(A, B) Cross section of immunostain for activated ERK in 12-ss (15hpf) embryo. (A) Activated ERK can be found in angioblast in the process of midline migration. (B) Merged view of migrating angioblasts, marked by GFP (green), and by activated ERK (yellow). By 12-ss, activated ERK is found preferentially in the “leading edge” angioblast subpopulation (yellow) that reaches the midline earlier than the rest (green). NC, notochord. Dorsal side is to the top. Figure reproduced or adapted from Hong et al.38 with permission form Cell Press.

(A) mid-somite stage embryo (12 to 14 hpf, 6 to 10 somites). At this stage, angioblasts are formed in the lateral plate mesoderm (LPM). In response to midline signaling by sonic hedgehog (Shh), secreted by notochord (NC), and VEGF, secreted by somites (S), the angioblasts migrate medially. (B) 24 hpf embryo. Upon completion of the migration, angioblasts situated dorsally adjacent to NC differentiate into the dorsal aorta (A) while ventral angioblast differentiate into the posterior cardinal vein (V). Dorsal is to the top. Schematic figure is based on findings presented in Gering and Patient.14

This model is based largely on findings in zebrafish, plus Foxc1/2 and COUP-TFII results in mice (noted by dashed arrows). Shh, expressed in the notochord and hypochord (on top), induces VEGF expression in nearby somites. By a process yet to be fully determined, VEGF activates the PLCγ-ERK/MAPK pathway only in the dorsal angioblasts (pink circle), which will develop into aortic endothelial cells. ERK activation results in Notch activation (indicated by Notch and Dll4 expression) via the transcriptional factors Foxc1/2. Notch signaling induces arterial differentiation (indicated by the arterial marker EphrinB2 expression). In the arterial progenitor cells, Grl, induced by Foxc1/2, blocks venous differentiation (indicated by the venous marker EphB4 expression. In the ventral angioblasts (blue circle), VEGF activates the PI3K/AKT pathway, which inhibits the PLCγ-ERK/MAPK pathway, possibly by a direct inhibition of Raf by AKT. AKT signaling is hypothesized to induce expression of COUP-TFII, which blocks arterial differentiation (also EphrinB2 expression). In this model, a gradient of VEGF along the dorsal-ventral axis is postulated to govern whether VEGF receptor activates PI3K or ERK signaling. For simplicity, the artery-vein specification is depicted here as occurring following the midline convergence of endothelial progenitors, but evidence suggests that this occurs earlier in zebrafish embryos. Schematic figure is based on discussions presented in Lamont and Childs.12

(A) Wholemount staining of 20-somite stage (ss) zebrafish embryo with antibody that recognizes di-phosphorylated (activated) ERK indicates high levels of ERK activation (indicated by arrows) in developing vasculature. (B) Longitudinal section and (C) cross section demonstrates the same. (D) In this cross section of a wild-type 20-ss embryo, ventral, venous angioblasts (V) are immunostained for vascular-specific GFP (green), and dorsal, arterial angioblasts (A) which immunostain for activated ERK are shown in yellow (green GFP merged with red phospho-ERK immunostaing). Nuclei are marked with DAPI (blue). (E) In embryos treated with a high dose of PI3K inhibitors, the proportion of angioblasts that immunostain for activated ERK (yellow) is greatly expanded. (F) In embryo treated with VEGF receptor inhibitor 676475, ERK activation in angioblasts is lost. (G) In this cross section of an in situ hybridization of 24-hpf wild-type embryo, the arterial marker ephrinB2a is clearly expressed in the dorsal aorta (A), but not in posterior cardinal vein (V). (H) In embryos treated with a high dose of PI3K inhibitors, the ephrin-B2a expressing dorsal aorta (A) is prominent, but the posterior cardinal vein is not observed. (I) In embryos treated with MEK inhibitor SL327, neither Ephrin-B2a expression nor dorsal aorta is observed. In A and B, the dorsal side is to the right. In C – I, the dorsal side is to the top. NC, notochord. Figures reproduced or adapted from Hong et al.38 with permission from Cell Press.