title: Fibroblast growth factor 2-induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay.
abstract: Angiogenesis plays a key role in tumour growth and metastasis. The teleost zebrafish (Danio rerio) represents a promising alternative model in cancer research. Here, we describe a zebrafish yolk membrane (ZFYM) angiogenesis assays based on the injection of 1-30 ng of human recombinant FGF2 (rFGF2) in the perivitelline space of zebrafish embryos in the proximity of developing subintestinal vein vessels (SIVs) at 48 hrs after fertilization. The rFGF2 induces a rapid and dose-dependent angiogenic response from the SIV basket, characterized by the ectopic growth of newly formed, alkaline phosphatase-positive blood vessels. These vessels are formed by proliferating cells that incorporate bromodeoxyuridine and express the endothelial cell markers vegfr2/kdr and fli1. Microangiography shows that rFGF2-induced vessels are patent and connected to the systemic circulation of the embryo. In keeping with these observations, fli1:EGFP(+) cells isolated from transgenic tg(fli1:EGFP)(y1) zebrafish embryos express the tyrosine kinase (TK) FGF receptor-1 (FGFR1) and activate extracellular signal-regulated kinase signalling when stimulated in vitro by rFGF2. The low molecular weight TK-FGFR1 inhibitor SU5402 and the high molecular weight FGF2 antagonist long-pentraxin 3 inhibit the angiogenic activity of rFGF2 when added to fish water or when co-injected with the growth factor, respectively. Moreover, similar to rFGF2, injection of the zebrafish form of vascular endothelial growth factor-A (VEGF-A) induces a significant angiogenic response in the ZFYM assay that is suppressed by the VEGF receptor-2/KDR TK inhibitor SU5416. The ZFYM assay represents a novel tool for testing the activity of low and high molecular weight inhibitors targeting a defined angiogenic growth factor in zebrafish. The assay may offer significant advantages when compared to other animal models.
authors.affiliation: General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia Medical School, Brescia, Italy.|Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Stefania
authors.initials: S
authors.lastname: Nicoli
authors.affiliation: General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia Medical School, Brescia, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: De Sena
authors.affiliation: General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia Medical School, Brescia, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Presta
mesh_terms: D000818:Animals; D016222:Fibroblast Growth Factor 2 / Q000502:physiology*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D015017:Yolk Sac / Q000098:blood supply*; D015027:Zebrafish
chemical_list: D016222:Fibroblast Growth Factor 2