Cilia in cardiac morphogenesis

描述(申请人提供)：先天性心脏病是最常见的严重出生缺陷，影响0.8%的活产婴儿。心脏的正常发育依赖于遗传因素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是必不可少的：然而，这些机械信号被感知和解释的机制仍然不清楚。纤毛通过其在胚胎组织者(节点)的功能在心脏LR不对称性的发展中也是必不可少的，最近被发现在其他管状的、充满液体的器官中作为机械传感器发挥作用，如肾脏。我们在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间隔的发育时间。这项建议的目的是明确心脏纤毛在心脏形态发生中直接作用的机制，独立于它们在LR不对称产生中的作用。纤毛不动但结构正常的小鼠，器官沿LR轴的位置异常。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，完全缺乏纤毛或纤毛感觉的小鼠有严重的心脏缺陷，其外显率为100%，导致妊娠中期胚胎死亡，与LR轴的发育无关。这些观察表明，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。我们假设心脏纤毛作为细胞外信号的感受器，如流量、心功能或影响形态发生的分泌配体。在等级库这个提议的目标1，我们将定义心脏纤毛的作用：它们是机械感受器、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。结构性纤毛突变对心脏发育的作用将通过分析具有导致纤毛运动、纤毛生物发生或纤毛机械感觉缺陷的突变的小鼠胚胎的心脏表型来评估。心脏缺陷和纤毛的分布将在突变导致心跳缺失的小鼠胚胎中进行评估。纤毛在LR发育中的作用将与其在放射内的功能区分开来。在等级库目的2，利用Cre-lox技术特异性地去除心外膜、心内膜、心包和心前区的纤毛，以确定心脏纤毛在哪里发挥作用。最后，在规范中。目的3我们将试图确定心脏纤毛如何引导形态发生。在这里，我们将通过分析睫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信号来研究连接纤毛感觉和心脏形态发生的下游信号通路(S)。公共卫生相关性：先天性心脏病影响所有活产婴儿的0.8%，是最常见的主要出生缺陷。纤毛是一种天线状结构，从几乎所有的细胞延伸出来，从细胞外环境中获取和解释信息，而异常的纤毛被认为是导致某些类型的复杂先天性心脏病的原因。这项建议调查纤毛如何在发育中的心脏中发挥作用，以指导正常的心脏发育。

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.