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Mechanical and Transcriptional Roles for Primary Cilia during Heart Development

原发纤毛在心脏发育过程中的机械和转录作用

基本信息

批准号：
10677718
负责人：
Kathryn Berg
金额：
$ 3.25万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10677718
关键词：
4D Imaging Address Affect Biological Blood flow Cardiac Cell Line Cilia Collaborations Complement Congenital Abnormality Congenital Heart Defects Cytoskeleton Data Data Analytics Defect Deterioration Development Developmental Biology Down-Regulation Embryo Endocardium Endothelium Event Functional disorder Genetic Genetic Transcription Genomics Goals Grant Heart Heart Abnormalities Heart Valves Human Hypoplastic Left Heart Syndrome Image Image Analysis Immunofluorescence Immunologic In Situ Hybridization In Vitro Left Link Live Birth Mechanics Mediating Mesenchymal Microscopy Modeling Mus National Heart, Lung, and Blood Institute Optics Pathogenicity Patients Process Promoter Regions Recording of previous events Regulation Reporter Research Personnel Rod Role Sampling Signal Transduction Testing Time Tissues Training Transgenic Organisms United States National Institutes of Health Up-Regulation Wild Type Mouse Work Zebrafish cardiogenesis cellular imaging ciliopathy de novo mutation experience genomic data heart function in vivo insight inter-institutional kinetosome mechanical force mechanotransduction migration molecular imaging mutant postnatal prevent progenitor programs promoter shear stress skills transcription factor

项目摘要

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.

翻译后摘要：先天性心脏病（CHD）是最常见的出生缺陷，影响所有活产的1%。的已知的CHD的遗传因素，致病机制是不确定的。我们的实验室在CHD患者中发现了Kruppel样因子2和4（KLF 2，4）的新生突变，左心发育不全综合征（HLHS）。Klf 2和Klf 4是机械敏感性转录因子，内皮细胞向间质细胞转化（EndoMT），这是缓冲（心脏瓣膜祖细胞）所必需的过程发展了解EndoMT如何通过机械调制在空间和时间上控制， Klf 2/4将提供对HLHS患者中存在的心脏瓣膜缺陷的见解。我们的实验室发现初级纤毛（非运动的，杆状机械传感器）在心脏发生中的内皮细胞（EC）上，在左右不对称中的作用。然而，EC初级纤毛是否将机械信号传递到 Klf 2/4在垫层发育过程中的调节还有待证实。这项提案的目的是发现在体心脏垫形成过程中机械力、EC初级纤毛和Klf 2/4之间的关系。根据我的初步结果，这个建议的总体假设是EC初级纤毛连接机械力和Klf 2/4转录在Ca 2+和细胞内的依赖性方式，体内内膜垫形成。为了解决这个假设，我们将利用小鼠和斑马鱼模型老鼠的心脏更接近人类（斑马鱼有两个腔室），但基因控制心脏发生的程序是保守的。静态鼠标示例中可能丢失的事件可能相反，由于它们的光学清晰度和外部特性，发展目的1将研究EC初级纤毛和Klf 2/4表达在心脏上的空间变化垫发展，利用原位杂交和免疫荧光在小鼠，和各种转基因标记活斑马鱼中Klf 2/4的内膜、纤毛和启动子区域的线。目标2将测试是否 Klf 2/4通过使用各种突变背景依赖于纤毛、纤毛Ca 2+和/或机械力，例如如Ift 20和Kif 3a（纤毛科斯），Ncx/沉默心脏吗啉代（分别为心跳KO，小鼠和斑马鱼），和Pkd 2（睫状体Ca 2 + KO）。我们的数据提供了对Klf 2/4机械敏感性控制机制的深入了解在心脏垫形成过程中，并强调了纤毛在心脏发生过程中作为机械传感器的作用。这该提案涉及NIH-NHLBI的目标1和2关于调查遗传和机械许多CHD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CHD患者经历的进行性瓣膜功能障碍。我对生活的利用，整个- 底座成像将超出静态、固定胚胎中可能的观察范围，并带来罕见的纤毛依赖的转录在体内的前景。机械力在发育过程中的作用是快速扩大的领域，我们的研究结果将有助于基础和翻译生物学的观点。