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(申请人提供)：流出道(OFT)的形成是心脏发生的一个重要方面：流出道的大小、方向和细分对于血液从心脏到外周的有效传输至关重要。OFT的发展始于组装一个小的心肌管，这随后为OFT的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(CMS)的胚胎起源引起了人们的极大兴趣。在小鼠和鸡胚胎中的一系列研究阐明了心脏前体细胞的两个主要来源，即第一心区(FHF)和第二心区(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附加在心脏的动脉极上。尽管已有几条信号通路参与调控SHF的分化，但对这些关键信号下游的哪些基因执行OFT组装，或者多个相关通路如何相互作用来设定OFT的尺寸，我们知之甚少。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OFT和羊膜OFT一样，是由SHF衍生的CMS种群构建而成的。此外，在斑马鱼中，就像在羊膜动物中一样，成纤维细胞生长因子信号是促进OFT CMS产生所必需的。然而，目前还不清楚哪些基因作用于成纤维细胞生长因子信号的下游，从而将适当数量的CMS招募到OFT中。我们的初步数据揭示了一组有趣的基因--细胞黏附分子4(Cadm4)、Cadm3和Cadm2a--受成纤维细胞生长因子信号的抑制，并在限制OFT心肌的形成中发挥重要作用。这些数据提示了一个有趣的模型，在该模型中，成纤维细胞生长因子信号通过限制CADM基因的表达从而改变SHF来源的祖细胞的关键细胞外相互作用来驱动OFT CMS的招募。在这个方案中，我们将通过建立斑马鱼OFT的起源，破译CADM功能的机制，并将成纤维细胞生长因子-CADM途径整合到多种影响的背景下来详细测试这一模型，这些影响汇聚在一起来定义OFT的大小。在目标1中，我们将使用命运映射、时间推移跟踪和心肌分化时间的分析来确定斑马鱼OFT心肌是否来自SHF等价物。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是否介导了抑制OFT CMS募集的细胞外相互作用。在目标3中，我们将确定抵消成纤维细胞生长因子-钙调素通路对OFT大小影响的信号，重点关注Notch、BMP和维甲酸信号在限制斑马鱼OFT大小中所起的作用。总之，这些实验可能揭示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，并阐明共同为胚胎OFT提供适当的心肌基础的通路网络。 与公共卫生相关：在100名活产儿中发现1例心脏缺陷，在10例死产中发现1例心脏缺陷，通常包括心脏流出道的形成问题。流出道的发育始于一个小的肌肉管的组装，其精确的尺寸对于其随后重塑成成熟的结构是必不可少的。因此，更好地理解控制肌肉最初投资到流出道的机制可能会阐明心脏出生缺陷的原因，并可能建议将多能细胞转化为心肌的策略。