Genetic Regulation of Outflow Tract Formation in Zebrafish

斑马鱼流出道形成的遗传调控

• 批准号: 8131347
• 负责人: DEBORAH YELON
• 金额: $ 35.9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131347
• 关键词: Animal Model; Biochemical; Biological Assay; Birth; Blood; Blood Circulation; Cardiac; Cardiac Myocytes; Cell Adhesion Molecule Gene; Cells; Chick Embryo; Comprehension; Congenital Abnormality; Congenital Heart Defects; Data; Development; Dimensions; Embryo; Foundations; Gene Expression; Genes; Genetic; Growth; Heart; Investments; Light; Live Birth; Maps; Mediating; Mediator of activation protein; Modeling; Molecular; Morphogenesis; Mus; Muscle; Myocardial; Myocardium; Nature; Pathway interactions; Play; Population; Production; Recruitment Activity; Regulation; Role; Series; Signal Pathway; Signal Transduction; Source; Staging; Stem cells; Structure; Testing; Time; Tretinoin; Tube; Undifferentiated; Work; Zebrafish; cardiogenesis; cell motility; congenital heart disorder; extracellular; gain of function; gene function; interest; loss of function; multipotent cell; notch protein; novel; progenitor; research study; trait

DESCRIPTION (provided by applicant): Formation of the outflow tract (OFT) is an essential aspect of cardiogenesis: the dimensions, orientation, and subdivision of the OFT are crucial for effective transport of blood from the heart to the periphery. OFT development initiates with the assembly of a small myocardial tube, which subsequently provides a vital foundation for OFT remodeling. Given the importance of establishing the OFT myocardium, the embryonic origins of OFT cardiomyocytes (CMs) have been of great interest. A series of studies in mouse and chick embryos have illuminated two major sources of cardiac progenitor cells, termed the first heart field (FHF) and the second heart field (SHF). Notably, the initial foundation of the OFT is built by SHF-derived CMs that are appended to the arterial pole of the heart. Although several signaling pathways have been implicated in regulating SHF differentiation, little is known about which genes function downstream of these key signals to execute OFT assembly or how the multiple relevant pathways interact to set the dimensions of the OFT. Here, we exploit the utility of the zebrafish as a model organism in order to identify novel regulators of OFT formation. Preliminary studies suggest that the zebrafish OFT, like the amniote OFT, is constructed from a population of SHF-derived CMs. Furthermore, in zebrafish, as in amniotes, Fgf signaling is required to promote the production of OFT CMs. However, it is unclear which genes act downstream of Fgf signaling to recruit the appropriate number of CMs into the OFT. Our preliminary data reveal an interesting set of genes - cell adhesion molecule 4 (cadm4), cadm3, and cadm2a - that are repressed by Fgf signaling and play essential roles in restricting the formation of OFT myocardium. These data suggest an intriguing model in which Fgf signaling drives the recruitment of OFT CMs by limiting the expression of cadm genes and thereby altering critical extracellular interactions of SHF-derived progenitor cells. In this proposal, we will test this model in detail by establishing the origins of the zebrafish OFT, deciphering the mechanisms of Cadm function, and integrating the Fgf-Cadm pathway into the context of the multiple influences that converge to define the size of the OFT. In Aim 1, we will employ fate mapping, time-lapse tracking, and assays for the timing of myocardial differentiation to determine whether the zebrafish OFT myocardium is derived from a SHF equivalent. In Aim 2, we will use loss-of-function, gain-of-function, structure-function, and biochemical analyses to test if Cadms mediate extracellular interactions that inhibit recruitment of OFT CMs. In Aim 3, we will identify signals that counterbalance the impact of the Fgf-Cadm pathway on OFT size, focusing on the roles played by Notch, Bmp, and retinoic acid signaling in limiting the dimensions of the zebrafish OFT. Together, these experiments are likely to reveal new mediators of OFT CM recruitment, to uncover a novel mechanism for regulating OFT size through modulation of extracellular interactions, and to shed light on the network of pathways that collaborate to insure an appropriate myocardial foundation for the embryonic OFT. PUBLIC HEALTH RELEVANCE: Cardiac defects are found in as many as 1 in 100 live births and 1 in 10 still births and frequently include problems with the formation of the cardiac outflow tract. Outflow tract development initiates with the assembly of a small tube of muscle, the precise dimensions of which are essential for its subsequent remodeling into a mature structure. Therefore, a better comprehension of the mechanisms controlling the initial investment of muscle into the outflow tract is likely to illuminate the causes of cardiac birth defects and may also suggest strategies for directing multipotent cells to become cardiac muscle.

