Cilia in cardiac morphogenesis

描述（由申请人提供）：先天性心脏病是最常见的严重出生缺陷，影响。8%的活产婴儿正常的心脏发育取决于遗传和表观遗传因素之间复杂的相互作用。特别是，血流和心脏功能对心脏形态发生至关重要，然而，这些机械信号被感知和解释的机制尚不清楚。纤毛通过其在胚胎组织器（淋巴结）的功能在心脏LR不对称的发展中也是必不可少的，最近发现纤毛在其他管状、充满液体的器官（如肾脏）中起机械传感器的作用。我们已经在e8.5 - e12.5的小鼠心脏中发现了一组纤毛，称为心脏纤毛，对应于从血流开始到瓣膜形成和分隔的发育时间。这一建议的目的是确定心脏纤毛在心脏形态发生中的直接作用机制，而不是它们在LR不对称产生中的作用。纤毛不动但结构正常的小鼠，其脏器沿LR轴的定位异常。虽然在7-50%的受影响小鼠中观察到心脏内缺陷，但相当多的小鼠存活到成年，心脏结构和功能正常。相比之下，完全没有纤毛或纤毛感应的小鼠有严重的心脏缺陷，其外显率为100%，导致妊娠中期胚胎致命，与LR轴发育无关。这些观察结果表明，纤毛在心脏发育中是必需的，独立于它们在LR发育中的功能。我们假设心脏纤毛的功能是作为细胞外信号的传感器，如血流、心功能或分泌的配体来影响形态发生。在本提案的Spec. Aim 1中，我们将定义心脏纤毛的作用：它们是机械传感器、刺猬受体还是运动结构？为此，将检查心脏纤毛的分布和组成。组成性纤毛突变对心脏发育的作用将通过分析小鼠胚胎的心脏表型来评估，这些突变导致纤毛运动缺陷、纤毛生物发生或纤毛机械感觉。心脏缺陷和纤毛的分布将在小鼠胚胎中进行评估，突变导致心跳消失。纤毛在LR发育中的作用将与其在桡动脉内的功能区分开来。在Spec. Aim 2中，我们将通过使用Cre-lox技术从心外膜、心内膜、心包和心前野特异性地去除纤毛，从而确定心脏纤毛发挥作用的位置。最后，在Spec. Aim 3中，我们将试图定义心脏纤毛如何直接形态发生。本文将通过分析纤毛功能和生物发生突变的小鼠胚胎心脏的上皮-间充质转化、增殖和hedgehog信号传导，研究连接纤毛感知与心脏形态发生的下游信号通路。公共卫生相关性：先天性心脏病的影响。占所有活产婴儿的8%，是最常见的主要出生缺陷。纤毛是天线状结构，从几乎所有细胞延伸，从细胞外环境获取和解释信息，已知异常纤毛可导致某些类型的复杂先天性心脏病。本研究旨在探讨纤毛在心脏发育过程中如何指导心脏的正常发育。

DESCRIPTION (provided by applicant): Congenital heart disease is the most common serious birth defect, affecting .8% of liveborn infants. Normal cardiac development depends on complex interplay between genetic and epigenetic factors. In particular, blood flow and cardiac function are essential for cardiac morphogenesis: however, the mechanism by which these mechanical signals are sensed and interpreted remains unclear. Cilia, which are also essential in the development of cardiac LR asymmetry via their function at the embryonic organizer (node), have recently been found to function as mechanosensors in other tubular, fluid-filled organs such as the kidney. We have identified a set of cilia, called cardiac cilia, in the mouse heart at e8.5 - e12.5, corresponding to the time in development extending from the onset of blood flow through valve formation and septation. The goal of this proposal is to define the mechanism by which cardiac cilia function directly in cardiac morphogenesis independent of their role in the generation of LR asymmetry. Mice with immotile, but structurally normal cilia have abnormal positioning of organs along the LR axis. Although intracardiac defects are observed in 7-50% of affected mice, a significant number survive to adulthood with structurally and functionally normal hearts. In contrast, mice with complete absence of cilia or ciliary sensing have severe cardiac defects with 100% penetrance that result in mid-gestational embryonic lethality independent of LR axis development. These observations suggest that cilia are required in cardiac development independently from their function in LR development. We hypothesize that cardiac cilia function as sensors for extracellular signlas such as flow, cardiac function or secreted ligands to affect morphogenesis. In Spec. Aim 1 of this proposal, we will define what cardiac cilia do: are they mechanosensors, hedgehog receptors or motile structures? To this end, the distribution and composition of cardiac cilia will be examined. The role of constitutive ciliary mutations on cardiac development will be evaluated by analysis of the cardiac phenotype of mouse embryos with mutations resulting in defective ciliary motility, ciliary biogenesis or ciliary mechanosensation. The cardiac defects and distribution of cilia will be evaluated in mouse embryos with a mutation resulting in an absent heart beat. The role of cilia in LR development will be distinguished from their intracradiac function. In Spec. Aim 2, we will identify where cardiac cilia exert their effect by using Cre-lox technology to delete cilia specifically from the epicardium, endocardium, pericardium and anterior heart field. Finally, in Spec. Aim 3 we will seek to define how cardiac cilia direct morphogenesis. Here, the downstream signaling pathway(s) connecting ciliary sensing with cardiac morphogenesis will be investigated by analyzing epithelial-mesenchymal transformation, proliferation and hedgehog signaling in mouse embryo hearts with mutations in ciliary function and biogenesis. PUBLIC HEALTH RELEVANCE: Congenital heart disease affects .8% of all liveborn infants, and is the most common major birth defect. Cilia are antenna-like structures that extend from almost all cells to obtain and interpret information from the extracellular environment, and abnormal cilia are known to cause some types of complex congenital heart disease. This proposal investigates how cilia function in the developing heart to instruct normal heart development.