Mechanisms of second heart field development regulated by Nkx genes

Nkx基因调控第二心区发育的机制

• 批准号: 10045275
• 负责人: KIMARA L TARGOFF
• 金额: $ 6.07万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045275
• 关键词: Accounting; Animal Model; Anterior; Atrial Heart Septal Defects; Biological Assay; Cardiac; Cardiac Myocytes; Cardiac development; Cell Count; Cell Differentiation process; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Heart Defects; Cues; Data; Defect; Development; Developmental Process; Diagnostic; Embryo; Embryonic Heart; Ensure; Etiology; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Genes; Genomics; Goals; Heart; Heart Abnormalities; Heart Atrium; Heat-Shock Response; Homologous Gene; Human; Invertebrates; Knowledge; Lead; Maintenance; Measures; Mediating; Methodology; Microscopy; Modeling; Molecular; Morphogenesis; Mutagenesis; Mutate; Mutation; Neonatal; Neonatal Mortality; Pathology; Pathway interactions; Patients; Pediatric Cardiac Genomics Consortium; Play; Population; Process; Publishing; Quantitative Reverse Transcriptase PCR; Regenerative Medicine; Regulation; Research; Role; Structure; Structure of sinus venosus of fetus; Testing; Tetralogy of Fallot; Therapeutic; Therapeutic Intervention; Time Series Analysis; Treatment Efficacy; Tube; Venous; Ventricular; Work; Zebrafish; chromatin immunoprecipitation; de novo mutation; exome sequencing; experimental study; fetal; gene function; genomic data; homeodomain; human genomics; improved; innovation; malformation; mutant; neonatal morbidity; novel; overexpression; progenitor; recruit; remediation; self-renewal; spatiotemporal; therapeutic development; tool; transcription factor; transcriptome sequencing

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.

项目摘要 同源域转录因子NKX2-5是最常见的突变基因 先天性心脏缺陷（CHD），占特定畸形的1-4％，偏爱 心脏动脉和静脉两极的异常。负责的基本分子机制 对于NKX2-5突变患者中发现的心脏畸形，知识率仍然很少。改进 在这些情况下，诊断和治疗措施，对早期心脏的更精确理解 流出（经常）和流入（IFT）的发展过程至关重要。胚胎心开始 作为源自第一心脏场（FHF）祖细胞的线性管，通过积聚发生膨胀 第二个心脏场（SHF）的晚期分化细胞与动脉和静脉两极。 NKX2-5表示 在FHF和SHF中，尽管FHF中NKX基因的重要功能已与心脏有关 规范和形态发生，对由NKX基因调节的不同机制知之甚少 前（ASHF）和后（PSHF）SHFS。通过利用斑马鱼模型的好处，我们最近 已发表的证据表明NKX2.5和NKX2.7，斑马鱼中表达的两个NKX2-5同源物 心脏，在维持心室身份并显示相似的室特异性功能中发挥重要作用 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 以前未欣赏到通过离散调节SHF祖先种群的关键功能 OFT和IFT的机制。我们发现NKX基因在动脉上促进ASHF祖细胞增强 极和限制ISL1，是鼻窦静脉的LIM同源域转录因子。在此提案中，我们测试 NKX基因需要招募ASHF祖细胞并限制PSHF所需要的新型假设 通过ISL1到IFT的祖细胞。在AIM 1中，我们将剖析NKX基因在中的细胞和时间作用 ASHF和PSHF中的规格，积聚，增殖和身份维护 NKX2.5的诱导过表达，时间序列分析，EDU融合研究，发育时机 测定和最先进的显微镜。在AIM 2中，我们将与CRISPR和CRISPR一起利用斑马鱼模型 CHIP方法学以检查先前未识别的NKX的直接和间接下游效应子 基因以及与人类冠心病相关的新候选人。结合斑马鱼中可用的工具 和人类基因组学数据，我们的研究将发现NKX基因调节的发展机制 负责SHF衍生的CHD的，这是一些最严重，最致命的畸形 NKX2-5突变。