Organogenesis of the Zebrafish Vasculature

Brant M. Weinstein, PhD, Head, Section on Vertebrate Organogenesis
Sumio Isogai, PhD, Visiting Scientist
Young Cha, PhD, Postdoctoral Fellow
Misato Fujita, PhD, Postdoctoral Fellow
Aniket Gore, PhD, Postdoctoral Fellow
Cathy McKinney, PhD, Postdoctoral Fellow
Mariana Melani, PhD, Postdoctoral Fellow¹
Matthew Swift, PhD, Postdoctoral Fellow
Josette Ungos, PhD, Postdoctoral Fellow
Karina Yaniv, PhD, Postdoctoral Fellow
Van N. Pham, BS, Scientific Technician
Brigid Lo, BS, Charles River Zebrafish Technician
Daniel Castranova, BS, Charles River Zebrafish Technician

We aim to understand how the elaborate networks of blood and lymphatic vessels arise during vertebrate embryogenesis. Blood vessels supply every tissue and organ with oxygen, nutrients, and cellular and humoral factors. Lymphatic vessels drain fluids and macromolecules from the interstitial spaces of tissues and then return them to the blood circulation; in addition, lymphatic vessels are essential participants in immune responses. Understanding the formation of blood and lymphatic vessels has become a subject of intense clinical interest because of the important roles played by both types of vessels in cancer and ischemia. The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that makes it particularly well suited for studying vessel formation. The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo. These features are highly advantageous for studying vascular development, permitting observation of every vessel in the living animal and simple, rapid screening for even subtle vascular-specific mutants. Major aims of our laboratory include (1) developing new tools for experimental analysis of vascular development in zebrafish, (2) forward-genetic analysis of vascular development, (3) analysis of vascular morphogenesis, (4) analysis of vascular patterning, and (5) analysis of lymphatic development.

Developing tools for experimental analysis of vascular development in the zebrafish

Cha, Fujita, Gore, McKinney, Swift, Yaniv; in collaboration with Davis, Dye

One important aim of our laboratory has been the development of new tools to facilitate vascular studies in the zebrafish. We have (1) established a microangiographic method for imaging patent blood vessels in the zebrafish and used the method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html); (2) generated a variety of transgenic zebrafish lines expressing various fluorescent proteins within vascular or lymphatic endothelial cells, allowing us to visualize vessel formation in intact, living embryos; and (3) developed methodologies for long-term multiphoton confocal time-lapse imaging of vascular development in transgenic fish. Recent technical advances have greatly facilitated the generation of new transgenic lines in the zebrafish. 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.

Cha Y-R, Weinstein BM. Visualization and experimental analysis of blood vessel formation using transgenic zebrafish. Birth Defects Res C Embryo Today 2007;81:286-296.
Gore AV, Lampugnani MG , Dye L, Dejana E, Weinstein BM. Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke. Dis Model Mech 2008;1:275-281.
Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, Weinstein BM. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 2006;442:6554-6559.

Genetic analysis of vascular development

Castranova, Cha, Gore, Lo, McKinney, Melani, Pham, Swift, Ungos, Yaniv; in collaboration with Dawid, Dye, Kennedy, Lawson, Liu, Roman

We use forward-genetic approaches to identify and characterize new zebrafish mutants that affect the formation of the developing vasculature. Using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels, we are carrying out an ongoing large-scale genetic screen for mutants induced by N-ethyl N-nitrosourea (ENU). To date, we have screened well over 2,000 genomes and have identified over 100 new vascular mutants with phenotypes that include loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. A bulked segregant mapping pipeline is in place to determine rapidly the rough position of newly identified mutants on the zebrafish genetic map. Fine mapping and molecular cloning are in progress for many mutants. We previously positionally cloned the defective genes from several vascular-specific mutants, including violet beauregarde (defective in Alk1/acvrl1), plcg1 (defective in phospholipase C-gamma 1), kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), and etsrp (defective in an ETS²-related transcription factor). We are currently focusing on several mutants affecting VEGF³-dependent and VEGF-independent vascular signaling pathways. Our ongoing mutant screens and positional cloning projects continue to yield a rich harvest of novel vascular mutants and genes, bringing to light new pathways regulating the formation of the developing vertebrate vasculature.

Alvarez Y, Cederlund ML, Cottell DC, Bill B, Ekker SC, Torres-Vazquez J, Weinstein BM, Hyde DR, Vihtelic T, Kennedy B. Genetic determinants of hyaloid and retinal vasculature in zebrafish. BMC Dev Biol 2007;7:114-131.
Anderson MJ, Pham VN, Vogel AM, Weinstein BM, Roman BL. Loss of unc45a precipitates arteriovenous shunting in the aortic arches. Dev Biol 2008;318:258-267.
Covassin LD, Siekmann AF, Kacergis MC, Laver E, Moore JC, Villefranc JA, Weinstein BM, Lawson ND. A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development. Blood, in press.
Covassin LD, Villefranc JA, Kacergis MC, Weinstein BM, Lawson ND. Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish. Proc Natl Acad Sci USA 2006;103:6554-6559.
Pham VN, Lawson ND, Mugford J, Dye L, Castranova DA, Lo BD, Weinstein BM. Combinatorial function of ETS transcription factors in the developing vasculature. Dev Biol 2007;303:772-783.

