The overall objective of the project is to understand the cellular and molecular mechanisms responsible for the specification, patterning, and differentiation of internal organs during development, or, more specifically, how the elaborate network of blood vessels arises during vertebrate embryogenesis. The regulation of blood vessel formation is currently a subject of intense research study, with profound potential implications for human health. Antiangiogenic and proangiogenic therapies show enormous promise for combating cancer and treating limb or cardiac ischemia, respectively. Efforts to develop these therapies, as well as targeted treatments for atherosclerosis, depend on a detailed understanding of the genetic mechanisms of normal blood vessel formation, about which relatively little is still known, as well as on the identification of new molecular therapeutic targets.

The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make it particularly well suited for the goals of our project. The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo. These features provide a tremendous advantage for studying vascular development because they permit direct, noninvasive observation of every blood vessel in a living embryo during its development and the isolation of mutants that cause defects in the formation of embryonic blood vessels. Major aims of the laboratory include studying the phenotypic and molecular basis for the defects in vascular patterning mutants, elucidating the molecular and morphogenetic mechanisms responsible for blood vessel formation in the embryonic trunk, and developing new experimental tools for studying vascular embryogenesis in the zebrafish.

Functional and Molecular Analysis of Vascular Patterning Mutants
Pham, Roman, Vogel, Weinstein
Our studies of zebrafish vascular mutants explore mutants that display disruption of cranial vessel formation, formation of abnormal arterial-venous connections, and localized blockage of circulation. By positional cloning, we have shown that the gridlock and violet beauregarde vascular patterning mutants are attributable to defects in a novel hairy-related bHLH factor expressed specifically in the dorsal aorta and in a zebrafish ortholog of the TGF-beta superfamily type 2 receptor ALK1, respectively. Interestingly, defects in the human ALK1 gene are responsible for hemorrhagic telangiectasia type 2 (HHT). HHT is a hereditary vascular disorder characterized by arterial-venous malformations that lead to a high incidence of hemorrhage and stroke. Further analysis of these and other mutants and genes is in progress.

Molecular Dissection of Arterial-Venous Development
Lawson, Mugford, Torrealday, Vogel, Weinstein
We have developed novel insights into molecular and morphogenetic mechanisms of trunk blood vessel formation. For example, we recently discovered previously unknown roles for two well-known signaling pathways, the Hedgehog and Notch pathways, in the specification and arterial-venous differentiation of developing blood vessels. By examining Hedgehog pathway mutants and experimentally manipulating Hedgehog signaling in vivo, we found that Sonic Hedgehog (Shh) signaling is necessary for specification of the trunk dorsal aorta. In the absence of Shh signaling, the dorsal aorta fails to form while ectopic activation of Shh signaling leads to arterialization of venous endothelial cells. We have used similar genetic and experimental methods to activate or repress Notch signaling in developing zebrafish embryos. Notch signaling is not necessary for specification of either the dorsal aorta or posterior cardinal vein, but it is essential for proper arterial-venous differentiation of these and other embryonic blood vessels.

Development of Tools for Experimental Analysis of Vascular Development in the Zebrafish
Isogai, Lawson, Subramanian, Weinstein
To exploit further the advantages of the zebrafish , we have begun to develop new experimental tools for studying the organism's blood vessel formation. Using a novel confocal microangiographic technique that we devised, we have prepared a detailed three-dimensional atlas of the complete vascular circuitry of the zebrafish embryo and early larva. An interactive, online version of the atlas is available at http://mgchd1.nichd.nih.gov:8000/zfatlas/Intro%20Page/intro1.html.

FIGURE 33

We have also generated transgenic zebrafish lines expressing green fluorescent protein (GFP) in blood vessels, making it possible for us to visualize blood vessel formation in intact, living embryos. In addition, we have developed methodologies for long-term multiphoton confocal time-lapse imaging of the dynamics of blood vessel formation in these transgenic zebrafish. By using these methods, we have made several unexpected observations about the morphogenetic mechanisms of blood vessel formation in developing animals. For example, we have elucidated a novel, two-step mechanism for formation and interconnection of the intersegmental vessels of the trunk. Taken together, our findings underscore both the complexity of the mechanisms guiding embryonic blood vessel formation and the power of the zebrafish for dissecting these mechanisms.