ORGANOGENESIS OF THE ZEBRAFISH VASCULATURE
, Head, Section on Vertebrate Organogenesis
Sumio Isogai,PhD, Visiting Scientist
Cathy McKinney, PhD, Postdoctoral Fellow
Matthew Swift, PhD, Postdoctoral Fellow
Josette Ungos, PhD, Postdoctoral Fellow
Young Cha, PhD, Visiting Fellow
Misato Fujita, PhD, Visiting Fellow
Aniket Gore, PhD, Visiting Fellow
Karina Yaniv, PhD, Visiting Fellow
Van N. Pham, BS, Scientific Technician
Daniel Castranova, BS, Charles River Zebrafish Technician
Brigid Lo, BS, Charles River Zebrafish Technician
We investigate how the elaborate networks of blood and lymphatic vessels arise during vertebrate embryogenesis. Blood vessels supply and sustain every tissue and organ with oxygen, nutrients, and cellular and humoral factors. Lymphatic vessels drain fluids and macromolecules from the interstitial spaces of tissues, returning them to the blood circulation, and play an important role in immune responses. Understanding the formation of blood and lymphatic vessels has become a subject of intense clinical interest because of the roles played by both types of vessel in cancer and ischemia. The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make 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.
Developing tools for experimental analysis of vascular development in the zebrafish
We previously established a microangiographic method for imaging patent blood vessels in the zebrafish and used that method to compile a comprehensive, staged atlas of the vascular anatomy of the developing fish (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We have also generated a variety of transgenic zebrafish lines expressing various fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize vessel formation in intact, living embryos. We have developed methodologies for long-term multiphoton confocal time-lapse imaging of vascular development in transgenic fish and used the methodologies to examine blood vessel patterning and lumenogenesis and the ontogeny of the lymphatic system. The recent development of Tol2-transposon–based vectors for transgenesis in zebrafish has greatly facilitated generation of new transgenic lines; we are continuing to develop 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. Embryo Today 2007, in press.
Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, Weinstein BM. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 2006;442:6554-9.
Kamei M, Weinstein BM. Long-term time-lapse fluorescence imaging of developing zebrafish. Zebrafish 2005;2:113-23.
Shaw KRM, Weinstein BM. Techniques and advances in vascular imaging in Danio rerio. In: Staton CA, ed. Angiogenesis Assays: a Critical Appraisal of Current Techniques. Wiley, 2006;311-26.
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.
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 N-ethyl-N-nitrosourea–induced mutants by using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. We have screened well over 2,000 genomes to date 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 is in progress for many mutants. We have already positionally cloned the defective genes from several vascular-specific mutants, including violet beauregarde (defective in Alk1/acvrl1), plcg1 (defective in phospholipase C-gamma 1), cds2 (defective in phosphoinositol signaling), kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), and etsrp (an ETS-related transcription factor, called ETS from erythroblast-transformation-specific domain). 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.
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;103:6554-9.
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;303:772-83.
Shaw KM, Castranova DA, Kamei M, Kidd KR, Lo BD, Torres-Vasquez J, Pham VN, Ruby A, Weinstein BM. fused-somites-like mutants exhibit defects in trunk vessel patterning. Dev Dyn 2006;7:1753-60.
Weinstein BM. The role of genetic predeterminants in regulating the phenotypic heterogeneity of the endothelium. In: Aird WC, ed. Endothelial Cells in Health and Disease. Taylor & Francis, 2005;133-47.
