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ORGANOGENESIS OF THE ZEBRAFISH VASCULATURE
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Brant M. Weinstein, PhD, Head, Unit on Vertebrate Organogenesis Moon-Kyoung Bae, PhD, Postdoctoral Fellowa |
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| The overall objective of our project is to understand the cellular and molecular mechanisms underlying organogenesis, in particular how the elaborate network of blood vessels arises during vertebrate embryogenesis. Blood vessels are ubiquitous and vital components of all vertebrate animals, innervating and supplying every tissue and organ with oxygen, nutrients, and cellular and humoral factors. They have become a subject of great clinical interest in recent years due in large part to the potential shown by antiangiogenic therapies for combating cancer. The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make it particularly well suited for studying blood vessel formation (Weinstein, 2002). The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo that permits easy observation of every vessel in the living animal and simple, rapid screening for even subtle vascular-specific mutants. We aim to develop experimental tools and resources to enhance the zebrafish as an experimental model for studying vascular embryogenesis, investigating the molecular basis for arterial-venous differentiation, exploring the basis for vascular patterning and morphogenesis, and identifying, by forward-genetic analysis, novel zebrafish genes critical for vascular development. Weinstein BM. Vascular cell biology in vivo-a new piscine paradigm? Trends Cell Biol 2002;12:439-445. Tools for experimental analysis of vascular development in the zebrafish Bae, Isogai, Kamei, Kidd, Lawson, Torres-Vazquez; in collaboration with Davis, Johnson To develop new experimental tools for studying blood vessel formation in the zebrafish, we previously established a microangiographic method for imaging patent zebrafish blood vessels and used it to compile a comprehensive staged atlas of the vascular anatomy of the developing fish. We also generated a number of different transgenic zebrafish lines expressing green fluorescent protein (GFP) in vascular endothelial cells (VEC), making it possible to visualize blood vessel formation in intact, living embryos (Lawson and Weinstein, 2002). We have developed methodologies for long-term multiphoton confocal time-lapse imaging of the dynamics of blood vessel formation in these transgenic fish and have used these methodologies to examine the morphogenesis of developing trunk (Isogai et al., in press; see "Analysis of vascular patterning and morphogenesis" below), cranial vessels (Lawson and Weinstein, 2002), and adult n blood vessels (Huang et al., in press). Our findings highlight the highly dynamic and unexpectedly growth cone-like behavior of growing angiogenic blood vessels and are reinforced by our analysis of the role of growth cone guidance factors in vascular patterning (see below). One of our current priorities is the development of additional transgenic lines that permit dynamic visualization of specific subsets of vessels such as arteries and veins or subcellular structures within vascular cells (see below) or that allow temporally regulated gene expression within the vasculature. We are also continuing to acquire and characterize new vascular-specific zebrafish genes and to develop antibody reagents for assaying their gene products (in our experience, antibodies directed against mammalian proteins generally do not cross-react well with zebrafish proteins; we have so far generated antibodies recognizing zebrafish k1, VE-cadherin, and plexinD1 and are in the process of preparing antibodies against additional vascular genes). Huang C-C, Lawson ND, Weinstein BM, Johnson SL. Independent requirement for reg6 in branching morphogenesis during distinct stages of blood vessel regeneration in zebrafish caudal fins. Dev Biol 2003;264:263-274. Hukreide N, Dawid IB, Weinstein BM. Embryologic, genetic, and molecular tools for investigating embryonic kidney development. In: Vize PD, Woolf AL, Bard JBL, eds. The Kidney: from Normal Development to Congenital Disease. London: Academic Press, 2003;119-137. Lawson NL, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 2002;248;307-318. Nishikawa K, Kobayashi M, Masumi A, Lyons SE, Weinstein BM, Liu PP, Yamamoto Y. Self-as- sociation of GATA-1 enhances the transcriptional activity in zebrafish embryo. Mol Cell Biol 2003;23:8295-8305. Subramanian R, Riemenschneider WK, Weinstein BM. Vascular smooth muscle investment of blood vessels in the zebrafish. Mech Dev; in press. Molecular dissection of arterial-venous development Bae, Kidd, Lawson, Mugford; in collaboration with Chitnis, Hoying We have detailed a molecular pathway regulating the acquisition of arterial-venous identity. Although the fundamental distinction between these two types of blood vessels has been appreciated for thousands of years, the fact that arterial and venous endothelial cells have distinct molecular