Specification And Patterning of Developing Blood Vessels

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

As described in the goals and objectives section of this report, this project consists of three 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 (see below and project HD008915-13). To further facilitate our increasing use of later-stage developing and adult animals for imaging, we recently published new methods addressing the challenges of comprehensive and accurate 3-D imaging and modeling of large, deep, and highly obscured fluorescent tissues and structures in fixed specimens of later-stage larval, juvenile, and adult zebrafish. Although tissue clearing methods have been used at these later stages, most of these methods have limited ability to clear dense tissues such as bone and cartilage, cause significant morphological distortion, and/or result in loss of fluorescent signal when used for imaging of fluorescent transgenes, dye stained animals, or specimens generated using immunofluorescence or fluorescence in situ hybridization methods. We used a new water-based high-refractive index clearing agent to achieve high-resolution 3D imaging of whole fluorescent larval or juvenile animals, even deep internal regions normally obscured by dense tissues such as cartilage.. 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. We recently published a study based on a mutant from our ENU screens with an ectopic vascular sprouting and branching phenotype (caused by a truncating mutation in the dynein cytoplasmic 1 light intermediate chain 1 (dync1li1, LIC1) gene. LIC1 is a core subunit of the dynein motor complex that interacts with cargo adaptors such as RILPL1 and 2 to regulate Rab GTPase-mediated endosomal recycling and lysosomal degradation57-60. Further in vivo and in vitro work showed that LIC1 and RILPL1 and 2 restrict angiogenesis by promoting degradation of recycling endosomes carrying the pro-angiogenic VEGFR2 receptor. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases endosomal recycling of the VEGFR2 receptor, increased cell surface SRC signaling, and excess angiogenesis. 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 projects include: (i) 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, (ii) studying the development of the vasculature of the testes and its role in proving a niche for germ cell development and maintenance and spermatogenesis, (iii) studying the development of the immune-vascular vasculature interface in the meninges and in the axillary lymphoid organ (ALO), a superficial lymph-node like organ we recently discovered in the zebrafish, (iv) studying the role of calcium signaling in regulating vascular development, and (v) in a collaboration with the Tzima lab at Cambridge University, studying the role of eif6 in flow-dependent gene regulation in endothelial cells.