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.