描述（由申请人提供）：流出道（OFT）的形成是心脏病的重要方面：OFT的维度，方向和细分对于有效地从心脏传播到周围的血液至关重要。经常发育会启动小型心肌管的组装，随后为经常重塑提供了重要的基础。鉴于建立经常心肌的重要性，经常心肌细胞（CMS）的胚胎起源引起了极大的兴趣。一系列在小鼠和雏鸡胚胎的研究中，阐明了两个主要的心脏祖细胞的主要来源，称为第一个心脏场（FHF）和第二心脏场（SHF）。值得注意的是，OFT的最初基础是由SHF衍生的CMS建立的，这些CMS附加到心脏的动脉极。尽管已经与调节SHF分化有关的几种信号通路与这些键信号下游的哪些基因在执行OFT组件的下游或多个相关途径如何相互作用以设置OFT的尺寸的方式知之甚少。 在这里，我们利用斑马鱼作为模型生物的效用，以识别FOST形成的新型调节剂。初步研究表明，斑马鱼像羊水一样，是羊膜炎，是由SHF衍生的CMS人群构建的。此外，在斑马鱼中，就像在羊膜中一样，FGF信号传导需要促进常见的CMS的产生。但是，尚不清楚哪些基因在FGF信号的下游作用，以募集适当数量的CMS中。我们的初步数据揭示了一组有趣的基因 - 细胞粘附分子4（CADM4），CADM3和CADM2A-通过FGF信号传导抑制，并在限制Oft心肌的形成中起着重要作用。这些数据表明了一个有趣的模型，其中FGF信号通过限制CADM基因的表达来驱动FFGF募集，从而改变了SHF衍生的祖细胞的关键细胞外相互作用。在此提案中，我们将通过建立斑马鱼OFT的起源，破译CADM函数的机理，并将FGF-CADM途径整合到融合以定义OFT的大小的多个影响的上下文中，从而详细测试该模型。在AIM 1中，我们将采用命运映射，延时跟踪以及心肌分化时机的测定法，以确定斑马鱼经常肌心肌是否来自SHF等效。在AIM 2中，我们将使用功能丧失，功能获得，结构功能和生化分析来测试CADM是否介导了抑制FST CMS的细胞外相互作用。在AIM 3中，我们将确定信号表明FGF-CADM途径对OFT大小的影响，重点是Notch，BMP和视黄酸信号传导在限制斑马鱼OFT的尺寸中所扮演的角色。总之，这些实验很可能揭示了FAST CM募集的新调解人，以发现一种通过调节细胞外相互作用来调节大小的新机制，并阐明了途径网络，以确保为胚胎oft的适当心肌基础提供合作的心肌基础。 公共卫生相关性：心脏缺陷在100个活产中有多达1个，十分之一的静物分娩中有1个，并且经常包括心脏流出道的形成问题。流出道的发展是通过一小管肌肉组装而启动的，其精确的尺寸对于随后的重塑为成熟的结构至关重要。因此，更好地理解控制肌肉对流出道的初始投资的机制可能会阐明心脏先天缺陷的原因，并且还可能提出指导多能细胞成为心脏肌肉的策略。