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.

项目总结 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。器官大小的破坏 控制机制导致先天性异常、器官动态平衡和组织修复不良，以及肿瘤。 成年斑马鱼的鳍可以完美地再生到它们原来的大小和形状，这就说明了这个根本的谜团。 无论受伤程度如何。因此，斑马鱼鳍再生是一个引人注目和易于处理的系统 审问“器官伸缩”机制。生物电，或离子流经细胞膜，长期与 同时控制器官大小和再生。然而，离子信号和它们的效应器之间的联系 决定器官大小的特定细胞行为是有限的。受扰动的离子信号，特别是电压升高- 门控性K+通道活动和抑制钙依赖的钙调神经磷酸酶信号，导致细胞急剧过度生长 再生斑马鱼的鳍。鳍再生内的远端成纤维细胞谱系池中的“龛”细胞 胚泡维持鳍的生长。随着增长放缓，生态位逐渐枯竭，可能是通过净再分配 分化为不促进生长的状态。我们最近发现了经典的long fint2突变表型 是由于Kcnh2a钾通道在成纤维细胞/生态位血统中的异位表达引起的。异位 Kcnh2a扰乱了生态位的有序枯竭，从而延长了外生期。KcnH2a可能阻断钙离子- 钙调神经磷酸酶信号转导，两者都在再生后期发挥独特作用。我们让一种钙离子响应 GCaMP6s转基因报告系，发现远端成纤维细胞/龛细胞表现出动态的钙离子流动。我们的 单细胞转录组发现了电压门控性上游钙离子通道。我们对基因进行了突变 对每个通道进行编码，生成显著拉长的鳍片的第一隐性模型。我们现在 假设由一组钙离子通道调节的小生境特异的钙信号，激活钙调神经磷酸酶以 促进从生态位到间质的状态转变。我们将追求三个具体目标来测试该模型和 确定离子信号与恢复鳍大小的细胞行为之间的联系机制：1)描述时空特征 野生型斑马鱼和长鳍斑马鱼细胞内钙动态和钙调神经磷酸酶活性，2)决定 电压门控钙通道调节FIN再生的钙动力学和FIN的生长，以及3)决定 钙动力学和钙调神经磷酸酶如何促进细胞停止生长。我们提议的研究将 相关电压门控钙通道调节的细胞内钙动力学，下游钙调神经磷酸酶信号， 以及一种新颖的“利基”状态转变，以回答在手指过程中强劲的器官伸缩这一经典谜题 再生。我们的研究的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。