Analysis of vascular morphogenesis
Classical studies dating back more than 100 years suggested a model for assembly of endothelial tubes via formation and fusion of vacuoles, but conclusive in vivo evidence for the model was lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Taking advantage of the favorable optical properties of the fish and the novel transgenic lines that we had developed, we used high-resolution time-lapse two-photon imaging to show that the formation and intra- and intercellular fusion of endothelial vacuoles drives vascular lumen formation. We are currently developing transgenic lines that permit us to visualize the dynamics of endothelial cell-cell junctions and intracellular cytoskeletal structures so that we may examine their role in the cellular rearrangements that occur during vascular sprouting, growth, and tube formation. At the same time, we are studying several genes required for vascular morphogenesis and vascular integrity, including pak2a and rap1b, in order to dissect the molecular regulatory mechanisms controlling vascular morphogenesis and maintenance of vascular integrity.
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;104:13996-4001.
Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, Weinstein BM. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 2006;442:6554-9.
Analysis of vascular patterning
We have used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish. Our ongoing studies are aimed at understand how the patterns arise and shedding light on the cues that guide vascular network assembly during development. Our earlier work 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 seek a further understanding of the role of additional factors guiding the patterning of developing vascular networks in vivo, of matrix proteins involved in vessel growth in vivo, and of signaling pathways involved in integrating the factors and signals in the endothelial cell.
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;103:6554-9.
Pollard SM, Parsons MJ, Kamei M, Kettleborough RNW, Thomas KA, Pham VN, Bae MK, Weinstein BM, Stemple DL. Redundant roles for laminin a1 and a4 in notochord and blood vessel formation. Dev Biol 2006;289:64-76.
Shaw KM, Castranova DA, Kamei M, Kidd KR, Lo BD, Torres-Vasquez J, Pham VN, Ruby A, Weinstein BM. fused-somites-like mutants exhibit defects in trunk vessel patterning. Dev Dyn 2006;7:1753-60.
Weinstein BM. Vessels and nerves: marching to the same tune. Cell 2005;120:299-302.
Analysis of lymphatic development
In recent years, the lymphatic system has become the subject of great interest 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. We have shown 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 and thus provides a superb, highly suitable model for imaging and studying lymphatic development. Using two-photon time-lapse imaging of transgenic zebrafish, we traced the migration and lineage of individual cells incorporating into the lymphatic endothelium, thereby generating the first conclusive in vivo evidence that early lymphatic endothelial cells are derived from primitive venous blood vessels. We are continuing to examine the assembly and origins of the lymphatic system of the zebrafish. We are developing new transgenic tools for imaging the developing lymphatic system and for forward-genetic screening. We are also studying the functional role of several genes implicated in lymphatic development. Our ongoing studies will provide new insights into the molecular regulation of lymphatic development.
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;139-51.
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.
Publications Related to Other Work
Hong S-K, Haldin CE, Lawson ND, Weinstein BM, Dawid IB, Hukriede NA. The zebrafish kohtalo/trap230 gene is required for the development of the brain, neural crest, and pronephric kidney. Proc Natl Acad Sci USA 2005;102:18473-8.
Kidd KR, Weinstein BM. Zebrafish: a model for studying microvascular development and function. In: Shepro D, ed. Microvascular Research: Biology and Pathology. Elsevier, 2006;165-71.
Ungos JM, Weinstein BM. Zebrafish vascular development. In: Bodmer R, ed. Cardiovascular Development. Elsevier, 2007;301-32.
Yaniv K, Weinstein BM. Blood vessel formation. In: Moody SA, ed. Principles of Developmental Genetics. Elsevier, 2007;721-54.
COLLABORATORS
Ajay Chitnis, MBBS, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
George Davis, PhD, Texas A&M Health Science Center, College Station, TX
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
Mark Fishman, MD, Massachusetts General Hospital, Boston, MA
Neil Hukreide, PhD, University of Pittsburgh School of Medicine, Pittsburgh, PA
Nathan Lawson, PhD, University of Massachusetts Medical School, Worcester, MA
Paul Liu, MD, PhD, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Susan Lyons, MD, PhD, University of Michigan, Ann Arbor, MI
Derek Stemple, PhD, Wellcome Trust Sanger Institute, Cambridge, UK
For further information, contact WeinsteB@mail.nih.gov.
