Specification And Patterning of Developing Blood Vessels

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

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Abstract

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.