Ephrinb2 regulates vascular loop formation. (A) Gross morphology following global ephrinb2 modulation. Both ephrinb2 MO and mRNA injections did not affect gross zebrafish morphology (white arrowheads). Scale bar: 100 μm. (B) Compared to the MO-injected controls, ephrinb2 MO (0.5–1 mM)-injected embryos had impaired loop formation (white arrow) altered the DA and SV morphology, immature CVP and DLAV at 4 dpa (white arrowheads). Transient overexpression of ephrinb2 mRNA restored DAPT, DN-Notch1b mRNA (10–20 pg/nl) and Gata1a MO-impaired loop formation (white arrows). Scale bar: 20 μm. (C) Quantification of the proportion of embryos exhibiting loop formation and normalized area of vascular loop (*p< 0.05 vs. control MO, n = 20 per group). (D,E) Representative images of Matrigel and human aortic endothelial cell (HAEC) migration assays following siScr or siephrinb2 transfection. (F) The density quantification of ephrinb2 expression following siephrinb2 transfection. TCL: total cell lysates (G,H)siephrinb2 transfection reduced both tube length and the number of branch points as compared to siScr-transfected HAEC (*p< 0.05, **p< 0.005 vs. siScr, n = 5 for tube length, n = 4 for branch point). (I)siephrinb2 transfection reduced the attenuated area of recovery (A.O.R) by ~45% at 24 h post scratch (**p< 0.005 vs. siScr, n = 3 per group).
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