Fibroblast growth factor 2‐induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay

• Published in: Journal of cellular and molecular medicine Vol. 13; no. 8b; pp. 2061 - 2068
• Main Authors: Nicoli, Stefania, De Sena, Giulia, Presta, Marco
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-08-2009; John Wiley & Sons, Inc
• Subjects: Alkaline phosphatase; Angiogenesis; Animal models; Animals; Antibodies; Blood vessels; Bromodeoxyuridine; Cancer research; Cell growth; Danio rerio; Digital cameras; embryo; Embryos; Endothelial cells; Extracellular signal-regulated kinase; Fertilization; FGF2; Fibroblast growth factor 2; Fibroblast Growth Factor 2 - physiology; Fibroblast growth factor receptor 1; Fibroblasts; inhibitors; Injection; Kinases; Mammals; Membranes; Metastases; Molecular Medicine; Molecular weight; Neovascularization, Physiologic - physiology; Oncology; Pentraxins; Protein-tyrosine kinase; Signal transduction; Tumors; Vascular endothelial growth factor; Yolk; Yolk Sac - blood supply; Zebrafish; United States > US; Germany
• ISSN: 1582-1838; 1582-4934
• DOI: 10.1111/j.1582-4934.2008.00432.x

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.