Fig. 1. ETV2 induces ectopic endothelial signature in neural stem cells in zebrafish brain.

a fli1a:GFP/kdrl:mCherry/hsp70i:etv2 embryo at 15 h post fertilization (hpf). Embryos heat shocked at 15 hpf exhibited strong ubiquitous Etv2-mCherry expression at 16 hpf (+HS+1 h). Embryos were then heat shocked at 48 hpf, and by 24 h post heat shock (72 hpf), robust ectopic GFP and mCherry expression (highlighted in dotted white circles) was present in the brain. The yellow dotted box in the zebrafish diagram indicates the imaging area, and areas in white boxes in the merged panels are magnified on the right. Scale bar: 100 μm. b The response to ETV2 overexpression is developmentally restricted. fli1a:GFP/kdrl:mCherry/hsp70i:etv2 embryos heat shocked (HS) at 24, 48, 72, and 96 hpf exhibited decreasing numbers of ectopic mCherry+ and GFP+ cells in the brain. Heat shock at 72 and 96 hpf did not induce ectopic mCherry+ or GFP+ expression in the brain. c GFAP+ neural stem cells responded to ETV2 expression. GFAP-GFP cells were isolated by FACS from 60 hpf GFAP:GFP/kdrl:mCherry/hsp70i:etv2 embryos (+HS+12 h). qPCR for kdrl, fli1a, tal1, erg, and CHD5 in the isolated GFAP+ cells versus the heat shocked control (GFAP:GFP/kdrl:mCherry). d Confocal images showing a kdrl-mCherry+/GFAP-GFP+ neural stem cell (arrow). Scale bar: 50 μm. e Ectopic fli1a-GFP+ cells shared the same pattern as the nestin-mCherry+ neural stem cells, and they did not co-localize with huc-mCherry+ mature neural cells. nestin:mCherry or huc:mCherry plasmids were injected into the fli1a:GFP/hsp70i:etv2 embryos at the one-cell stage, and the embryos were then heat shock at 48 hpf and imaged at 72 hpf. Dashed circles highlight ectopic GFP cells; arrows indicate partial nestin-mCherry+/fli1a-GFP+ cells. Scale bar: 100 μm