Molecular mechanisms of atrial development and regeneration

项目摘要/摘要 先天性心脏病是最常见的先天性畸形。5%的CHD包括 房室间隔缺损(AVSD)。然而，大多数aVSD背后的分子病因学并不是 明白了。此外，冠心病会在晚年导致心血管疾病，导致心律失常， 中风和过早死亡。为了开发能够预防冠心病和治愈特定疾病的新疗法 心血管组织，关键是要了解指导正常 心腔发育和再生。因此，我们实验室的长期目标是了解 指导个体心腔发育的保守机制和特殊心腔 脊椎动物的再生机制。已知需要的信号很少，特别是指向心房的信号 目前尚不清楚心房再生的具体调控因素。这样做的具体目的是 建议阐明同线长非编码rna(LncRNA)as-oca限制的机制 抑制Nr2f1a翻译对斑马鱼心房发育和再生的影响这方面的研究 建议与人类健康有关，因为最近的基因组分析表明，孤儿的突变 核受体Nr2f2与人类的房间隔缺损相关。而Nr2f2基因敲除小鼠和体外研究 人类干细胞的研究揭示了心房发育对Nr2f1和Nr2f2的需求， Nr2f蛋白引导心房正常发育的机制尚不完全清楚。 重要的是，目前还不了解表观遗传依赖的lncRNA调节nr2f的机制。 蛋白质。我们对斑马鱼新突变体橡子虫(ACO)的初步分析表明，过量 AS-ocA的表达特异性地限制了后来分化的第二心区(SHF)来源的增加 心房细胞。在目标1中，我们将使用囊胚细胞移植，体内心肌细胞分化试验，以及 基因组编辑以确定心房缺陷潜在的细胞需求和AS增加的原因- OCA在ACO突变体中的表达。我们不知道AS-OCA是如何抑制ACO突变体中的Nr2f1a翻译的。在……里面 目的2，我们将使用RNA和核糖体结合技术和功能丧失方法来确定AS- OCA通过与nr2f1a 3‘非翻译区相互作用抑制Nr2f1a的翻译。除了他们的 在发育过程中，我们发现AS-ocA和nr2f1a在 成年斑马鱼。在目标3中，我们将测试AS-OCA对胚胎和成年心脏模型的影响。 再生。由于Nr2f转录因子在所有脊椎动物的心房发育中发挥着保守的作用， 这些研究将极大地提高我们对转录后调节正常的机制的理解 脊椎动物心房发育与aVSD和ASDS的分子病因学。最终，这些 研究将为能够有效预防儿童先天性心脏病的改进疗法奠定基础 修复成年人受伤的心脏。

Project Summary/Abstract Congenital heart defects (CHDs) are the most common congenital malformations. 5% of CHDs comprise atrioventricular septal defects (AVSDs). However, the molecular etiology underlying most AVSDs are not understood. Furthermore, CHDs can cause cardiovascular diseases later in life, resulting in arrhythmias, stroke, and premature death. In order to develop novel therapies able to prevent CHDs and heal specific cardiovascular tissues, it is critical to garner understanding of fundamental mechanisms directing normal cardiac chamber development and regeneration. Therefore, long-term goals of our lab are to understand conserved mechanisms that direct the development of the individual cardiac chambers and chamber-specific mechanisms of regeneration in vertebrates. Few signals are known to be required that specifically direct atrial development, with specific regulators of atrial regeneration not being understood. The specific aims of this proposal are to elucidate the mechanisms by which a syntenic long non-coding RNA (lncRNA) as-oca restricts atrial development and regeneration through inhibition of Nr2f1a translation in zebrafish. The studies in this proposal are relevant to human health as recent genomic analysis indicates that mutations in the orphan nuclear receptor Nr2f2 are associated with AVSDs in humans. While Nr2f2 knockout mice and in vitro studies with human stem cells have revealed requirements for both Nr2f1 and Nr2f2 in atrial development, the mechanisms by which Nr2f proteins direct proper atrial development are not completely understood. Importantly, there is currently no understanding of epigenetic lncRNA-dependent mechanisms regulating Nr2f proteins. Our preliminary analysis of the novel zebrafish mutant acorn worm (aco) indicate that excess expression of as-oca specifically restricts the addition of later differentiating second heart field (SHF)-derived atrial cells. In Aim 1, we will use blastula cell transplantation, in vivo cardiomyocyte differentiation assays, and genome editing to determine the cellular requirements underlying the atrial defects and cause of increased as- oca expression in aco mutants. We do not understand how as-oca inhibits Nr2f1a translation in aco mutants. In Aim 2, we will use RNA and ribosomal association techniques and loss of function methods to determine if as- oca inhibits Nr2f1a translation through interactions with the nr2f1a 3' untranslated region. In addition to their requirements during development, we find that as-oca and nr2f1a are specifically expressed in the atria of adult zebrafish. In Aim 3, we will test the effects of as-oca on embryonic and adult models of cardiac regeneration. Because Nr2f transcription factors play conserved roles in atrial development of all vertebrates, these studies will dramatically improve our understanding of posttranscriptional mechanisms regulating normal vertebrate atrial development and the molecular etiology of AVSDs and ASDs in humans. Ultimately, these studies will generate a foundation for improved therapies capable of preventing CHDs in children and efficiently repairing injured hearts in adults.