Open AccessFibroblast growth factor 2‐induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay
Author(s) -
Nicoli Stefania,
De Sena Giulia,
Presta Marco
Publication year - 2009
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/j.1582-4934.2008.00432.x
Subject(s) - zebrafish , angiogenesis , fibroblast growth factor , microbiology and biotechnology , basic fibroblast growth factor , yolk , biology , fibroblast , growth factor , chemistry , cancer research , biochemistry , fishery , in vitro , gene , receptor
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.