Several drugs which do not selectively target VEGF have indeed shown anti-angiogenic efficacy in eyes. Pazopanib, PKC412 and TG100572 are small molecule inhibitors that each targets multiple kinases [6,9,14]. Pazopanib (ArmalaH) which blocks PDGFRs, c- Kit, FGFR and c-fms, and TG100572, which inhibits FGF, PDGF and VEGF, both suppress choroidal neovascularisation in mouse models [6,9]. TG100572 has improved delivery features as it is effective following local administration as eye drops [6]. PKC412 blocks PKC, VEGF-R, PDGF-R and SCF-R isoforms, and in diabetics is reported to reduce macular oedema [14]. However, liver toxicity was observed following oral administration. Another approach is to selectively target a distinct pro- angiogenic molecule. Indeed recent studies report that the broad-spectrum PI3K inhibitor LY294002 and the avb3/avb5 integrin antagonist EMD478761 suppress retinal or choroidal neovascularisation following intraocular injection in rodents [15,16]. A combination of both approaches involves targeting multiple foci of pro-angiogenic pathways with several selective inhibitors. Dorrell et al. report that combination angiostatic therapy with a VEGF aptamer, an integrin antagonist and a proteolytic fragment of tryptophan tRNA synthetase inhibits ocular angiogenesis [11]. Zebrafish Intraocular Vasculature and Chemical Screens We recently demonstrated that the inner retina of zebrafish is nourished by an intricate vascular network which shares many features with human hyaloid and retinal vasculatures [17]. Initially this intraocular vasculature presents as a hyaloid vasculature more tightly associated with the lens which in adults has migrated to firmly attach to the inner retina. We identified a cohort of genes necessary for normal hyaloid vasculature formation in zebrafish [17]. In addition to amenability towards genetic screens, zebrafish are also suitable for pharmacological screens [18,19]. In relation to intraocular vasculature, Cao et al. recently show that exposure of adult zebrafish to hypoxia combined with treatment with a notch signaling inhibitor is sufficient to induce retinal neovascularisation [20]. In a random screen of small molecules, Kitambi et al. recently identified novel molecules inhibiting hyaloid angiogenesis in zebrafish larvae [21]. Summary Here, we conduct a targeted screen of known regulators of angiogenesis to identify drugs that inhibit developmental angio- genesis in the zebrafish eye. LY294002, a small molecule inhibitor of PI3 kinase was identified as an effective inhibitor of developmental angiogenesis in the eye. LY294002 inhibits new vessel growth without any apparent effect on existing vessels. The anti-angiogenic effect of LY294002 is dose-dependent and can be phenocopied using an inhibitor to AKT, which itself is known to act downstream of PI3K. Importantly, intraocular administration of LY294002 selectively inhibits angiogenesis in the eye but does not diminish visual function. Methods Ethics Statement All experiments were carried out under ethical approval granted by the UCD animal research ethics committee (http://www.ucd. ie/researchethics/arec.html). Drug Treatment Tg(fli1:EGFP) zebrafish were maintained according to standard procedures on a 14 h light/10 h dark cycle at 28uC. Embryos were obtained by natural spawning and developmental stages PI3K in Retinal Angiogenesis established by time and morphological criteria. At 24 hpf, 5 embryos per well were placed in 400 ml of Embryo Medium/1% DMSO and incubated with drug at 28uC on a 14 h light/10 h dark cycle [22]. Larvae were euthanised, and fixed in 4% PFA at 4uC overnight before analysis. Quantification of Primary Branch Number Prior to analysis of the intraocular vasculature, control- and treated-larvae were screened under an Olympus SZX16 micro- scope for general malformations. The overall patterning of the fin, gut and intersegmental vasculature was examined. Larval lenses from right eyes were dissected, transferred to depression slides and observed by epi-fluorescence microscopy using an Olympus SZX16, or placed in petri-dishes with cover-slip bottoms for confocal microscopy using a Zeiss UV510 META LSM. To orientate, lenses were embedded in 10% methyl-cellulose and manipulated with a 0.5 mm tungsten needle. Number of primary branches radiating from optic disc at the back of lens was determined for each specimen (see white arrows in Fig. 1). Regenerative Angiogenesis Assay Adult Tg(fli1:EGFP) zebrafish were anesthetised in 0.02% Tricaine (Sigma) and caudal, dorsal or anal fins amputated with a scalpel below the second branch points. Fish were incubated in tank water or tank water/10 mM LY294002 (Sigma) for 9 days, euthanised in 0.04% Tricaine, decapitated and fixed in 4% PFA (Sigma) for 24 hours at 4uC. Fins were mounted on glass slides in VectaShield (Vector) and visualised on an Olympus S2X12 fluorescent microscope. Images were acquired using a Hamamatsu ORCA-ER camera with OpenLab capturing software and adapted for publication in Photoshop CS2 9.02. Electroretinography Electroretinograms (ERGs) were performed on Tg(fli1:EGFP) zebrafish treated from 2–5 dpf with 10 mM LY294002 (n = 15) or 1% DMSO vehicle (n = 13). ERGs were performed in vivo during the afternoon as described by Fleisch et al., with a few modifications [23]. Larvae were paralysed with 0.5 mg/ml mivacurium chloride (MivacronH) and the recording electrode filled with 0.9% saline. A Kodak projector containing a 300W halogen light source was used for light stimulation. The unattenuated irradiance stimulus was 2.86103 mW/cm 2 . Three optical density filters were used to produce flash intensities of 23.0 log, 22.0 log and 21.0 log together with the unattenuated stimulus, 20 log. Flash duration was 100 milliseconds for all intensities, with an interstimulus interval of between 10 seconds and 1 minute, depending on flash intensity. To digitise and visualise data, a NIDAQ 6024E board (National Instruments) and Windows Whole Cell Programme software (WinWCP V3.9.1; courtesy of John Dempster, Strathclyde University, Scotland) were used. Data shown are signal averages of 2–10 responses. The a- wave was measured from baseline to the trough of the a-wave, and the b-wave was measured from the trough of the a-wave to the peak of the b-wave. Raw data from the sample groups were compared using an independent 2-sample t-test with unequal variances. Visual Behaviour Assay Visual function was assessed by the optokinetic-response (OKR) assay [24]. Briefly, 5 dpf larvae in Embryo Medium/9% methylcel- lulose were placed inside a black and white striped drum. The drum was rotated for 30 seconds in clockwise then counterclockwise directions and the number of eye saccades counted. PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7867