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Genetic Analysis of Vascular Development in Zebrafish

Final Report Summary - VASCDEVZEB (Genetic Analysis of Vascular Development in Zebrafish)

• A summary description of the project objectives:

The long-term goal of my research is to elucidate the cellular and molecular mechanisms underlying the formation of blood and lymphatic vessels during vertebrate embryogenesis. To this end we suggested the following specific aims:
Specific Aim 1: Uncover novel genes required during early lymphangiogenesis. The main purpose of this aim was to take advantage of the imaging and genetic capabilities of the zebrafish, to study the early stages of lymphatic formation. By doing so, we expected to unravel novel genes and mechanisms underlying the different steps of lymphangiogenesis in the developing embryo.
Specific Aim 2: Characterize novel regulators of blood vessel growth. Specifically in this aim, we proposed to characterize the interaction between lipoproteins and the endothelium during embryonic vascular development, with the goal of understanding the link between lipid metabolism and angiogenesis under pathological conditions. To this end we concentrated on the study of novel zebrafish mutants in combination with hyperlipidemic mice and culture endothelial cells.

• A description of the main results obtained during the course of the project:

Specific Aim 1: Uncover novel genes required during early lymphangiogenesis. This part of the project was expanded beyond the originally proposed research. A detailed description of the aberrant lymphatic growth in y114 mutants required that we first carry out a careful characterization of the same events in normal, wild type embryos. To this end we used long-term, time-lapse imaging of living zebrafish embryos to follow the formation of the first lymphatic vessels in the fish- the PACs. By doing so, we were able to identify a restricted subpopulation within the main axial vein- the Posterior Cardinal Vein (PCV)-, which gives rise to lymphatic endothelial precursors. Furthermore we found that these cells represent a previously uncharacterized pool of multipotent angioblasts, located in the floor of the CV. Using lymphatic-specific transgenic zebrafish, in combination with endothelial photoconvertible reporters, and long-term imaging, we demonstrated that these multipotent angioblasts can generate not only lymphatic, but also arterious, and venous fates. We further revealed that the underlying endoderm serves as a source of Wnt5b, which acts as a lymphatic inductive signal, promoting the angioblast-to-lymphatic transition. Results from this project have been compiled and are currently under revision for publication:

Nicenboim J., Malkinson G., Lupo T., Asaf, L., Mayseless O., Gibbs-Bar L., Grassme K., Hofi R., Almog G., Bochner F., Astin J.W. Golani O., Ben-Dor S., Crosier P.S. Herzog W., and Yaniv K. Lymphatic vessels arise from a niche of multipotent angioblasts within the floor of the cardinal vein (under revision)

Specific Aim 2: Characterize novel regulators of blood vessel growth. We have essentially fulfilled most of the objectives set for this aim. A large bulk of the data accumulated in this research project has been published in Avraham-Davidi I., Nature Medicine 18, 967–973 (2012). During the second reporting period, we expanded our studies and performed a comparative analysis of RNA expression in endothelial cells derived form normal-, lipid-depleted-, and lipid overloaded- zebrafish. The results generated provide the first gene expression panel of endothelial cells facing different lipoprotein levels in vivo. Among the genes whose expression changed in an opposite manner between the different models, we chose to focus on ATX- a pro-angiogenic factor, previously implicated in cancer and embryonic development. We analyzed atx expression in the developing zebrafish and found it to be tightly regulated by lipoprotein levels. Further epistatic analyses highlight ATX, as a novel candidate to serve as mediator of lipoprotein effects on endothelial cells.

• Socio-economic impact of the project

During the course of this project we generated novel transgenic reporters that were made available to the whole zebrafish community. In addition, the identification of a novel player required for normal lymphatic development would contribute to the understanding of the etiology of lymphatic pathogeneses.
Altogether our proposal aimed at identifying and characterizing novel players involved in the formation of blood and lymphatic vessels during embryonic development. Studies of vascular development have tremendous potential medical relevance. Circulatory system-associated mortality is the leading cause of death in the western world. Many of the proteins being targeted in pro-, and anti-angiogenic and lymphangiogenic therapies are the same ones that play major roles in developmental vessel formation, and developmental studies such as those carried out in this research project will likely unearth additional medically useful molecules. Furthermore, understanding how these genes act in their normal developmental contexts is important in instructing whether and how they are to be used in therapeutic settings. Altogether results from this research grant shed light on the mechanisms regulating lipid metabolism in endothelial cells, and uncovered novel players involved in the induction of lymphatic fate. Better understanding of these pathways may provide novel therapeutic targets for the treatment of metabolic related cardiovascular disease, and tumor-related angiogenesis and lymphangiogenesis.