Figure 4

WSS promotes Notch-mediated arterial network. (A) A representative image of tail vasculature in the transgenic Tg(flt1:tdtomatoe; flt4: yfp) zebrafish line at 3 dpf. flt1⁺: arterial vascular endothelium, flt4⁺: venous vascular endothelium. flt1⁺ & flt4⁺ DLAV: flt1⁺ or flt4⁺ regenerated from the DLAV. flt4⁺ PCV: flt4⁺ regenerated from the PCV. Scale bar: 100 μm. (B) In response to DMSO treatment, flt1⁺ arteries preferentially formed an initial dorsal to ventral connection between the DLAV (flt1⁺ DLAV) and the DA in the amputated site (white arrowhead). Between 1–2 dpa, flt4⁺ veins from the DLAV (flt4⁺ DLAV) regenerated toward the DA exhibiting collateral arterial phenotype (overlapped yellow, white arrowheads). While regeneration of flt4⁺ veins occurred in the PCV (flt4⁺ PCV) and DLAV (flt4⁺ DLAV) for loop formation between 2–3 dpa, the distal segmental network exhibited enhanced flt1 expression at 3 dpa. flt1⁺ arteries extended dorsally from SeA at 4 dpa and formed a vascular loop with flt4⁺ PCV (white arrow). Conversely, DAPT treatment inhibited the initial connections of both flt1⁺ arteries and flt4⁺ veins at 2 dpa and partially inhibited both flt4⁺ DLAV and CVP at 3 dpa (white arrowheads). At 4 dpa, DAPT treatment inhibited flt1⁺network (white arrowheads) and attenuated loop formation at 4 dpa (white arrows, n = 5 per group). Scale bar: 20 μm. (C) Following tail amputation, Gata1a MO injection or BDM treatment diminished flt1⁺ in the amputated site (white arrowheads) and partially attenuated flt4⁺ DLAV and PCV (*asterisk, n = 20 per group). Increase in WSS (epo mRNA, isoproterenol, 6% hydroxyethyl hetastarch) enhanced flt1⁺ network (white arrowheads) during loop formation (white arrows) as compared to MO-injected controls. (n = 20 per group). Scale bar: 20 μm. (D) Schematic representations and 3-dimensional (3D) overview of WSS-mediated arterial- and venous- regeneration. Black arrowheads depict regenerated flt1⁺ (Red) and flt4⁺ network (Blue) in response to changes in WSS.