Specification And Patterning of Developing Blood Vessels

如本报告的目标和目的一节所述，该项目包括四个具体目标： 斑马鱼血管发育实验分析工具的开发 开发新的工具来促进斑马鱼血管研究一直是该项目的一个重要目标。 在以前的工作中，我们（i）开发了一种广泛使用的共聚焦微血管造影方法（ii）编制了发育中斑马鱼血管的解剖图谱，（iii）产生了多种在血管或淋巴管内皮细胞内表达不同荧光蛋白的转基因斑马鱼系，使我们能够可视化完整的活胚胎中的血管形成，和（iv）开发了转基因鱼血管发育的长期多光子共聚焦时移成像方法。 我们目前正在开发许多新的转基因株系，可用于体内血管成像以及体内血液或淋巴管内皮特异性功能操纵的信号通路参与血管的规格，图案化和形态发生。 值得注意的是，我们已经产生了RiboTag和AgoTag转基因斑马鱼，用于基因和microRNA表达的高通量全基因组组织特异性分析，并且我们已经证明这些转基因系可用于进行血管信号传导途径的体内分析。 我们还在开发用于脉管系统中组织特异性蛋白质组分析的转基因工具。 血管发育的遗传学分析 在此之前，我们已经在转基因斑马鱼中使用正向遗传ENU诱变筛选来产生、鉴定和表征许多影响发育中血管形成的新的斑马鱼突变体。 我们鉴定并定位克隆了具有表型的突变体，包括大多数血管或血管亚群的丢失、增加的发芽/分支和血管错误图案化。 这些突变体导致了许多重要的发现，涉及内皮细胞特化，动脉分化，血管图案，和VEGF信号，仅举几例。 最近的出版物包括对RECK和影响VEGF信号传导和血管生成的氨酰-tRNA合成酶的突变体的研究，对Rac信号传导在血管生成中的作用的研究，以及对作为抗血管生成癌症方法的磷酸肌醇再循环的靶向研究。 我们目前的许多努力是针对理解我们已经获得的影响血液或淋巴管形成或血管完整性的突变体中的缺陷的细胞和分子基础。 血管规格、图案化和形态发生的分析 我们以前使用多光子延时成像来表征整个发育中的斑马鱼的血管组装模式，并使用分子和实验分析来了解这种模式是如何产生的，以及在发育过程中引导血管规格，分化和网络组装的线索。 我们的发现包括证据表明，神经元的指导因素发挥了重要的以前未知的作用，血管的指导和血管图案。 我们目前的工作包括：（a）利用新开发的转基因工具研究斑马鱼血管平滑肌的特化、分化和模式化;（B）了解细胞内信号底物在调节血管内皮信号中的作用;（c）探索RhoA信号在血管形成和血管完整性中的作用;（d）描述胸鳍脉管系统的复杂组合和模式，胸鳍是与哺乳动物前肢（臂）类似的结构。

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. Notably, we have generated RiboTag and AgoTag transgenic zebrafish for high-throughput whole genome tissue-specific profiling of gene and microRNA expression, and we have demonstrated that these transgenic lines can be used to perform in vivo profiling of vascular signaling pathways. We are also developing transgenic tools for tissue-specific proteomic profiling in the vasculature. 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 discoveries related to endothelial specification, arterial differentiation, vascular patterning, and VEGF signaling, to mention only a few. Recent publications include studies on mutants in RECK and aminoacyl-tRNA synthetases affecting VEGF signaling and angiogenesis, on the role of Rac signaling in angiogenesis, and on targeting of phosphoinositide recycling as an anti-angiogenic cancer approach. Many of our current efforts are directed at understanding the cellular and molecular basis for the defects in mutants we have obtained affecting blood or lymphatic vessel formation or vascular integrity. 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 newly developed transgenic tools, (b) understanding the role of intracellular signaling substrates in regulating vascular endothelial signaling, (c) exploring the role of RhoA signaling in vessel formation and vessel integrity, (d) characterizing the complex assembly and patterning of the vasculature of the pectoral fin, an analogous structure to mammalian forelimbs (arms).