Genetic regulation of cardiac proliferation during development and in regeneration

 描述（由申请人提供）：了解驱动多能祖细胞分化为特定细胞类型的分子机制与器官发生的研究密切相关，并且在再生医学中具有重要意义。虽然心脏发育中的关键心脏特异性转录因子的作用已被仔细研究，但参与心脏细胞形成中表观遗传调节或转录延伸的蛋白质复合物的重要性才刚刚被揭示。在这个提议中，我们的目标是研究RNA聚合酶II-相关因子1复合物（PAF 1C）控制心脏发育的分子机制。多种模式生物的生化和遗传研究表明，PAF 1C作为关键信号事件所需的转录平台发挥作用。然而，它在心脏形成中的作用尚不清楚。我们最近的遗传学研究发现了一个新的和重要的作用，Rtf 1，一个组成部分的PAF 1C，在心肌细胞的形成和增殖。我们发现，斑马鱼rtf 1缺陷胚胎缺乏整个群体的心脏祖细胞，这表明Rtf 1是心脏发育所必需的。我们从结构-功能分析和功能丧失和获得研究中获得的初步数据使我们假设Rtf 1通过PAF 1C相关的表观遗传修饰和PAF 1C独立的转录调节来控制心脏基因表达。我们将在斑马鱼中检验这一假设，并评估这些机制如何影响心脏祖细胞的形成和心肌细胞的增殖（目的1）。我们发现，Rtf 1促进斑马鱼胚胎中胚层的心脏分化。我们将使用小鼠ES细胞作为体外分化模型来研究这种机制在哺乳动物中是否是保守的。我们还将创建心脏特异性条件性基因敲除小鼠，以评估Rtf 1在小鼠心脏发育中的作用（目的2）。最后，许多发育调控基因在心脏再生过程中被“重新利用”，并且心室切除后，Rtf 1下游转录因子Tbx 20上调。因此，我们建议使用成年斑马鱼和新生小鼠心脏再生模型来检查Rtf 1-Tbx 20通路是否参与心脏再生（目的3）。拟议项目的成功完成将为发育和再生过程中心脏祖细胞形成和心肌细胞增殖的调节提供新的机制见解。

 DESCRIPTION (provided by applicant): Understanding molecular mechanisms driving a pluripotent progenitor cell to differentiate into a specific cell type is germane to the study of organogenesis and has important significance in regenerative medicine. While the roles of key cardiac-specific transcription factors in heart development have been carefully studied, the importance for protein complexes involved in epigenetic regulation or transcription elongation in cardiac cell formation is just being revealed. In this proposal, we aim to investigate molecular mechanisms by which the RNA Polymerase II- Associated Factor 1 Complex (PAF1C) controls heart development. Biochemical and genetic studies in multiple model organisms suggest that PAF1C functions as a transcription platform required for critical signaling events. However, its roles in heart formation are not known. Our recent genetic studies discovered a novel and essential role for Rtf1, a component of the PAF1C, in the formation and proliferation of cardiomyocytes. We found that zebrafish rtf1 deficient embryos lack the entire population of cardiac progenitor cells, demonstrating that Rtf1 is absolutely required for heart development. Our preliminary data obtained from structure-function analysis and loss- and gain-of- function studies lead us to hypothesize that Rtf1 controls cardiac gene expression by PAF1C- associated epigenetic modification and PAF1C-independent transcription regulation. We will examine this hypothesis in zebrafish and evaluate how these mechanisms influence the formation of cardiac progenitor cells and the proliferation of cardiomyocytes (Aim 1). We showed that Rtf1 promotes cardiac differentiation from embryonic mesoderm in zebrafish. We will examine whether this mechanism is conserved in mammals using mouse ES cells as an in vitro differentiation model. We will also create cardiac-specific conditional knockout mice to assess the role of Rtf1 in mouse heart development (Aim 2). Finally, many developmentally regulated genes are "reutilized" during heart regeneration and Tbx20, an Rtf1 downstream transcription factor, is upregulated after ventricular resection. We thus propose to examine whether the Rtf1-Tbx20 pathway is involved in heart regeneration using both adult zebrafish and neonatal mouse heart regeneration models (Aim 3). Successful completion of the proposed projects will provide new mechanistic insights into the regulation of cardiac progenitor cell formation and cardiomyocyte proliferation during development and in regeneration.