Mechanical and Transcriptional Roles for Primary Cilia during Heart Development

摘要：先天性心脏病是最常见的出生缺陷，占活产儿总数的1%。的 已知的遗传因素是CHD的致病因素，但大多数发病机制尚不清楚。我们的实验室有 冠心病患者中发现Kruppel样因子2和4(KLF2，4)的新基因突变 左心发育不良综合征(HLHS)。KLF2和4是机械敏感的转录因子，调节 内皮细胞向间充质转化(EndoMT)，这是缓冲(心脏瓣膜祖细胞)所必需的过程 发展。了解EndoMT如何在空间和时间上受到机械调制的控制 KLF2/4将提供对HLHS患者存在的心脏瓣膜缺陷的见解。我们的实验室牵连到 心内膜(EC)上的初级纤毛(非运动性、杆状机械传感器) 已知在左右不对称中扮演的角色。然而，EC初级纤毛是否将机械信号传递到 KLF2/4在缓冲发育过程中的调控尚未显示出来。这项提议的目标是发现 在体心垫形成过程中机械力、EC初级纤毛和KLF2/4的关系。 根据我的初步结果，这项提议的总体假设是EC初级纤毛联系 钙离子和细胞骨架依赖的机械力与KLF2/4转录 在体心内膜垫形成。为了解决这一假设，我们将同时利用老鼠和斑马鱼 模特们。老鼠的心脏更像人类(斑马鱼有两个腔室)，但基因 管理心脏生成的程序是保守的。静态鼠标样本中可能丢失的事件可能 取而代之的是使用活斑马鱼在延时显微镜下观察，因为它们的光学清晰度和外部 发展。目标1将研究EC初级纤毛和KLF2/4在心脏上的表达在空间上的变化 利用原位杂交和免疫荧光在小鼠体内的垫状发育，以及各种转基因 标记活斑马鱼的心内膜、纤毛和KLF2/4启动子区域的线条。Aim 2将测试 KLF2/4依赖于纤毛、纤毛钙和/或机械力，通过使用各种突变背景，如 作为Ift20和Kif3a(纤毛KO)，NCX/沉默的心脏吗啡(分别是心跳KO、老鼠和斑马鱼)， 和PKD2(睫状钙KO)。我们的数据为控制KLF2/4机械敏感性的机制提供了洞察力 在心脏软垫形成过程中，突出纤毛在心脏发生过程中作为机械传感器的作用。这 该提案解决了NIH-NHLBI关于研究遗传和机械的目标1和2 许多冠心病患者存在瓣膜缺陷背后的机制。我们的发现也可能导致治疗 以改善许多冠心病患者所经历的进行性瓣膜功能障碍。我用活的，完整的- 安装成像将扩展到静态、固定胚胎中可能观察到的范围，并带来罕见的 活体纤毛依赖转录的前景。机械力在发展过程中的作用是 我们的研究成果将有助于从基础生物学和翻译生物学的角度进行研究。

Abstract: Congenital heart defects (CHD) are the most common birth defects, affecting 1% of all live births. Of the known genetic contributors to CHD, the pathogenic mechanism is undetermined for most. Our lab has identified de novo mutations in kruppel-like factors 2 and 4 (KLF2, 4) in CHD patients presenting with hypoplastic left heart syndrome (HLHS). Klf2 and 4 are mechanosensitive transcription factors that regulate endothelial to mesenchymal transition (EndoMT), a process necessary for cushion (heart valve progenitor) development. Understanding how EndoMT is spatially and temporally controlled by mechanical modulation of Klf2/4 will provide insights into the heart valve defects present in HLHS patients. Our lab has implicated primary cilia (non-motile, rod-like mechanosensors) on the endocardium (EC) in cardiogenesis beyond their known role in left-right asymmetry. However, whether EC primary cilia transduce mechanical signals into regulation of Klf2/4 during cushion development has yet to be shown. The goal of this proposal is to discover the relationship between mechanical forces, EC primary cilia and Klf2/4 during heart cushion formation in vivo. Based on my preliminary results, the overall hypothesis of this proposal is that EC primary cilia link mechanical forces and Klf2/4 transcription in a Ca2+ and cytoskeletal-dependent manner during endocardial cushion formation in vivo. To address this hypothesis, we will utilize both mouse and zebrafish models. Mouse hearts are more comparable to humans (zebrafish have two chambers), but the genetic programs governing cardiogenesis are conserved. Events that may be lost in static mouse samples can instead be observed using live zebrafish in time-lapse microscopy due to their optical clarity and external development. Aim 1 will investigate how EC primary cilia and Klf2/4 expression change spatially over heart cushion development, utilizing in situ hybridization and immunofluorescence in mice, and various transgenic lines marking endocardium, cilia, and the promoter regions of Klf2/4 in live zebrafish. Aim 2 will test whether Klf2/4 depend on cilia, ciliary Ca2+ and/or mechanical forces through use of various mutant backgrounds, such as Ift20 and Kif3a (cilia KOs), Ncx/silent heart morpholino (heartbeat KO, mouse and zebrafish, respectively), and Pkd2 (ciliary Ca2+ KO). Our data provides insight into the mechanism controlling Klf2/4 mechanosensitivity during heart cushion formation, and highlight a ciliary role as mechanosensors during cardiogenesis. This proposal addresses the NIH-NHLBI's objectives 1 and 2 in regards to investigating the genetic and mechanical mechanism behind the valve defects present in many CHD patients. Our findings could also lead to treatments to ameliorate the progressive valve dysfunction experienced by many CHD patients. My use of live, whole- mount imaging will extend beyond the observations possible in static, fixed embryos and bring a rare perspective of ciliary-dependent transcription in vivo. The role of mechanical forces during development is a rapidly expanding field, and our results will contribute to both a basic and translational biological perspective.