(A) Suppression of the gridlock phenotype (atresia of the proximal aorta in grl^m145 mutants) by GS4898 [2-(4-methylphenyl)-4H-chromen-4-one]. Above, amicroangiogram of an untreated 60 hpf grl^m145 mutant, in which arterial circulation to the tail is disrupted, is shown. Below, amicroangiogram shows the restoration of tail circulation in a grl^m145 mutant by GS4898 treatment.
(B) The gridlock phenotype is suppressed by GS4012, GS4898, LY294002, and wortmannin, but not PD98059 and quercetin. GS4898 and the PI3K inhibitors are enclosed in dashed boxes.
(C) Quantitative western analysis of activating AKT phosphorylation (at Ser472) in 20-somite-stage (ss) zebrafish embryos shows dramatic reduction in AKT phosphorylation by GS4898 (25 μM) or wortmannin (0.5 μM) treatment, starting at 10 hpf. Results were normalized from three independent experiments and achieved significance at p < 0.004 for each condition versus untreated. Error bars represent standard error.
(D) Dose response for GS4898, LY294002, and wortmannin. Compounds were added at 12 hpf and washed out at 27 hpf. Suppression (%) represents percentage of treated embryos that have tail circulation at 48 hpf. Number on top of each bar represents actual number of embryos with normal tail flow over the total number treated.
(E) Model for two VEGF-receptor-dependent signaling branches with opposing effects on arterial specification. The PLC-γ/ERK branch mediates arterial cell specification, whereas the PI3K branch exerts a negative effect on the PLC-γ/ERK branch, possibly via direct inhibition of Raf by Akt. By blocking PI3K, GS4898 lifts PI3K’s inhibition of the PLC-γ/ERK pathway, leading to increased ERK activation and arterial specification.
(F) Synergy between GS4898 or PI3K inhibitors and GS4012. Single treatment involving a subeffective dose of GS4898 (0.75 μM), LY294002 (3 μM), wortmannin (0.075 μM), or GS4012 (0.7 μg/mL) does not suppress the gridlock phenotype, but cotreatment with GS4012 and GS4898, LY294002, or wortmannin at the same doses leads to significant suppression of the gridlock phenotype.

(A) A 30 hpf embryo. Red line indicates dorsal aorta (DA). Blue line indicates posterior cardinal vein (PCV), which terminates at the common cardinal vein (CCV). Yellow box indicates the region shown in (B). Green vertical line indicates cross-sections in (C)–(F).
(B) In situ hybridization of 30 hpf embryos with the arterial marker ephrin-B2a. At left, normal ephrin-B2a expression (arrowhead) in untreated embryos is shown. In the middle, intact ephrin-B2a expression in an embryo treated with high-dose (15 μM) GS4898 is shown. At right, ephrin-B2a expression is lost in SL327-treated (60 μM) embryos.
(C) Cross-sections of embryos shown in (B). At left, in untreated embryos, ephrin-B2a is expressed in the DA (black arrowhead), but not in the PCV (white arrowhead). In the middle, in GS4898-treated embryos, the ephrin-B2a expressingDA is prominent, but the PCV is not visible. At right, in SL327-treated embryos, neither ephrin-B2a expression nor the DA is observed.
(D–F) Cross-sections of 48 hpf embryos immunostained for GFP (brown) expressed in endothelial cells under the vascular-specific fli-1 promoter. Arrowheads indicate the aorta. (D) In untreated embryos, both the dorsal aorta (A) and the posterior cardinal vein (V) are prominent. (E) In embryos treated with 15 μM GS4898, duplication of the aorta is observed. (F) In embryos treated with 100 μM U0126, the aorta is greatly reduced, whereas the vein (V) is enlarged. (G) At top, fluorescent images of 48 hpf embryos expressing GFP in endothelial cells are shown. Dorsal view of the torso at the DA bifurcation and the PCV terminating at the CCV is shown. The head is above, and the tail is below. At bottom, cartoon representations of the fluorescent images are shown. At left, in untreated embryos, the CCV, via which blood from the tail drains to the heart, is situated lateral to the DA, which delivers blood to the tail. At the levels of the lower trunk and tail, the DA is situated dorsal to the PCV. In the middle, in GS4898-treated embryos, the DA is partially duplicated, and the CCV, situated lateral to the DA, is greatly reduced. At right, in SL327-treated embryos, the DA is missing and the CCV is significantly enlarged.

