Brant M. Weinstein, PhD, Head, Section on Vertebrate Organogenesis
Young Cha, PhD, Postdoctoral Fellow 1
Makoto Kamei, PhD, Postdoctoral Fellow
Kameha Kidd, PhD, Postdoctoral Fellow 2
Jesus Torres-Vazquez, PhD, Postdoctoral Fellow 2
Josette Ungos, PhD, Postdoctoral Fellow
Karina Yaniv, PhD, Postdoctoral Fellow
Van N. Pham, BS, Scientific Technician
Daniel Castranova, BS, Charles River Zebrafish Technician
Brigid Lo, BS, Charles River Zebrafish Technician
Sumio Isogai, PhD, Guest Researcher
Our overall objective is to understand how the elaborate networks of blood and lymphatic vessels arise during vertebrate embryogenesis. Blood vessels innervate and 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, 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 both types of vessel play important roles 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 allowing simple, rapid screening for even subtle vascular-specific mutants.
Tools for experimental analysis of vascular development in the zebrafish
Isogai, Kamei, Kidd, Pham, Torres-Vazquez, Shaw,3 Yaniv; in collaboration with Davis, Dye
We previously established a microangiographic method for imaging patent blood vessels in the zebrafish and have 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). More recently, we generated a variety of transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible to visualize vessel formation in intact, living embryos. We developed methods for long-term multiphoton confocal time-lapse imaging of vascular development in transgenic fish and recently used these methods to examine blood vessel lumenogenesis and the ontogeny of the lymphatic system. We are continuing to develop new lines useful for vascular imaging in vivo.
Genetic analysis of vascular development
Castranova, Cha, Kamei, Kidd, Lo, Pham, Shaw, 3 Torres-Vazquez, Ungos, Yaniv; in collaboration with Dawid, Lawson, Liu
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 ENU-induced mutants. 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 are in progress for many mutants. We have already positionally cloned the defective genes from several vascular patterning mutants, including violet beauregarde (defective in Alk1/acvrl1), y10 (defective in phospholipase C-gamma 1), kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), and etsrp (encoding an ETS-related transcription factor). These mutant screens will continue to yield a rich harvest of novel vascular-specific mutants and bring to light new pathways regulating the formation of the developing vertebrate vasculature.
Analysis of vascular morphogenesis
Kamei; in collaboration with Davis, Dye
Classical studies dating back more than 100 years have suggested a model for assembly of endothelial tubes via formation and fusion of vacuoles, but conclusive in vivo evidence for this model has been lacking as a result 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 novel transgenic lines that we have developed, we used high-resolution time-lapse two-photon imaging to visualize how the formation and intra- and intercellular fusion of endothelial vacuoles drives vascular lumen formation. Ongoing studies are aimed at further characterizing the nature of this process.
Analysis of vascular patterning
Isogai, Torres-Vazquez, Cha; 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, and ongoing studies in the laboratory aim at understanding how this pattern arises and elucidating the cues that guide vascular network assembly during development. We recently demonstrated that well-known neuronal guidance factors play an important, previously unknown role in vascular guidance and vascular patterning. We showed that Semaphorin-Plexin signaling is an essential determinant of trunk vessel patterning. We thus uncovered a novel plexin gene that is expressed specifically in the vasculature and showed that loss of function of this gene causes dramatic mispatterning of trunk vessels. Ongoing studies focus on further understanding the role of Semaphorin-Plexin signaling in the vasculature and identifying and characterizing additional signals and receptors that function in guidance and patterning during vascular network assembly.
Analysis of lymphatic development
Yaniv, Isogai; 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 system's origins and early development has been hampered by difficulties in observing lymphatic cells in vivo and performing defined genetic and experimental manipulation of the system in currently available model organisms. We recently showed 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 superb new model for imaging and studying lymphatic development. Using two-photon time-lapse imaging of transgenic zebrafish, we also traced the migration and lineage of individual cells incorporating into the lymphatic endothelium, providing the first conclusive in vivo evidence establishing that early lymphatic endothelial cells are derived from primitive venous blood vessels. In studies that should provide new insights into the molecular regulation of lymphatic development, we are continuing to examine the assembly and origins of the lymphatic system of the zebrafish.
Additional Publications
1 Arrived during FY2006.
2 Left during FY2006.
3 Kenna Shaw, PhD, former Postdoctoral Fellow
COLLABORATORS
Sarah Childs, PhD, University of Calgary, Calgary, Canada
George Davis, PhD, Texas A&M Health Science Center, College Station, TX
Igor Dawid, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda, MD
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
Jay Hoying, PhD, Vascular Research Group, University of Arizona, Tucson, AZ_
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
Derek Stemple, PhD, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
Benjamin White, PhD, Laboratory of Molecular Biology, NIMH, Bethesda, MD
For further information, contact flyingfish2@nbih.gov.