Vegfd modulates SoxF transcription factor activity through the MEK-ERK signaling pathway. (A) Quantitative data showing the percentage of embryos with arteriovenous fusion phenotypes in the presence of inhibitors and MOs. Uninjected (black bars), MO-sox18 (gray bars), or MO-sox7 (white bars) injected embryos were treated with inhibitors, including tyrosine kinase inhibitor (SU5416, 0.55 μM), VEGFR3 inhibitor (MAZ51, 5 μM), PI3K inhibitors (Wortmannin, 250 nM; LY294002, 10 μM), and MEK inhibitors (U0126, 10 μM; PD98059, 15 μM). The arteriovenous fusion phenotype can be observed in combinations of MO-sox18 or MO-sox7 injection with either SU5416 inhibitor or MEK inhibitors (U0126, PD98059), but not in combination with MAZ51 or PI3K inhibitors (Wortmannin, LY294002). (B) Transverse sections of the trunk region confirmed the arteriovenous fusion phenotypes. (C) Percentage of HUVECs with SOX18 nuclear condensation after inhibitor treatments in the presence or absence of VEGFD. (D) Immunofluorescence staining on HUVECs for endogenous SOX18 (red), phalloidin (green) and DAPI (blue). VEGFD-treated cells showed nuclear condensation of SOX18 (asterisk), which was blocked by SU5416 and U0126 inhibitors, but not by MAZ51 inhibitor treatment. ***P < .001, **P < .01, *P < .05 by 1-way ANOVA test. Scale bars represent 20 μm (B) and 50 μm (D).