Specification And Patterning of Developing Blood Vessels

发育中血管的规格和模式

• 批准号: 8941454
• 负责人: Brant Weinstein
• 金额: $ 103.19万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8941454
• 关键词: Affect; Anatomy; Angiogenesis Inhibitors; Angiogenic Factor; Animals; Atlases; Blood; Blood Cells; Blood Vessels; Cell Lineage; Cells; Clinical; Cues; Development; Embryo; Embryonic Development; Endothelium; Epigenetic Process; Ethylnitrosourea; Family; Fishes; Fresh Water; Gene Expression; Genes; Genetic; Genetic Screening; Genome; Goals; Growth; Hematopoietic; Image; Life; Ligands; Lymphatic; Lymphatic Endothelial Cells; Malignant Neoplasms; Methodology; Methods; Molecular; Molecular Analysis; Morphogenesis; Mutagenesis; Nutrient; Online Systems; Organ; Oxygen; Pathway interactions; Pattern; Phenotype; Play; Proteins; Reporting; Role; Seeds; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism Map; Smooth Muscle Myocytes; Stem cells; Time; Tissues; Transgenic Organisms; Vascular Endothelial Growth Factors; Vascular Smooth Muscle; Vertebrates; Work; Zebrafish; base; cell type; combat; exome sequencing; experimental analysis; genetic analysis; in vivo; insight; interest; microangiography; mutant; neuronal guidance; novel; screening; stem; tool; Affect; Anatomy; Angiogenesis Inhibitors; Angiogenic Factor; Animals; Atlases; Blood; Blood Cells; Blood Vessels; Cell Lineage; Cells; Clinical; Cues; Development; Embryo; Embryonic Development; Endothelium; Epigenetic Process; Ethylnitrosourea; Family; Fishes; Fresh Water; Gene Expression; Genes; Genetic; Genetic Screening; Genome; Goals; Growth; Hematopoietic; Image; Life; Ligands; Lymphatic; Lymphatic Endothelial Cells; Malignant Neoplasms; Methodology; Methods; Molecular; Molecular Analysis; Morphogenesis; Mutagenesis; Nutrient; Online Systems; Organ; Oxygen; Pathway interactions; Pattern; Phenotype; Play; Proteins; Reporting; Role; Seeds; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism Map; Smooth Muscle Myocytes; Stem cells; Time; Tissues; Transgenic Organisms; Vascular Endothelial Growth Factors; Vascular Smooth Muscle; Vertebrates; Work; Zebrafish; base; cell type; combat; exome sequencing; experimental analysis; genetic analysis; in vivo; insight; interest; microangiography; mutant; neuronal guidance; novel; screening; stem; tool

As described in the goals and objectives section of this report, this project consists of four specific aims: Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish The development of new tools to facilitate vascular studies in the zebrafish has been an important ongoing aim of this project. In previous work we (i) developed a widely used confocal microangiography method (ii) compiled an atlas of the anatomy of the developing zebrafish vasculature, (iii) generated a variety of transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize vessel formation in intact, living embryos, and (iv) developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish. We are currently developing many new transgenic lines useful for in vivo vascular imaging as well as for in vivo blood or lymphatic endothelial-specific functional manipulation of signaling pathways involved in vascular specification, patterning, and morphogenesis. We are also developing new transgenic tools to facilitate high-throughput whole genome profiling of gene expression in selected cell types, in particular blood and lymphatic vascular endothelial and smooth muscle cells. Genetic Analysis of Vascular Development Previously, we have used forward-genetic ENU mutagenesis screens in transgenic zebrafish to generate, identify, and characterize many new zebrafish mutants affecting the formation of the developing vasculature. We identified and positionally cloned mutants with phenotypes including loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. These mutants have resulted in numerous important dscoveries related to endothelial specification, arterial differentiation, vascular patterning, and VEGF signalling, to mention only a few. In our current studies we are focusing on several mutants affecting VEGF-dependent and VEGF-independent vascular signaling pathways. We are also carrying out additional genetic screens focusing on vascular smooth muscle. As part of separate project we recently developed a SNP map for the zebrafish and web-based tools that allow us to use whole-genome and whole-exome sequencing to greatly accelerate discovery of the defective genes from identified mutants. Analysis of Vascular Specification, Patterning, and Morphogenesis We have previously used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and used molecular and experimental analysis understand how this pattern arises and what cues guide vascular specification, differentiation, and network assembly during development. Our discoveries have included evidence that neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning. Our current work includes projects aimed at (a) studying the specification, differentiation, and patterning of vascular smooth muscle in the zebrafish, making use of newlty developed transgenic tools, (b) understanding the role of intracellular signalling substrates in regulating vascular endothelial signalling, (c) exploring the role of BMP family ligands in modulating vessel growth and vascular integrity, (d) analyzing additional pro- or anti-angiogenic factors. Emergence of Hematopoietic Stem and Progenitor Cells from the Endothelium We have recently developed an additional aim based on our identification of a novel epigenetic mechanism regulating the emergence of hematopoietic stem and progenitor cells (HSPC) from the endothelium. HSPC are critical cells that emerge during early vertebrate development and subsequently seed the development of all blood cell lineages for the life of the animal. We have discovered a novel epigenetic mechanism intersecting with genetic pathways regulating the specification of these cells from endothelium. We are currently characterizing this epigenetic mechanism in detail.

