Dissecting injury-responsive gene expression during zebrafish heart regeneration

项目摘要/摘要 成年哺乳动物很难再生受伤的心脏。相比之下，成年斑马鱼具有非凡的能力 使受损的心脏再生。与现有的遗传工具相结合，这种能力使斑马鱼成为一种强大的 用于破译心脏再生机制的系统。在受伤时，心内膜和 心外膜被迅速激活，分泌旁分泌因子，促进心脏再生。心脏再生 研究主要集中在识别触发再生程序的秘密因素上，但很少有人这样做 已知控制其表达的调控机制，这是再生过程中的一个最近的步骤。 对这些基因进行适当的时空调控对于复杂和紧密协调的过程是必不可少的。 心脏再生的基础。因此，阐明调控伤害反应的基因调控机制 基因表达将为基于刺激旁分泌的潜在治疗策略提供见解 效应器。在此之前，我们发现瘦素b(Leptin B)是一种由心内膜分泌的损伤诱导因子。 重要的是，我们证明了与瘦素相关的心脏再生增强剂(LEN)是由 心脏损伤，在再生期间保持活动，然后在完成 再生。我们还鉴定了一种与白细胞介素11a(Il11a)相关的心脏损伤反应增强子，它 编码一种在结构上类似于Lepb的促再生细胞因子。我们发现斑马鱼对伤害有反应 增强剂驱动受损小鼠心脏中报告基因的表达，表明介导 损伤诱导的增强子功能在进化上是保守的。基于这些发现，我们提出了两个目标 破译心脏再生的基因调控机制。中心假说是心脏 损伤反应增强子建立编码旁分泌因子的基因的转录状态，从而促进 心脏修复。AIM 1将确定心脏损伤反应增强子的转录机制 活动。我们发现，心脏Len(Clen)不仅被损伤所激活，而且在损伤后被主动抑制。 没有受伤。我们将使用转基因检测、遗传和药理学方法以及 表观基因组学和计算分析，以检验抑制和激活机制的假设 共同确定心脏损伤反应增强子功能以授予损伤依赖的心脏基因 抄写。目标2将使用功能丧失和功能获得研究来确定il11a是如何控制心脏的 并对其损伤反应增强子进行剖析。阐明转录机制和 心脏损伤时分泌的关键增强子调节因子的功能将如何改变人们对 损伤信号被转导以促进心脏再生。

PROJECT SUMMARY/ABSTRACT Adult mammals poorly regenerate injured hearts. In contrast, adult zebrafish possess a remarkable capacity to regenerate damaged hearts. Combined with available genetic tools, this capacity makes zebrafish a powerful system for deciphering the mechanisms underlying heart regeneration. Upon injury, the endocardium and epicardium are rapidly activated to secrete paracrine factors that facilitate heart regeneration. Heart regeneration research has focused mostly on identifying the secreted factors that trigger regenerative programs, yet little is known about the regulatory mechanisms controlling their expression, a proximal step in the regenerative process. Proper spatiotemporal regulation of these genes is essential for the intricate and tightly coordinated processes underlying cardiac regeneration. Thus, elucidating gene regulatory mechanisms governing injury-responsive gene expression will provide insights into potential therapeutic strategies based on stimulating paracrine effectors. Previously, we identified leptin b (lepb) as an injury-induced factor secreted by the endocardium. Importantly, we showed that the cardiac regeneration enhancer linked to lepb (LEN) is robustly activated by cardiac injury, maintains activity during regeneration, and then returns to a naïve state upon completion of regeneration. We also identified a cardiac injury-responsive enhancer linked to interleukin 11a (il11a), which encodes a proregenerative cytokine structurally similar to lepb. We showed that zebrafish injury-responsive enhancers drive reporter-gene expression in injured mouse hearts, indicating that the mechanisms mediating injury-induced enhancer function are evolutionarily conserved. Based on these findings, we propose two aims to decipher gene regulatory mechanisms governing heart regeneration. The central hypothesis is that cardiac injury-responsive enhancers establish the transcriptional state of genes encoding paracrine factors that facilitate heart repair. Aim 1 will determine the transcriptional mechanisms underlying cardiac injury-responsive enhancer activity. We discovered that cardiac LEN (cLEN) is not only activated by injury but also actively repressed in the absence of injury. We will employ transgenic assays, genetic and pharmacological approaches, as well as epigenomic and computational analyses to test the hypothesis that repression and activation mechanisms collectively determine cardiac injury-responsive enhancer function to confer injury-dependent cardiac gene transcription. Aim 2 will use loss-of-function and gain-of-function studies to define how il11a controls heart regeneration and to dissect its injury-responsive enhancer. Elucidating the transcriptional mechanisms and function of crucial enhancer-regulated factors secreted upon cardiac injury will transform knowledge on how injury signals are transduced to facilitate heart regeneration.