Genetic Regulation of Outflow Tract Formation

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：流出道的尺寸、方向和细分对于血液从心脏到外周的有效运输至关重要。OFT的发展始于一个小心肌管的组装，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（CMs）的胚胎起源已经引起了极大的兴趣。对小鼠和鸡胚胎的一系列研究已经阐明了心脏祖细胞的两个主要来源，称为第一心脏野（FHF）和第二心脏野（SHF）。值得注意的是，OFT的初始基础是由附着在心脏动脉极的shf衍生的CMs构建的。虽然有几种信号通路与调节SHF分化有关，但对于哪些基因在这些关键信号的下游发挥作用来执行OFT组装，以及多种相关通路如何相互作用来设置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的效用，以确定OFT形成的新调节因子。初步研究表明，斑马鱼的OFT，像羊膜动物的OFT一样，是由shf衍生的CMs群体构建的。此外，与羊膜动物一样，斑马鱼也需要Fgf信号来促进OFT cm的产生。然而，目前尚不清楚哪些基因在Fgf信号的下游作用，以招募适当数量的CMs进入OFT。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 -被Fgf信号抑制，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，其中Fgf信号通过限制cadm基因的表达来驱动OFT CMs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功能的机制，并将Fgf-Cadm途径整合到聚合以定义OFT大小的多重影响的背景中来详细测试该模型。在Aim 1中，我们将采用命运图、延时跟踪和心肌分化时间测定来确定斑马鱼OFT心肌是否来源于SHF当量。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 CMs募集的细胞外相互作用。在Aim 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尺寸方面所起的作用。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调节OFT大小的新机制，并阐明合作确保胚胎OFT适当心肌基础的途径网络。

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.