from 2–3 dpf is sufficient to result in a statistically significant inhibition of hyaloid angiogenesis. These results are consistent with LY294002 blocking developmental angiogenesis of new hyaloid vessels but exerting no effect on existing intraocular vessels. The PI3K-Akt Pathway Controls Hyaloid Angiogenesis To corroborate the anti-angiogenic activity of LY294002 on hyaloid vasculature being mediated via inhibition of PI3K signalling, we selectively inhibited the activity of Akt/PKB, a downstream target of PI3K [30]. Analysis of hyaloid vessels at 5 dpf demonstrates that SH6, a PKB/Akt inhibitor, can phenocopy LY294002. Both drugs at 10 mM, result in a striking difference in the patterning of hyaloid vessels (Fig. 4 A–C). Interestingly, LY294002, but not SH6, also significantly reduces the number of primary hyaloid vessels (Fig. 4 D). LY294002 Attenuates Ectopic and Regenerative Angiogenesis The ability of LY294002 to inhibit extraneous angiogenesis was determined in out of bounds (obd) mutants. As a consequence of a recessive mutation in plexin D, obd2/2 larvae present with ectopic developmental angiogenesis [17,31]. Treatment of obd2/2 larvae with 10 mM LY294002 inhibits ectopic hyaloid angiogenesis and can reduce branch patterning to near wild type levels (Fig. 5). The anti-angiogenic efficacy and safety of LY294002 in the context of adult tissues was determined using a fin regeneration assay [32]. The extent of angiogenesis in regenerating fins was determined in adult fish following caudal, dorsal or anal fin clipping. Compared to controls, LY294002 significantly inhibits regenerative angiogenesis (Fig. 6 and Fig S2). LY294002 also inhibits regeneration length, which is reported to be angiogenesis- dependent [32] (Fig. 6 and Fig S2). Otherwise, no toxic effects were observed, and LY294002- and control-treated adults were indistinguishable in relation to behaviour, ventilation rate and swimming patterns. LY294002 Inhibits Retinal Angiogenesis without Affecting Retinal Function The electroretinogram (ERG) is a diagnostic method for quantifying outer retinal function. ERGs from larvae treated with LY294002 or vehicle from 2–5 dpf show equivalent responses at 4 flash intensities (Fig. 7A–C). At maximum flash intensity, the a-wave measure of photoreceptor hyperpolarisa- tion has amplitudes of 22865.8 mV or 23163.5 mV, and implicit times of 6167.6 milliseconds (ms) or 6867.7 ms in control and drug-treated larvae, respectively. Similarly, the b- wave which reflects signal transmission to bipolar cells has amplitudes of 199620.5 mV or 208628 mV and implicit times of 157610.5 ms or 169610.1 ms in control and drug-treated larvae, respectively (Fig. 7A–C). In agreement, the retinal histology of larvae treated with LY294002 from 2–5 dpf is indistinguishable from controls. Both samples consist of laminated retinas with prominent optic nerves and transparent lenses (Fig. 7D). Retinal histology in larvae treated earlier from 1–5 dpf does feature apoptotic nuclei and tissue vacuolation (Fig. 7D). This is consistent with a loss of visual function in larvae treated with LY294002 for just 24 hours beginning at 1 dpf and suggests a role for PI3K in early retinal development, independent of angiogenesis. In summary, treatment with LY294002 can block hyaloid angiogenesis with no detrimental effect on retinal function. PI3K in Retinal Angiogenesis Intraocular LY294002 Selectively Inhibits Angiogenesis and not Visual Behaviour Finally, we determined if localised delivery of LY294002 can inhibit intraocular angiogenesis, and do so without systemic side-effects, or effects on retinal function. Compared to larvae injected with vehicle alone, larvae injected with 10 mM LY294002 do display significant inhibition of hyaloid vessel angiogenesis (Fig. 8 A, C). Unlike the mild oedema observed in larvae treated systemically with LY294002, intraocular injection of LY294002 has no obvious systemic side-effects. Visual function in larvae injected intraocularly with LY294002 was analysed by the optokinetic response (OKR) [24]. In this Figure 6. LY294002 attenuates Regenerative Angiogenesis in Adult Zebrafish. The caudal fins of adult Tg(fli1:EGFP) zebrafish were amputated and left to regenerate in either tank water or tank water supplemented with 10 mM LY294002. Bight field micrographs (A) or fluorescent micrographs (B) of representative fins at 9 days post- amputation are shown. The maximal, average blood vessel length in the regenerated tissue was quantified at the third fin ray in either fin lobe and the results are depicted in (C). LY294002 significantly inhibits regenerative angiogenesis in the tail fin (** p,0.01, t-test). Yellow lines in B indicate the amputation plane. n = 8 fin rays in 4 fish. Scale bars = 500 mm. doi:10.1371/journal.pone.0007867.g006 PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7867