如本报告的目标和目标部分所述，该项目包括四个具体目标： 开发用于斑马鱼血管发育的实验分析的工具 开发新工具以促进斑马鱼中的血管研究一直是该项目的重要目标。 在先前的工作中，我们（i）开发了一种广泛使用的共焦微传导方法（II），编译了发展中斑马鱼脉管系统解剖结构的地图，（III）产生了各种表达转基因的斑马鱼线，表达不同的荧光蛋白，从长期多光子共聚焦时间解体成像的方法学对转基因鱼的血管发育成像。 我们目前正在开发许多新的转基因线，可用于体内血管成像以及体内血液或淋巴内皮特异性功能操纵，对参与血管规范，图案和形态发生的信号通路的功能操纵。 我们还开发了新的转基因工具，以促进选定细胞类型中基因表达的高通量全基因组分析，尤其是血液和淋巴血管内皮和平滑肌细胞。 血管发育的遗传分析 以前，我们已经在转基因斑马鱼中使用了前遗传ENU诱变筛选，以生成，识别和表征许多影响发育中脉管系统形成的新的斑马鱼突变体。 我们鉴定出具有表型的和位置克隆的突变体，包括大多数血管或血管子集的损失，发芽/分支的增加以及船只误会。 这些突变体导致了许多与内皮规范，动脉分化，血管模式和VEGF信号传导相关的重要D尺度。 在目前的研究中，我们着重于几个影响依赖VEGF和veGF的血管信号通路的突变体。 我们还将执行以血管平滑肌为本的其他遗传筛选。 作为单独项目的一部分，我们最近为斑马鱼和基于Web的工具开发了SNP图，该工具使我们能够使用全基因组和全外观测序，以极大地加速了来自已识别突变体的有缺陷基因的发现。 血管规范，图案和形态发生的分析 我们以前已经使用多光子的延时成像来表征整个发育中的斑马鱼的船只组装模式，并使用分子和实验分析来理解这种模式的产生以及什么提示指导在发育过程中的血管规范，分化和网络组件。 我们的发现包括证据表明，神经元引导因素在血管指导和血管模式中起重要作用。 Our current work includes projects aimed at (a) studying the specification, differentiation, and patterning of vascular smooth muscle in the zebrafish, making use of newlty developed transgenic tools, (b) understanding the role of intracellular signalling substrates in regulating vascular endothelial signalling, (c) exploring the role of BMP family ligands in modulating vessel growth and vascular integrity, (d) analyzing additional亲或抗血管生成因素。 来自内皮的造血干和祖细胞的出现 我们最近基于我们对一种新的表观遗传机制的鉴定，开发了一个额外的目标，该机制调节了内皮细胞中造血干细胞和祖细胞（HSPC）的出现。 HSPC是在早期脊椎动物发育过程中出现的关键细胞，随后播种了动物生命中所有血细胞谱系的发育。 我们发现了一种新型的表观遗传机制，该机制与调节内皮细胞规范的遗传途径相交。 我们目前正在详细介绍这种表观遗传机制。