Analysis of vascular morphogenesis

Gore; in collaboration with Davis, Dejana, Dye, Ginsburg, Kennedy, Lyons

In previous studies, we used high-resolution time-lapse two-photon imaging to show that the formation and intra- and intercellular fusion of endothelial vacuoles drive vascular lumen formation in vivo. We are currently examining the formation and maintenance of vascular junctions, which are important in stroke. To begin dissecting the molecular regulatory mechanisms controlling vascular morphogenesis and the maintenance of vascular integrity, we are studying a variety of genes required for vascular morphogenesis and vascular integrity, including the pak2a and rap1b genes, about which we recently reported. 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.

Buchner DA, Su F, Yamaoka JS, Kamei M, Shavit JA, McGee B, Hanosh AW, Kim S, Jagadeeswaran P, Weinstein BM, Ginsburg D, Lyons SE. pak2a mutations cause cerebral hemorrhage in redhead zebrafish. Proc Natl Acad Sci USA 2007;104:13996-14001.
Gore AV, Lampugnani MG , Dye L, Dejana E, Weinstein BM. Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke. Dis Model Mech 2008;1:275-281.

Analysis of vascular patterning

Cha, Fujita, Isogai, McKinney, Swift; in collaboration with Childs, Epstein, Fishman, Lawson, Stemple

We have used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish. Ongoing studies in the laboratory aim to understand how the patterns arise and 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 is an essential determinant of trunk vessel patterning. Current studies are further elucidating our understanding of the role of additional factors that guide 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.

Alvarez Y, Cederlund ML, Cottell DC, Bill B, Ekker SC, Torres-Vazquez J, Weinstein BM, Hyde DR, Vihtelic T, Kennedy B. Genetic determinants of hyaloid and retinal vasculature in zebrafish. BMC Dev Biol 2007;7:114-131.
Anderson MJ, Pham VN, Vogel AM, Weinstein BM, Roman BL. Loss of unc45a precipitates arteriovenous shunting in the aortic arches. Dev Biol 2008;318:258-267.

Analysis of lymphatic development

Cha, Isogai, Yaniv; in collaboration with Dye

The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes, but progress in understanding the origins and early development of the system has been hampered by difficulties in observing lymphatic cells in vivo and performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. Recently, we demonstrated for the first time that the zebrafish possesses a lymphatic system that shares many of the morphological, molecular, and functional characteristics of the lymphatic vessels found in other vertebrates, providing a powerful new model for imaging and studying lymphatic development. As we continue to examine the origins and assembly of the lymphatic system of the zebrafish, we are developing new transgenic tools for imaging the development of the lymphatic system and for forward-genetic screening for lymphatic mutants. Our genetic analysis has already identified several novel genes involved in lymphatic development. We are also studying the functional role of several previously identified genes implicated in lymphatic development. Our ongoing studies will provide new insights into the molecular regulation of lymphatic development.

Isogai S, Hitomi J, Yaniv K, Weinstein BM. Zebrafish as a new animal model to study lymphangiogenesis. Anat Sci Int 2008, in press.
Yaniv K, Isogai S, Castranova D, Dye L, Hitomi J, Weinstein BM. Imaging the developing lymphatic system using the zebrafish. In: Chadwick D, Goode J, eds. Vascular Development. Wiley, 2007;139-151.
Yaniv K, Isogai S, Castranova D, Dye L, Hitomi J, Weinstein BM. Live imaging of lymphatic development in the zebrafish. Nat Med 2006;12:711-6.

Additional Publications

Ungos JM, Weinstein BM. Vascular development in the zebrafish. In: Bodmer R, ed. Cardiovascular Development. Elsevier Press, 2007;301-332.
Yaniv K, Weinstein BM. Blood vessel formation. In: Moody S, ed. Principles of Developmental Genetics. Elsevier Press, 2007;721-754.

¹Arrived during 2008.
²E26 transformation-specific
³Vascular endothelial growth factor

Collaborators

Chi-Bin Chien, PhD, University of Utah, Salt Lake City, UT
Sarah Childs, PhD, University of Calgary, Calgary, Canada
George Davis, PhD, University of Missouri-Columbia, Columbia, MO
Igor Dawid, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Elisabetta Dejana, PhD, The FIRC Institute of Molecular Oncology Foundation, Milan, Italy
Louis Dye, BS, Microscopy and Imaging Core, NICHD, Bethesda, MD
Jonathan Epstein, MS, University of Pennsylvania, Philadelphia, PA
Mark Fishman, MD, Massachusetts General Hospital, Boston, MA
David Ginsburg, MD, Howard Hughes Medical Institute and University of Michigan, Ann Arbor, MI
Allan Goldstein, MD, Massachusetts General Hospital, Boston, MA
Jiro Hitomi, MD, Iwate Medical University, Morioka, Japan
Breandán Kennedy, PhD, UCD Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
Nathan Lawson, PhD, University of Massachusetts, Worcester, MA
Paul Liu, MD, PhD, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Susan Lyons, MD, PhD, University of Michigan, Ann Arbor, MI
Beth Roman, PhD, University of Pittsburgh, Pittsburgh, PA
Radu Stan, MD, Dartmouth Medical School, Lebanon, NH
Derek Stemple, PhD, Wellcome Trust Sanger Institute, Cambridge, UK

For further information, contact flyingfish2@nih.gov or visit http://uvo.nichd.nih.gov.