We study the embryonic origins of the vertebrate vascular system, using the zebrafish. Genetic and experimental analysis of the formation of this early-developing, essential organ system is difficult in most vertebrates, but the zebrafish offers important advantages for these studies. We make full use of these advantages in our research, which is aimed at understanding what guides the specification, differentiation, patterning, and morphogenesis of blood vessels during embryogenesis.
Studies of vascular development have tremendous potential medical relevance. Circulatory system-associated mortality is the leading cause of death in the western world. Many of the developmental processes we study are important in human congenital and acquired vascular diseases, and some of the mutations we have already characterized model known human congenital disorders. Recently, there has also been tremendous interest in using antiangiogenic therapies to combat cancer by "starving" tumors of their blood supply. Proangiogenic therapies also show promise for treating or preventing limb and cardiovascular ischemia. Many of the proteins being targeted for these therapies are the same ones that play major roles in developmental blood vessel formation. and developmental studies such as those carried out in our laboratory will likely unearth additional medically useful molecules. Furthermore, understanding how these genes act in their normal developmental contexts will be an important in instructing whether and how they are to be used in therapeutic settings.
Zebrafish are small tropical fish native to southeast Asia. They have a unique combination of genetic and experimental embryologic advantages that make them ideal for studying early development, particularly the embryogenesis of the circulatory system:
Adult zebrafish are small (aproximately one inch long as adults), have a short generation time (3-4 months), and can lay hundreds of eggs at weekly intervals, making them amenable to large scale mutant screens. Thousands of mutants affecting many different aspects of early development have already been recovered from mutagenesis screens. The ability to collect thousands of progeny from a single pair of adult zebrafish also makes it easy to map mutations and cloned genes to less than 0.1 cM resolution.
Unlike the embryos of many other vertebrates, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development. This facilitates experimental techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis, and single cell transplantation. We have pioneered additional novel experimental embryologic methods for studying the circulatory system, including confocal microangiography and multiphoton timelapse imaging of transgenic fish.
The optical clarity of zebrafish embryos provides perhaps the greatest advantage for studies of vascular development. Every blood vessel in a living embryo can be seen using nothing more than a low-power dissecting microscope. One can easily visually screen for organotypic defects or analyze living embryos at single-cell resolution over long periods of time. The optical clarity and physical accessibility of zebrafish embryos also make this an ideal system to exploit the advantages of transgenic animals expressing fluorescent proteins such as GFP.
An important aim of the laboratory has been to develop new experimental tools in orderfor studying blood vessel formation in this organism. We devised a confocal microangiography 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. We generated a variety of different transgenic zebrafish lines expressing fluorescent proteins in vascular endothelial cells (VEC) to help visualize the blood vessel formation in intact, living embryos, and worked out methods for extremely long-term multiphoton confocal timelapse imaging. Current priorities inclide preparing additional transgenic lines that permit dynamic visualization of specific subsets of vessels such as arteries and veins, or subcellular structures within vascular cells, or that permit temporally regulated and/or localized gene expression within the vasculature. See selected referenceson Developing Experimental Tools for Studying Blood Vessel Formation in the Zebrafish.
Figure 1. Confocal micrographs of blood vessels in the trunk (left) and head (right) of a Fli-EGFP transgenic zebrafish. 630X magnification.
We use mutant screens to identify and characterize zebrafish mutants that affect the formation of developing blood vessels, in order to uncover genes important for vascular development,. We previously cloned the defective genes from violet beauregarde (defective in Alk1/acvrl1), plcg1-y10 (defective in phospholipase C-gamma 1), and kurzschluss (defective in a novel chaperonin) mutants. We are currently carrying out an ongoing large-scale genetic screen for ENU-induced mutants using Fli-EGFP transgenic zebrafish and have identified dozens of new vascular-specific mutants. Genetic mapping and and molecular cloning is carried out on the novel mutants. These mutants bring to light new pathways regulating the specification, differentiation, and patterning of the developing vertebrate vasculature. Below: plcg1-y10 mutant lacking the angiogenic primary intersegmental trunk vessels.
We have uncovered a molecular pathway regulating the acquisition of arterial-venous identity consisting of sonic hedgehog (SHH), vascular endothelial growth factor (VEGF), and notch signaling acting in series. Subsequent studies from other labs have supported a similar role for this novel pathway during mammalian arterial differentiation. Using genetic screening and positional/candidate cloning methods we identified 'plcg1-y10, a mutant in phospholipase C gamma-1 with angiogenesis and arterial differentiation defects, and used this to demonstrate that this gene is a major downstream effector of VEGF signaling in vivo. We are continuing efforts to dissect arterial-venous development using genetic screens, microarrays, and novel transgenic tools. Our ongoing screens have identified new loci that are required for proper arterial or venous vascular development, and cloning and molecular characterization of these new mutants is in progress. We have established microarray technology in the laboratory, and are screening animals with gain- or loss- of function in different steps of the previously characterized arterial differentiation pathway. Finally, we are establishing arterial- and venous-specific transgenic lines that will allow us to easily assay for arterial and venous differentiation states in vivo. See selected references on Molecular Dissection of Arterial-Venous Development.
In the Weinstein Lab we also study the mechanisms and molecular basis for vascular patterning and morphogenesis during development. We are interested in how vascular networks assemble with a defined, stereotypic anatomical pattern during development, and what cues guide this reproducible patterning. We use multiphoton time-lapse imaging to examine how angiogenic networks assemble in vivo, and experimental snd genetic studies to elucidate molecular mechanisms directing this. Our studies have revealed that at least some axonal guidance factors play analogous novel roles in guiding and directing the proper assembly of vascular networks in vivo. Semaphorin-plexin signaling is critical for specifying the proper segmentally arranged growth of trunk angiogenic blood vessels. We are currently using additional genetic and experimental studies to elucidate downstream platers in semaphorin-plexin vascular signaling and identify new mechanisms for vessel guidance. See selected references on Analysis of Vascular Patterning and Morphogenesis.
Figure 3. Mispatterned trunk intersegmental blood vessels in zebrafish with loss of plexinD1, a vascular-specific sem,aphorin receptor required for proper vessel guidance. Left panel, plexinD1+ animal. Right panel, plexinD1- animal.
Isogai S, Horiguchi M, Weinstein BM. (2001) The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol 230(2):278
Lawson ND, Weinstein BM. (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol. 248(2):307-18.
Isogai S, Lawson ND, Torrealday S, Horiguchi M, Weinstein BM. (2003) Angiogenic network formation in the developing vertebrate trunk. Development. 2003 Nov;130(21):5281-90.
Torres-Vazquez J, Gitler AD, Fraser SD, Berk JD, Van N Pham, Fishman MC, Childs S, Epstein JA, Weinstein BM. (2004) Semaphorin-plexin signaling guides patterning of the developing vasculature. Dev Cell 7(1):117-23.
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