(A–C) Immunostain for activated (diphosphorylated) ERK in 20-ss zebrafish embryos. Arrowhead shows activated ERK within the developing vasculature. In (B), dorsal side is to the right, and in (C) and in all other cross-sections, dorsal is to the top. NC denotes notochord, which lies dorsal to the angioblasts.
(D–F) Cross-sections of immunostain for activated ERK in 12-ss (15 hpf) embryos (D and E) and in a 24 hpf embryo (F). (E) shows a merged view of angioblasts, marked by GFP (green) expressed under the fli-1 vascular-specific promoter [26] and by activated ERK (yellow). At around 12 ss, activated ERK (yellow) is preferentially detectable in angioblasts that reach the midline earlier than the rest of the angioblasts (green). (F) By around 24 hpf, activated ERK is no longer detected in angioblasts.
(G–I) Cross-section of a 20-ss embryo. (G) shows angioblasts marked by vascular-specific GFP (green). (H) shows immunostain for activated ERK (red). (I) Merged view of activated ERK and GFP, with overlap in yellow. At this stage, detectable ERK activation is restricted to the dorsal-most angioblasts (yellow).
(J and M) Quantitative western analysis of ERK phosphorylation in 20-ss embryos. Error bars represent standard error. (J) Inhibition of ERK phosphorylation by cyclopamine (50 μM) or 676475 (25 μg/mL) treatment, starting at 10 hpf. Results were normalized from three independent experiments and achieved significance at p = 0.03 for cyclopamine and p < 0.0001 for 676475 versus untreated. (M) shows enhancement of ERK phosphorylation by GS4898 (10 μM) or LY294002 (15 μM) treatment, starting at 10 hpf. Results were normalized from seven independent experiments and achieved significance at p = 0.04 for GS4898 and p = 0.009 for LY294002 versus untreated.
(K and L) Merged view of activated ERK and GFP in embryos treated with cyclopamine (K) or 676475 (L). Note the loss of ERK activation with cyclopamine and 676475 treatments.
(N and O) Merged view of activated ERK and GFP in embryos treated with GS4898 (N) and LY294002 (O) showing the relative expansion in the overlap (yellow) between angioblasts and activated ERK.

(A–C) Overlay of GFP-fluorescence image onto bright-field image of a live 48 hp embryo injected with pAdTrackCMV.AA-AKT (dominant-negative AKT), pAdTrackCMV.myr-AKT (myristoylated AKT), or pAdTrackCMV (GFP alone). (A) Tail region of a plasmid-injected embryo expressing GFP in a small subset of cells is shown. Yellow box indicates the region represented in (B) and (C). (B) A high-magnification overlay image of a typical GFP-positive patch scored as arterial (red asterisk) is shown. It overlaps with the aorta (A), with a robust arterial flow (red arrow). (C) An overlay image of a typical GFP+ patch scored as venous (blue asterisk) is shown. It overlaps with the caudal vein, with a robust venous flow.
(D) Numbers of GFP-positive patches in the aorta and in the caudal vein in embryos injected with pAdTrackCMV.AA-AKT (DN-AKT), pAdTrackCMV.myr-AKT (myr-AKT), and pAdTrackCMV (GFP alone). A significant skewing of vascular GFP-positive cells in favor of aorta is noted in DN-AKT-injected embryos (p = 0.028, versus GFP alone), whereas a significant skewing of vascular GFP-positive cells in favor of vein is noted in myr-AKT-injected embryos (p = 0.0039, versus GFP alone).