Fig. 4

Upregulated vegfaa expression in ptena^-/-ptenb^-/- mutants is diminished by LY294002 and enhanced by sunitinib. (A,B) Quantitative PCR was performed to determine vegfaa expression levels in ptena^-/-ptenb^-/- mutants compared with wild type at 4 dpf (A) and at 1, 2 and 3 dpf (B). Rescue experiments were done by microinjection of ptena mRNA at the one-cell stage or by treatment with 25 μM LY294002 (LY) or 20 μM sunitinib from 72 hpf onwards. Wild-type control was set to 1 and all values were determined relative to the wild-type control at 3 dpf. Three embryos were pooled per condition and the data represent the results of at least two independent experiments. Statistical analysis (Kruskal-Wallis with Dunn’s post-hoc test) was performed using Excel and SPSS 20 (IBM); significance is indicated: **P<0.01; ***P<0.001. Note that the y-axis is discontinuous to accommodate the 40-fold increase in vegfaa expression upon sunitinib treatment of ptena^-/-ptenb^-/- mutants. (C–F) In situ hybridization was performed with a vegfaa-specific probe on 3-dpf or 4-dpf wild-type and ptena^-/-ptenb^-/- mutant embryos, as indicated. The number of embryos analyzed is: wild type 3 dpf, 6; wild type 4 dpf, 4; ptena^-/-ptenb^-/- 3 dpf, 4; ptena^-/-ptenb^-/- 4 dpf, 5. Representative pictures are depicted here. Pictures were taken with a 4.5× objective.