Nr2f1a suppresses bulbous arteriosus identity in the zebrafish atrium

项目摘要/摘要 脊椎动物心脏的正常发育需要保持适当的心腔比例。不适当 维持心肌细胞(CM)的特性是许多先天性心脏病(CHD)的基础，这些先天性心脏病可能 会导致新生儿以及整个成年期的心肌病和病态。Nr2f孤儿核激素 受体是脊椎动物心房发育的保守调节因子。NR2F2突变与 人类的CHDS。我们最近的工作表明斑马鱼Nr2f1aa在功能上等同于哺乳动物 Nr2f2与早期心房室发育有关，因为它在心房中特异表达，对 适当的房性分化。在这里，我们使用了一种新的ENU诱导的突变名为橡子蠕虫(ACO)，它表现出 Nr2f1a表达缺失。虽然经过改造的nr2f1a突变胚胎在我们手中是早期致命的，但我们已经 发现一些ACO突变体可以存活到成年期，这为研究 Nr2f1a在成熟心脏中的长期丢失。成人ACO心脏形成一个非收缩的房室，这是 周围是不透明的结缔组织。令人惊讶的是，与小鼠条件性Nr2f2的预测相反 KOS缺乏Nr2f2的房性CMS将具有心室CM特征，应用AFOG进行组织学分析 染色显示成人ACO心房缺乏梳状或小梁结构，而是有胶原基质。 类似于流出道球状动脉(BA)。分离的ACO心脏的转录组分析 Chambers和原位杂交证实，ACO突变的心房在上调的同时表达BA特异性基因 维甲酸(RA)信号成分。我们发现，心房的类BA标识首先在静脉处被观察到 从受精后3周开始(WPF)。在目标1中，我们将使用复杂的遗传谱系追踪 系统永久标记心房CMS，并阐明ACO突变成人中BA样房室的来源。 在目标2中，我们将使用新的转基因工具在2WPF开始的心房CMS中特异性地恢复nr2f1a到 确定Nr2f1a抑制BA样心房的发育窗口。在目标3中，我们将使用 药理学方法调节维甲酸信号(RA)信号水平2-3WPF以阐明 在ACO突变体中，Nr2f1a下游过量的RA信号促进了BA样房室的形成。 因此，我们的发现强调了Nr2f1a在抑制心房内BA同一性方面出人意料的作用，这可能 对儿童和成人中发现的先天性心脏病的分子和细胞病因学产生新的见解。

Project Summary/Abstract Normal vertebrate heart development involves the preservation of proper cardiac chamber proportions. Improper maintenance of cardiomyocyte (CM) identity underlies numerous congenital heart defects (CHDs), which can lead to cardiomyopathy and morbity in newborns as well as throughout adulthood. Nr2f orphan nuclear hormone receptors are conserved regulators of atrial development in vertebrates. NR2F2 mutations are associated with CHDs in humans. Our recent work demonstrates that zebrafish Nr2f1ais the functional equivalent of mammalian Nr2f2 with respect to early chamber development, as it is specifically expressed in the atrium and is vital for proper atrial differentiation. Here, we use a novel ENU-induced mutant named acorn worm (aco), which exhibits a loss of Nr2f1a expression. While engineered nr2f1a mutant embryos are early lethal in our hands, we have found that some aco mutants are viable to adulthood, affording a unique opportunity to investigate the effects of prolonged Nr2f1a loss in the mature heart. Adult aco hearts develop a non-contractile atrial chamber, which is surrounded by opaque connective tissue. Surprisingly, in contrast to the prediction from mouse conditional Nr2f2 KOs that atrial CMs lacking Nr2f2 would take on ventricular CM identity, histological analysis using AFOG staining revealed the adult aco atria lack pectinate or trabecular structures and instead have collagenous matrix similar to the bulbous arteriosus (BA) of the outflow tract. Transcriptome analyses from isolated aco cardiac chambers and in situ hybridization affirmed that aco mutant atria express BA-specific genes while upregulating retinoic acid (RA) signaling components. We find the BA-like identity of the atrium is first observed at the venous pole starting at 3 weeks post fertilization (wpf). In Aim 1, we will use a sophisticated genetic lineage tracing system to permanently label atrial CMs and elucidate the origin of the BA-like atrial chamber in aco mutant adults. In Aim 2, we will use novel transgenic tools to restore nr2f1a specifically in atrial CMs beginning at 2 wpf to determine the developmental window when Nr2f1a is critical to suppress the BA-like atria. In Aim 3, we will use pharmacological methods to modulate retinoic acid signaling (RA) signaling levels from 2 to 3 wpf to elucidate excess RA signaling downstream of Nr2f1a in aco mutants promotes the formation of the BA-like atrial chamber. Therefore, our findings highlight an unexpected role for Nr2f1a in repressing BA-identity within atria, which may yield novel insight into the molecular and cellular etiology of CHDs found in children and adults.