and functional identities has only recently become apparent, and the mechanisms responsible for establishing such identity have not been elucidated. We have now shown that sonic hedgehog (SHH), vascular endothelial growth factor (VEGF), and notch signaling act in series to determine arterial-venous identity (Lawson et al., 2002). Accordingly, we used different combinations of drug treatments, mutants, morpholinos, and mRNA injections to activate or repress the activity of each of these signals in vivo, either alone or in combination. Subsequent studies from other laboratories have supported a similar role for this novel pathway during mammalian arterial differentiation. Using genetic screening and positional/candidate cloning methods, we recently identified y10, a mutant in phospholipase C gamma-1, and demonstrated that the gene is a major downstream effector of VEGF signaling in vivo (Lawson et al., 2003). We are continuing to identify potential mutants in genes in this pathway in our ongoing genetic screen. We have also recently begun microarray screening by using the available genetic and experimental tools to activate or inhibit arterial differentiation at different steps. With these two complementary approaches, we will continue to fill in the pathway we have elucidated with additional genes. Lawson ND, Mugford J, Diamond B, Weinstein BM. phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev 2003;17:1346. Lawson ND, Vogel AM, Weinstein BM. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch signaling pathway during arterial endothelial cell differentiation. Dev Cell 2002;3:127-136. Lawson ND, Weinstein BM. Arteries and veins--making a difference with zebrafish. Nat Rev Genet 2002;3:674-682. Torres-Vazquez J, Kamei M, Weinstein BM. Molecular distinction between arteries and veins. Cell Tissue Res 2003;314:43-59. Weinstein BM, Lawson ND. Arteries, veins, Notch, and Vegf. In: The Cardiovascular System. Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor, NY: Cold Spring Harbor Press, 2002;67:155-162. Analysis of vascular patterning and morphogenesis Berk, Isogai, Lawson, Torres-Vazquez; in collaboration with Chien, Childs, Davis, Epstein, Fishman, Li We have begun to study the mechanisms and molecular basis for vascular patterning and morphogenesis during development, in particular how vascular networks assemble with a defined, stereotypic anatomical pattern and how vessels undergo lumen formation. We have used multiphoton time-lapse imaging of the developing trunk to obtain important new insights into how angiogenic networks assemble in vivo, including a novel two-step model for vascular network formation and evidence for genetic determination of vascular pattern (Isogai et al., in press). We have also defined some of these genetic pattern determination factors, showing that well-known neuronal guidance factors also play an important role in vascular guidance and vascular patterning. Slit-Robo and semaphorin-plexin ligand-receptor pairs play important roles in axonal pathfinding, mostly via repulsive interactions between Slit or semaphorin ligands and Robo- or plexin-bearing neuronal growth cones. In collaboration with researchers studying related murine genes, we have uncovered novel Robo and plexin receptors expressed in zebrafish blood vessels and examined their functional roles. Our analysis of the role of semaphorin-plexin signaling has shown that it is an essential determinant of trunk vessel pattern. The plxnD1 gene is expressed in an entirely vascular-specific fashion in both mice and zebrafish. In the developing trunk, angiogenic intersegmental vessels extend near somite boundaries. Loss of plxnD1 function via morpholino injection or in the zebrafish out of bounds (obd) mutant causes dramatic mispatterning of these vessels, which are no longer restricted to growth near intersomitic boundaries. Somites flanking intersegmental vessels express semaphorins, and reducing the function of these semaphorins also causes intersegmental vessel mispatterning. The results indicate that the establishment of anatomical pattern in the developing vasculature is directed in part by cues and mechanisms similar to those used to pattern the developing nervous system, including semaphorin-plexin signaling. We are currently attempting to extend these studies and uncover the signal transduction machinery that transmits semaphorin-plexin signaling. Using genetic screening methods, we are also identifying additional loci that influence vascular patterning in vivo. In separate studies, we have generated a transgenic line that should aid us in visualizing the process of tubular morphogenesis during vessel formation in vivo. A variety of studies have suggested that vessels may form lumens via intra- and intercellular fusion of large vacuolar organelles within angioblasts and endothelial cells, but definitive in vivo evidence for this idea has been lacking. Using high-sensitivity multiphoton time-lapse imaging of a transgenic line that preferentially highlights these organelles, we are attempting to provide the first dynamic in