Activation of Notch signaling represses venous cell identity. (A,B) Expression of grl in hsp70:Gal4;UAS:notch1a-intra heat shocked embryos. (A) grl expression in heat shocked embryos not expressing Notch1a-intra. (B) grl is not ectopically induced within the vasculature by Notch1a-intra. (C-F) flt4 expression in hsp70:Gal4; UAS:notch1a-intra embryos heat shocked at the 18- somite stage. flt4 is expressed within venous vessels of the head (C) and trunk (E) at the 28- to 30-somite stages in embryos that do not express Notch1a-intra. (D,F) Notch1a-intra represses flt4 expression in venous blood vessels of the head (D) and trunk (F). (G,H) Targeted expression of Notch3 ICD to the endothelium using the fli1 promoter, represses flt4 expression. (G) An embryo injected with pfliMTN3ICD showing normal flt4 expression within the midcerebral vein (blue arrowhead) in the absence of ectopic Notch3 ICD; anterior is to the right. (H) Opposite side of embryo in G, showing a mycpositive cell (blue arrowhead) within the midcerebral vein that fails to express flt4; anterior is to the left. (I-L) Expression of tie1 in hsp70:Gal4;UAS:notch1a-intra heat shocked embryos. (I,K) In embryos not expressing ectopic Notch1a-intra, tie1 is expressed in all blood vessels at the 28- to 30-somite stage. (J,L) Tie1 expression within all vessels is maintained in embryos expressing Notch1a-intra. (C, D) Embryos are shown prior to staining with anti-myc antibody. (A,B,E-L) Embryos were subjected to whole-mount immunostaining with an anti-myc antibody to visualize ectopic expression of the (A,B,E,F,I-L) Notch1-intra or (G,H) Notch 3 ICD. (E,F,I,J) Arrows and arrowheads indicate the positions of the primordial midbrain channel and mid-cerebral vein, respectively. (A,B,K,L) Red arrowheads and blue arrows indicate the position of the DA and PCV, respectively. Scale bar (A,B,K,L) 50 μm, (C,D,I,J) 100 μm, (G,H) 200 μm.