Mechanisms of second heart field development regulated by Nkx genes

项目总结 同源结构域转录因子NKX2-5是最常见的与 先天性心脏缺陷(CHD)，占特定畸形的1-4%，易患 心脏动脉和静脉两极的异常。潜在的分子机制 对于在NKX2-5突变患者中发现的心脏畸形，人们仍然知之甚少。为了提高 在这些病例的诊断和治疗措施中，更准确地了解早期心脏 流出(OFT)和流入(IFT)区的发育过程是至关重要的。胚胎心脏开始 作为源于第一心区(FHF)前体细胞的线形管，其扩张发生在 第二心区(SHF)的晚期分化细胞向动脉和静脉两极分化。表达了Nkx2-5 在FHF和SHF中，虽然FHF中Nkx基因的重要功能与心脏 Nkx基因调控的不同机制目前知之甚少。 前(ASHF)和后(PSHF)SHF。通过利用斑马鱼模式的好处，我们最近 已公布的证据表明，NKX2.5和NKX2.7这两个NKX2-5同系物在斑马鱼中表达 心脏在维持心室特性方面起着至关重要的作用，并在 SHF心肌细胞。此外，我们的初步数据提供了nkx基因表现出的新证据。 以前未被认识到的，在调节SHF祖细胞群体中的关键作用，通过离散 OFT和IFT的机制。我们发现nkx基因促进了动脉中aSHF前体细胞的增强。 将LIM同源结构域转录因子Isl1极化并限制于静脉窦。在这个提案中，我们测试 新的假设，即nkx基因需要招募aSHF祖细胞到OFT并限制pSHF 先祖通过ISL1到达IFT。在目标1中，我们将剖析Nkx基因在细胞和时间上的作用 利用热激的aSHF和pSHF的规范、增长、扩散和身份保持 NKX2.5诱导过表达，时间序列分析，EDU掺入研究，发育时序 化验和最先进的显微镜。在目标2中，我们将利用斑马鱼模型以及CRISPR和 检查以前未识别的Nkx直接和间接下游效应的芯片方法学 与人类冠心病相关的基因和新的候选基因。结合斑马鱼中可用的工具 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衍生的先天性心脏病，一些最严重和致命的畸形与 Nkx2-5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.