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）的形成是心脏发生的一个重要方面：OFT 的尺寸、方向和细分对于血液从心脏到外周的有效输送至关重要。 OFT 的发育始于小心肌管的组装，随后为 OFT 重塑提供了重要的基础。鉴于建立 OFT 心肌的重要性，OFT 心肌细胞 (CM) 的胚胎起源引起了极大的兴趣。对小鼠和鸡胚胎的一系列研究阐明了心脏祖细胞的两个主要来源，称为第一心脏区域（FHF）和第二心脏区域（SHF）。值得注意的是，OFT 的最初基础是由附加到心脏动脉极的 SHF 衍生的 CM 构建的。尽管一些信号通路与调节 SHF 分化有关，但我们对哪些基因在这些关键信号下游发挥作用以执行 OFT 组装或多个相关通路如何相互作用以设定 OFT 的尺寸知之甚少。 在这里，我们利用斑马鱼作为模式生物来识别 OFT 形成的新调节因子。初步研究表明，斑马鱼的 OFT 与羊膜动物的 OFT 一样，都是由 SHF 衍生的 CM 群体构建而成。此外，在斑马鱼中，就像在羊膜动物中一样，Fgf 信号传导是促进 OFT CM 产生所必需的。然而，尚不清楚哪些基因在 Fgf 信号下游发挥作用，以将适当数量的 CM 招募到 OFT 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 - 受到 Fgf 信号传导的抑制，在限制 OFT 心肌的形成中发挥重要作用。这些数据表明了一个有趣的模型，其中 Fgf 信号传导通过限制 cadm 基因的表达来驱动 OFT CM 的募集，从而改变 SHF 衍生祖细胞的关键细胞外相互作用。在本提案中，我们将通过建立斑马鱼 OFT 的起源、破译 Cadm 功能机制以及将 Fgf-Cadm 通路整合到汇聚以确定 OFT 大小的多重影响的背景中来详细测试该模型。在目标 1 中，我们将采用命运图谱、延时跟踪和心肌分化时间测定来确定斑马鱼 OFT 心肌是否源自 SHF 等效物。在目标 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 Cadms 是否介导抑制 OFT CM 募集的细胞外相互作用。在目标 3 中，我们将识别抵消 Fgf-Cadm 通路对 OFT 大小影响的信号，重点关注 Notch、Bmp 和视黄酸信号在限制斑马鱼 OFT 尺寸中所起的作用。总之，这些实验可能会揭示 OFT CM 募集的新介质，揭示通过调节细胞外相互作用来调节 OFT 大小的新机制，并揭示协同确保胚胎发育适当的心肌基础的途径网络。奥夫特。 公共卫生相关性：多达百分之一的活产儿和十分之一的死产儿发现心脏缺陷，并且经常包括心脏流出道形成的问题。流出道的发育始于一小块肌肉的组装，其精确的尺寸对于其随后重塑为成熟的结构至关重要。因此，更好地理解控制肌肉初始投入流出道的机制可能会阐明心脏出生缺陷的原因，也可能提出指导多能细胞成为心肌的策略。