Mechanisms of second heart field development regulated by Nkx genes

项目摘要 同源结构域转录因子NKX 2 -5是最常见的突变基因， 先天性心脏病（CHD），占特定畸形的1-4%， 心脏的动脉和静脉极的异常。潜在的分子机制 对于NKX 2 -5突变患者中发现的心脏畸形，仍然知之甚少。提高 在这些情况下，诊断和治疗措施，更准确地了解早期心脏病 流出道（OFT）和流入道（IFT）的发育过程至关重要。胚胎心脏开始 作为源自第一心场（FHF）祖细胞的线性管，通过 第二心脏领域（SHF）的晚期分化细胞的动脉和静脉极。Nkx 2 -5表达 在FHF和SHF中，虽然Nkx基因在FHF中的重要功能与心脏功能有关， 由于Nkx基因在细胞的分化、分化和形态发生中起着重要的作用，因此对Nkx基因在细胞分化和形态发生中的不同调控机制知之甚少。 前（aSHF）和后（pSHF）SHF。通过利用斑马鱼模型的好处，我们最近 已发表的证据表明，斑马鱼中表达的两种NKX 2 -5同源物nkx2.5和nkx2.7 心脏在维持心室特性中起重要作用，并在 SHF心肌细胞。此外，我们的初步数据提供了新的证据，nkx基因表现出 以前未被认识到的是，通过离散的细胞因子调节SHF祖细胞群体的关键功能， OFT和IFT的机制。我们发现Nkx基因促进动脉中aSHF祖细胞扩增， pole和restrict isl 1，一个LIM同源域转录因子，静脉窦。在本提案中，我们测试 新的假设是nkx基因是募集aSHF祖细胞到OFT和限制pSHF所必需的， 祖细胞通过isl 1进入IFT。在目标1中，我们将剖析Nkx基因在细胞和时间中的作用， 在采用热休克的aSHF和pSHF中的特化、增长、增殖和同一性维持 诱导型nkx2.5过表达，时间序列分析，EdU掺入研究，发育时间 分析和最先进的显微镜。在目标2中，我们将利用斑马鱼模型沿着CRISPR， ChIP方法用于检查先前未识别的Nkx直接和间接下游效应物 基因以及与人类CHD相关的新候选基因。结合斑马鱼可用的工具 和人类基因组学数据，我们的研究将揭示Nkx基因调控的发育机制 导致SHF衍生的CHD，一些与SHF相关的最严重和致命的畸形 NKX 2 -5突变。

PROJECT SUMMARY The homeodomain transcription factor, NKX2-5, is the most commonly mutated gene associated with congenital heart defects (CHDs), accounting for 1-4% of specific malformations with a predilection for abnormalities at the arterial and venous poles of the heart. The underlying molecular mechanisms responsible for cardiac malformations found in patients with NKX2-5 mutations remain poorly understood. To improve diagnostic and therapeutic measures in these cases, a more precise understanding of early cardiac developmental processes at the outflow (OFT) and inflow (IFT) tracts is critical. The embryonic heart begins as a linear tube derived from first heart field (FHF) progenitors, with expansion occurring through accretion of late-differentiating cells of the second heart field (SHF) to the arterial and venous poles. Nkx2-5 is expressed in both the FHF and SHF and, while vital functions of Nkx genes in the FHF have been implicated in cardiac specification and morphogenesis, little is known about the distinct mechanisms regulated by Nkx genes in the anterior (aSHF) and posterior (pSHF) SHFs. By exploiting benefits of the zebrafish model, we recently published evidence demonstrating that nkx2.5 and nkx2.7, two NKX2-5 homologs expressed in the zebrafish heart, play essential roles in maintaining ventricular identity and display similar chamber-specific functions in SHF cardiomyocytes. Furthermore, our preliminary data provide new evidence that nkx genes exhibit previously unappreciated, crucial functions in regulating SHF progenitor populations through discrete mechanisms at OFT and IFT. We find that Nkx genes promote aSHF progenitor augmentation at the arterial pole and restrict isl1, a LIM homeodomain transcription factor, to the sinus venosus. In this proposal, we test the novel hypothesis that nkx genes are required to recruit aSHF progenitors to the OFT and to restrict pSHF progenitors via isl1 to the IFT. In Aim 1, we will dissect the cellular and temporal roles of Nkx genes in specification, accretion, proliferation, and identity maintenance in the aSHF and pSHF employing heat-shock inducible overexpression of nkx2.5, time series analysis, EdU incorporation studies, developmental timing assays, and state-of-the-art microscopy. In Aim 2, we will utilize the zebrafish model along with CRISPR and ChIP methodologies to examine previously unrecognized direct and indirect downstream effectors of Nkx genes and also new candidates associated with CHD in humans. Combining the tools available in zebrafish and human genomics data, our research will uncover the developmental mechanisms regulated by Nkx genes that are responsible for SHF-derived CHDs, some of the most severe and lethal malformations associated with NKX2-5 mutations.