vivo look at how lumen formation occurs. Isogai S, Lawson ND, Torrealday S, Horiguchi M, Weinstein BM. Angiogenic vascular network formation in the developing vertebrate trunk. Development 2003;130:5281-5290. Weinstein BM. Angiogenesis. In: McGraw-Hill Encyclopedia and Yearbook of Science & Technology. McGraw-Hill Press; in press. Weinstein BM. Blood vessels under construction. Cell 2002;111:456-458. Weinstein BM. Plumbing the mysteries of vascular development using the zebrafish. Semin Cell Dev Biol 2002;13:515-522. Genetic analysis of vascular development Bae, Berk, Kamei, Kidd, Lawson, McElwain, Mugford, Pham, Roman, Torres-Vazquez; in collaboration with Childs, Dawid, Liu, Moon, Stainier To dissect the genetics of vascular development and the molecular pathways that regulate it, we employ unbiased, forward-genetic mutational screening approaches that allow us to identify and then phenotypically and molecularly characterize zebrafish mutants that affect the formation of the developing vasculature. We have positionally cloned the defective genes from a number of different vascular patterning mutants. As a result of defects in a zebrafish ortholog of the TGF-beta superfamily receptor acvrl1 (Roman et al., 2002), violet beauregarde mutants have abnormal cranial vascular patterning and circulation. Defects in human acvrl1 cause hereditary hemorrhagic telangiectasia type 2 (HHT), an inherited vascular disorder characterized by arterial-venous malformations with a high incidence of hemorrhage and stroke. As noted above, y10 mutants have defects in the plcg1 gene (Lawson et al., 2003, see above). Another mutation, kurzschluss, has defects in the posterior aortic arches caused by defects in smap1, which encodes a chaperonin that is expressed in the mesenchyme surrounding the arch vessels and that may be involved in regulating myosin assembly. In collaborative studies, we have also determined the molecular basis for silent heart, a defect in cardiac troponin T, that results in a morphologically normal but nonbeating heart (Sehnert et al., 2002); vlad tepes, a defect in gata1 that causes a bloodless phenotype (Lyons et al., 2002); and out of bounds (see above), a mutant in plxnD1. Molecular characterization of additional mutants obtained in ongoing screens is in progress. We have successfully performed screens for new vascular-specific mutants by using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. We identified 11 new vascular-specific mutants in a pilot screen (approximately 500 genomes screened) of haploid progeny of ENU-mutagenized animals that was performed with the Dawid and Liu laboratories (members of the laboratories independently screened for hematopoietic and neuronal defects), identifying mutants in plcg1, k1, and several additional as yet undetermined genes. In collaboration with members of the Dawid laboratory, a larger-scale F3 diploid screen of ENU-mutagenized animals is now in progress (currently up to approximately 700 mutagenized genomes) to screen for mutants affecting both intersegmental vessel formation and later vascular patterning events that cannot be examined in haploid animals. To date, our F3 screen has yielded an additional 15 vascular-specific mutants with phenotypes that include loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. Molecular characterization of these mutants is in progress. Our experience so far suggests that ongoing mutant screens should continue to yield a rich harvest of novel vascular-specific mutants and bring to light new pathways regulating the specification, differentiation, and patterning of the developing vertebrate vasculature. Kidd KR, Weinstein BM. Fishing for novel angiogenesis therapies. Brit J Pharmacol 2003;140:585-594. Lyons SE, Lawson ND, Lei L, Bennett PE, Weinstein BM, Liu PP. A nonsense mutation in gata1 causes the bloodless phenotype in vlad tepes. Proc Natl Acad Sci USA 2002;99:5454-5459. Roman BL, Pham VN, Childs S, Kulik M, Lawson ND, Lekven AC, Neubaum D, Fishman MC, Lechleider RJ, Moon RT, Weinstein BM. Disruption of acvrl1 increases endothelial cell number in zebrafish Sehnert AJ, Huq A, Weinstein BM, Walker C, Fishman MC, Stainier DYR. Cardiac troponin T plays essential roles in sarcomere assembly and contraction. Nat Genet 2002;31:106-110. Yelon D, Weinstein BM, Fishman MC. Cardiovascular system. In: Solnica-Krezel L, ed. Pattern Formation in Zebrafish. COLLABORATORS Chi-Bin Chien, MD, PhD, Department of Anatomy & Neurobiology, University of Utah, Salt Lake City UT Sarah Childs, PhD, Department of Biochemistry & Molecular Biology, University of Calgary, Canada Ajay Chitnis, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda MD George Davis, PhD, Department of Pathology, Texas A&M Health Sciences Center, College Station TX Igor Dawid, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda MD Dean Li, MD, PhD, Human Molecular Biology & Genetics, University of Utah, Salt Lake City UT bDeparted during 2003 cMorioka University, Japan For further information, contact bmw7@box-b.nih.gov |
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