Specification And Patterning of Developing Blood Vessels

如本报告的目标和目的部分所述，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研制 开发新的工具来促进斑马鱼的血管研究一直是该项目正在进行的一个重要目标。在之前的工作中，我们(I)开发了一种广泛使用的共聚焦显微血管成像方法，(Ii)利用该方法编制了斑马鱼发育中血管的解剖学图谱，(Iii)获得了在血管或淋巴管内皮细胞中表达不同荧光蛋白的各种转基因斑马鱼系，从而使我们有可能可视化完整的活胚胎中的血管形成，(Iv)开发了对斑马鱼发育的长期延时成像的方法，(V)开发了针对细胞类型的RiboTag工具，用于体内转录蛋白质组的剖析。 我们正在继续开发许多新的转基因株系，用于体内成像、转录和蛋白质组学分析，以及特定血管细胞群体内的基因表达和基因破坏。我们最近还开发并积极使用对成年斑马鱼进行插管和长期成像的方法，使我们能够将斑马鱼模型用于复杂的高分辨率成像扩展到成年阶段，并研究成年动物的血管修复和再生(见下文)。 血管发育的遗传学分析 以前，我们在转基因斑马鱼中使用正向遗传ENU突变筛选来产生、鉴定和表征许多影响发育中的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，发芽/分枝增加，以及血管错构化。这些突变导致了许多与内皮指定、动脉分化、血管构型和血管内皮生长因子信号相关的重要发现，仅举几例。我们目前的许多努力都是为了了解我们获得的影响血管或淋巴管形成或血管完整性的突变缺陷的细胞和分子基础。我们的表观遗传突变筛选(在HD008977-04项目中描述)也幸运地导致了第一个已知的动静脉发育表观遗传调节因子的发现。 血管规格化、图案化和形态发生分析 在这个项目的整个生命周期中，我们一直使用尖端、复杂的显微成像工具和方法来表征发育中的斑马鱼的血管组装模式，然后使用分子和实验分析来了解这种模式是如何产生的，以及在发育过程中是什么线索指导着血管网络的规范、分化和组装。我们的发现包括一些首次证据表明，许多神经元引导因子在血管引导和血管构型中也发挥着以前未被认识到的关键作用。 我们正在进行的研究包括：(I)描述胸鳍血管系统的复杂组装和图案特征，这是一种类似哺乳动物前肢(手臂)的结构；(Ii)研究斑马鱼鳃的发育，这是一种与哺乳动物肺功能相似的气体交换器官，以及对其功能至关重要的独特而高度专业化的内皮细胞；(Iii)研究睾丸血管系统的发育及其在证明生殖细胞发育、维持和精子发生中的作用；(Iv)探讨RhoA信号在血管形成和血管完整性中的作用(另见HD008915-11项目)。 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规格、分化和模式；(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)表征胸鳍血管系统的复杂组装和模式，胸鳍是一种类似于哺乳动物前肢(ARM)的结构。 研究血管修复和再生 我们最近开发出了对成年斑马鱼进行插管和长期成像的新方法，使我们能够对成年斑马鱼进行血管修复和再生的实验研究。血管再生和修复对于伤口愈合是必不可少的，但在衰老和糖尿病等病理情况下，血管再生和修复明显受损。我们已经开发了皮肤伤口愈合的斑马鱼模型，现在正在使用这些模型来研究指导成人血管修复和再生的解剖学、细胞和分子机制，使用我们拥有的高分辨率成像和血管基因表达谱工具。我们的目标是确定在伤口愈合过程中对新生血管生成至关重要的关键信号通路和分子，长期目标是确定潜在的新治疗靶点。

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) used this method to compile 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 timelapse imaging of developing zebrafish, (v) developed RiboTag tools for cell type-specific in vivo translatome profiling. We are continuing to develop many new transgenic lines useful for in vivo imaging and transcriptomic and proteomic profiling of, and for gene expression and gene disruption within, specific vascular cell populations. We have also recently developed and are actively using methods for intubation and long-term imaging of adult zebrafish, allowing us to extend the usefulness of the zebrafish model for sophisticated high-resolution imaging into adult stages and study vascular repair and regeneration in adult animals (see below). Genetic Analysis of Vascular Development Previously, we 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. 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. Our epigenetic mutant screen (described in project HD008977-04) has also fortuitously led to the discovery of the first known epigenetic regulator of arterial-venous development. Analysis of Vascular Specification, Patterning, and Morphogenesis Throughout the life of this project we have employed cutting-edge, sophisticated microscopic imaging tools and methods to characterize patterns of vessel assembly in the developing zebrafish, and then used molecular and experimental analysis understand how this pattern arises and what cues guide the specification, differentiation, and assembly of vascular networks during development. Our discoveries have included some of the first evidence that many neuronal guidance factors also play critical, previously unappreciated roles in vascular guidance and vascular patterning. Our ongoing studies include (i) characterizing the complex assembly and patterning of the vasculature of the pectoral fin, an analogous structure to mammalian forelimbs (arms), (ii) studying the development of the zebrafish gills, a gas-exchange organ with strong parallels to the mammalian lung, and the unique and highly specialized endothelial cells vital for its function, (iii) studying the development of the vasculature of the testes and its role in proving a niche for germ cell development and maintenance and spermatogenesis, (iv) exploring the role of RhoA signaling in vessel formation and vessel integrity (see also project HD008915-11). 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). Studying vascular repair and regeneration Our recent development of new methods for intubation and long-term imaging of adult zebrafish has made it possible for us to carry out experimental studies of vascular repair and regeneration in adult zebrafish. Vascular regeneration and repair is essential for wound healing, but is significantly impaired in aging and in pathologies such as diabetes. We have developed zebrafish models of cutaneous wound healing and are now using these models to study the anatomical, cellular, and molecular mechanisms guiding vascular repair and regeneration in adults, using the high-resolution imaging and vascular gene expression profiling tools we have at our disposal. Our goal is to identify key signaling pathways and molecules essential for neoangiogenesis during wound healing, with the long-term goal of identifying potential novel therapeutic targets.