确定生物电的概念和机制联系 决定器官大小和形状的特定分子和细胞行为。最后，研究健壮的成年人 斑马鱼骨骼再生将为人类骨骼疾病的再生医学方法提供信息。