PI3K in Retinal Angiogenesis Figure 3. Time-dependent inhibition of intraocular, developmental angiogenesis by LY294002. Tg(fli1:EGFP) larvae were treated with 10 mM LY294002 for the indicated time frames and the number of primary branches of hyaloid vessels determined at 5 dpf. The data is plotted as the average and standard deviation of 3 replicate experiments. 10 mM LY294002 is added for 24 hours (A) or 48 hours (B) starting at 1, 2, 3 or 4 dpf. In C 10 mM LY294002 is added at 1 dpf for 96, 72, 48 or 24 hours. In any experimental variation, the presence of LY294002 from 2–3 dpf is sufficient to result in a statistically significant inhibition of developmental angiogenesis of hyaloid vessels (*p,0.05; ***p,0.001, t-test). doi:10.1371/journal.pone.0007867.g003 However, the small molecule drug LY294002 significantly reduces the number of primary branches of hyaloid vasculature, indicative of anti-angiogenic activity (Fig. 1A). Analysis of wholemount larvae suggests that LY294002 treatment has no significant effect on the intersegmental or trunk vessels (Fig. 1B), which develop earlier than the hyaloid vasculature [17,29]. Confocal analysis Figure 4. The Akt inhibitor SH6 phenocopies the PI3K inhibitor LY294002. Comparison of hyaloid vasculature patterning in DMSO-treated control larvae (A), 10 mM LY294002 treated larvae (B) and larvae treated with the Akt inhibitor SH6 at 10 mM (C). All larvae are from the Tg(fli1:EGFP) line and were treated from 1–5 dpf. Like LY294002, SH6 affects angiogenesis of the hyaloid vessels. Abnormal patterning of the hyaloid vasculature is observed with SH6 (C) resembling LY294002-treated larvae (B), though no significant reduction in primary vessel number is observed with SH6 (D). The data shown in panel D is plotted as the average and standard deviation of 3 replicate experiments (***p,0.001, t-test). doi:10.1371/journal.pone.0007867.g004 PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7867