Figure 1. Screens reveal LY294002 to inhibit developmental angiogenesis in the eye. A. Known regulators of vasculature formation were screened for effects on developmental angiogenesis of the hyaloid vasculature in vivo. 1 day post fertilisation (dpf) Tg(fli1:EGFP) larvae were treated with the indicated drugs and the number of primary branches of hyaloid vessels determined at 5 dpf. The drugs used are: recombinant VEGF (VEGF), 50 ng/ml; Tumor Necrosis Factor (TNF), 20 ng/ml; Adalimumab - HumiraH (Hum), 5 mg/ ml; Infliximab (Inflix), 5 mg/ml; anti-EGF (a-EGF), 5 mg/ml; Bevacizumab- AvastinH, 5 mg/ml; Oncostatin M (OSM), 10 ng/ml; Interleukin 1 beta (IL- 1B), 10 ng/ml; Nacyselyn (NAL), 10 mM; and LY294002 (LY), 10 mM. The data is plotted as the average and standard deviation of 3 replicate experiments. LY294002 results in a statistically significant inhibition of hyaloid vasculature development (***p,0.001, t-test). B–C. Fluorescent images illustrating the integrity of GFP-positive blood vessels at 5 dpf in LY294002-treated and DMSO-treated controls. No differences are observed in the morphology of the trunk vessels (B), whereas the hyaloid vessel branching pattern is abnormal and the branch number is reduced (C). White arrows in C indicate the primary branches radiating from the optic disc at the back of the lens. doi:10.1371/journal.pone.0007867.g001 Retinal Light Microscopy Larvae were euthanised with a lethal dose of Tricaine and fixed in 0.1 M Sorensen phosphate buffer (pH 7.3)/4% PFA/2.5% glutaraldehyde overnight at RT. Larvae were post-fixed in 0.1 M Sorensen phosphate buffer/1% osmium tetroxide for 1 hour at PI3K in Retinal Angiogenesis RT, dehydrated in ascending concentrations of alcohol to 100% and embedded in Epon resin using standard methods [25]. 1 mm sections were stained for 20 seconds with toluidine-blue, and examined by light microscopy using a Leica DMLB bright field illumination microscope and a Leica DFC 480 camera. Intraocular Drug Treatments Tg(fli1:EGFP) larvae were anesthetised with 0.02% Tricaine, dechorionated and transferred to 3 cm Petri dishes containing 1 ml of cold-liquid CyGELTM (Biostatus). Larvae were immobi- lised in CyGEL at room temperature with the left eye facing upwards for injection (Fig. S1). Immobilised larvae were injected in the left eye at 1 or 2 dpf with 10 mM LY294002 or 1% DMSO diluted in ddH2O/1% FastRed tracer dye using heat pulled glass capillaries (bore diameter OD/ID: 1/0.58 mm). After intraocular injection, larvae were retrieved by placing the dish on a chilled ice pad to melt the CyGEL and immediately transferred to Embryo Medium at RT for recovery. Larvae were maintained at 28uC until 5 dpf and hyaloid vasculature analysed as described above. Results LY294002 Inhibits Developmental Angiogenesis of the Eye To identify drugs that significantly alter development of new blood vessels in the eye, we performed a small pharmacological screen in zebrafish. The drugs screened (Table S1) are known to control growth factor-, cytokine- or inflammatory-mediated angiogenesis in other tissues, and several are used clinically to treat AMD, cancer or rheumatoid arthritis [26–28]. Tg(fli1:EGFP) larvae which specifically express EGFP in all blood vessels [29] were immersed in drug from 1–5 days post fertilisation (dpf) and the integrity of the hyaloid vasculature determined by fluorescent microscopy of dissected lenses. None of the growth factors, cytokines or antibodies affect the number of primary branches of hyaloid vasculature (Fig. 1A). Figure 2. LY294002 results in dose-dependent inhibition of intraocular angiogenesis. 1 dpf Tg(fli1:EGFP) larvae were treated with LY294002 at concentrations ranging from 2.5–20 mM and the number of primary branches of hyaloid vessels determined at 5 dpf. The data graphed is plotted as the average and standard deviation of 3 replicate experiments. LY294002 results in a statistically significant inhibition of hyaloid vasculature development (***p,0.001, t-test). doi:10.1371/journal.pone.0007867.g002 PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7867