Specification And Patterning of Developing Blood Vessels

发育中血管的规格和模式

• 批准号: 7968553
• 负责人: Brant Weinstein
• 金额: $ 88.96万
• 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/7968553
• 关键词: Affect; Anatomy; Angiogenesis Inhibitors; Animals; Atlases; Blood Vessels; Brain hemorrhage; Chromosome Mapping; Clinical; Cues; DNA Sequence Rearrangement; Development; Embryo; Embryonic Development; Endothelial Cells; Eye; Fishes; Fresh Water; Generations; Genes; Genetic; Genetic Screening; Genome; Goals; Green Fluorescent Proteins; Growth; Harvest; Image; Intercellular Junctions; Laboratories; Legal patent; Life; Light; Locales; Lymphatic Endothelial Cells; Maintenance; Malignant Neoplasms; Maps; Methodology; Methods; Molecular; Molecular Cloning; Morphogenesis; Nutrient; Organ; Oxygen; PLC gamma1; Pathway interactions; Pattern; Phenotype; Play; Positioning Attribute; Proteins; Publishing; Reporting; Resolution; Role; Screening procedure; Signal Pathway; Signal Transduction; Somites; Staging; Stroke; Structure; Susceptibility Gene; Time; Tissues; Transgenic Organisms; Tube; Vacuole; Vascular Endothelial Growth Factors; Vertebrates; Viola; Zebrafish; aortic arch; chaperonin; combat; experimental analysis; genetic analysis; hindbrain; imaging modality; in vivo; insight; interest; mutant; neuronal guidance; novel; positional cloning; tool; transcription factor; two-photon; vascular bed

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. Previously, we (i) established a microangiographic method for imaging patent blood vessels in the zebrafish and used this method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html), (ii) 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 (iii) developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish. Recent technical advances have greatly facilitated generation of new transgenic lines in the fish, and we are currently developing many new lines useful for in vivo vascular imaging as well as for in vivo endothelial-specific functional manipulation of signaling pathways involved in vascular specification, patterning, and morphogenesis. Genetic Analysis of Vascular Development We use forward-genetic approaches to identify and characterize new zebrafish mutants that affect the formation of the developing vasculature. We are carrying out an ongoing large-scale genetic screen for ENU-induced mutants using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. We have screened well over 2000 genomes to date, and identified over 100 new vascular mutants with phenotypes including loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. We have recently initiated a new genetic screen to identify hemorrhagic stroke susceptibility genes. A bulked segregant mapping pipeline is in place to rapidly determine the rough position of newly identified mutants on the zebrafish genetic map, and fine mapping and molecular cloning is in progress for many mutants. We have previously positionally cloned the defective genes from a number of vascular-specific mutants, including violet beauregarde (defective in Alk1/acvrl1), plcg1 (defective in phospholipase C-gamma 1), and kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), etsrp (an ETS-related transcription factor). We are currently focusing on several mutants mutants affecting VEGF-dependent and VEGF-independent vascular signaling pathways. Ongoing mutant screens and positional cloning projects in the lab are continuing to yield a rich harvest of novel vascular mutants and genes, bringing to light new pathways regulating the formation of the developing vertebrate vasculature. Analysis of Vascular Morphogenesis and Vascular Integrity In previous studies we used high-resolution time-lapse two-photon imaging to show that the formation and intra- and inter-cellular fusion of endothelial vacuoles drives vascular lumen formation in vivo. We are currently carrying out studies to examine the formation and maintenance of vascular junctions, important in stroke. We are studying a variety of genes required for vascular morphogenesis and vascular integrity, including the pak2a and rap1b genes we recently published studies on, in order to begin to dissect the molecular regulatory mechanisms controlling vascular morphogenesis and the maintenance of vascular integrity. We are also developing transgenic lines that permit us to visualize the dynamics of endothelial cell-cell junctions and intracellular cytoskeletal structures in order to examine their role in the cellular rearrangements that occur during vascular sprouting and growth and vascular tube formation. Analysis of Vascular Patterning We have used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and ongoing studies in the laboratory are aimed at understanding how this pattern arises and the what cues guide vascular network assembly during development. We previously demonstrated that known neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning, showing that Semaphorin signaling it is an essential determinant of trunk vessel patterning. Current studies are aimed at further understanding the role of additional factors guiding the patterning of developing vascular networks in vivo, both in the trunk and in vascular beds in the eye, aortic arches, hindbrain, and other anatomical locales.

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。 Previously, we (i) established a microangiographic method for imaging patent blood vessels in the zebrafish and used this method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html), (ii) generated a variety of transgenic zebrafish lines expressing different血管或淋巴内皮细胞中的荧光蛋白，使我们有可能在完整的胚胎中可视化血管形成，而（iii）（iii）开发了长期多光子共凝聚时间间隔成像的方法论，是转基因鱼类中血管发育的血管发育。 最近的技术进步极大地促进了鱼类中新的转基因线的产生，目前，我们正在开发许多新线，可用于体内血管成像以及体内内皮内皮特异性功能操纵涉及血管规格，图案，图案和形成的信号途径的功能操纵。 血管发育的遗传分析 我们使用前瞻性遗传方法来识别和表征影响发展脉管系统形成的新斑马鱼突变体。我们正在使用血管中的转基因斑马鱼（GPF）进行ENU诱导的突变体的持续大规模遗传筛选。迄今为止，我们已经筛选了超过2000个基因组，并确定了100多种具有表型的新血管突变体，包括损失大多数血管或血管子集，增加的发芽/分支以及船只误会。我们最近启动了一个新的遗传筛选，以鉴定出血性中风敏感性基因。 建立了大量的分离映射管道，可以快速确定新近鉴定的突变体在斑马鱼遗传图上的粗糙位置，并且许多突变体都在进行精细的映射和分子克隆。 We have previously positionally cloned the defective genes from a number of vascular-specific mutants, including violet beauregarde (defective in Alk1/acvrl1), plcg1 (defective in phospholipase C-gamma 1), and kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), etsrp (an ETS-related转录因子）。 我们目前正在关注几个影响依赖VEGF和vegf独立的血管信号通路的突变体突变体。 实验室中正在进行的突变体筛选和位置克隆项目继续产生丰富的新型血管突变体和基因，从而揭示了调节发育中脊椎动物脉管系统形成的新途径。 血管形态发生和血管完整性的分析 在先前的研究中，我们使用高分辨率的延时两光子成像来表明内皮液泡的形成和细胞内和细胞间融合驱动体内的血管管腔形成。 我们目前正在进行研究，以研究中风重要的血管连接的形成和维护。 我们正在研究血管形态发生和血管完整性所需的多种基因，包括我们最近发表的研究的PAK2A和RAP1B基因，以便开始剖析控制血管形态发生的分子调节机制以及维持血管完整性的。 我们还开发了转基因线，使我们能够可视化内皮细胞 - 细胞连接和细胞内细胞骨架结构的动力学，以便检查它们在血管发芽和血管生长和血管管形成过程中发生的细胞重排中的作用。 血管图案分析 我们已经使用了多光子的延时成像来表征整个发育中的斑马鱼中血管组装模式的模式，实验室中正在进行的研究旨在了解这种模式的产生以及在开发过程中指导血管网络组装的方法。 我们先前证明，已知的神经元引导因素在血管引导和血管模式中起着重要的先前未知作用，表明闪光蛋白信号传导它是树干容器模式的重要决定因素。 目前的研究旨在进一步理解其他因素的作用，指导体内发展血管网络的模式，无论是在树干还是在眼睛的血管床中，主动脉弓，后脑脑和其他解剖层。