PI3K in Retinal Angiogenesis clearly demonstrates that treatment with 10 mM LY294002 reduces the branch number and patterning of the hyaloid vasculature (Fig. 1C). This result suggests that the PI3K inhibitor, LY294002, inhibits the formation of new vessels but does not reduce the number of existing vessels systemically. Dose- and Time-Dependent Inhibition of Intraocular Angiogenesis by LY294002 Treating larvae from 1–5 dpf, with a range of LY294002 concentrations, reveals a dose-dependent inhibition of hyaloid vessel angiogenesis with an IC50 of ,12 mM (Fig. 2). At concentrations from 7.5–12.5 mM LY294002, statistically significant inhibition of hyaloid angiogenesis is observed in the absence of gross morphological defects. Larvae incubated in 10 mM LY294002 occasionally display mild oedema indicative of systemic side effects. At concentrations of 17.5 mM LY294002 and above, systemic toxic effects including oedema, bent body axis and craniofacial abnormalities are observed. Thus, 10 mM LY294002 was utilised for all additional experiments. To determine if LY294002 inhibits hyaloid angiogenesis during a specific developmental time-frame, 1–4 dpf Tg(fli1:EGFP) larvae were treated for 24, 48, 72 or 96 hours, and hyaloid vasculature analysed at 5 dpf (Fig. 3). If LY294002 is added to 1 or 4 dpf larvae for 24 hours, development of the hyaloid branches is unaffected (Fig. 3 A & C). However, the presence of LY294002 Figure 5. LY294002 attenuates ectopic developmental angiogenesis in obd mutants. Wildtype Tg(fli1:EGFP) larvae (wt) or out of bounds mutants in the Tg(fli1:EGFP) background (obd) were treated with 10 mM LY294002 from 1–5 dpf before dissecting lenses and analysing hyaloid vasculature development. LY294002 inhibits normal developmental angiogenesis in wildtype Tg(fli1:EGFP) larvae as seen by the reduced vessel patterning in lateral views of the lens (A, D). LY294002 also inhibits the extraneous hyaloid branch patterning (B) and branch number (C) observed in obd mutants as observed by reduction of branch patterning to near normal levels (E and F). Arrows depict the orientation of the lens, pointing in the direction from the optic disk to lens. G) Quantification of the reduction in the extraneous primary hyaloid branches number in LY294002-treated obd mutants. The data is plotted as the average and standard deviation of larvae (obd2/2 n = 7, obd2/2 treated: n = 8) from 2 independent experiments (***p,0.001, t-test). OD: Optic Disc. doi:10.1371/journal.pone.0007867.g005 PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7867