Fig. 4

Vegf Signaling Is Required for tgfb1a and tgfb1b Expression in the Dorsal Aorta

(A and B) Wild-type embryos were treated from 10 hpf with DMSO (control), Vegf inhibitor DMH4 (20 µM), and Notch inhibitor DAPM (100 µM) and collected at 22 hpf or 28 hpf. Embryos were collected and analyzed for (A) kdrl expression at 22 hpf and (B) runx1 expression at 28 hpf.

(C) Tg(Fli1:EGFP) embryos were treated from 10 to 26 hpf with DMSO or DMH4 (20 µM). DMH4-treated embryos showed a severe loss of intersomitic vessels but ECs are still present in the trunk, and circulation was detected in a majority of embryos at 48 hpf (data not shown).

(D) Expression of kdrl, tgfb1a, tgfb1b by qPCR in 26 hpf sorted Fli1:EGFP⁺ ECs. All three genes were downregulated after DMH4 treatment. Results are shown as averages ± SD of 4-5 biological replicates.

(E) Wild-type embryos were treated from 10 hpf with DMSO (control), Vegf inhibitor DMH4 (20 µM), and Notch inhibitor DAPM (100 µM) and collected at 22 hpf for analysis of tgfb1a, tgfb1b, tgfb3, and tgfbR2 by in situ hybridization at 22 hpf.

(F) Schematic representation of the experimental results.

Black arrows indicate the location of the DA; yellow arrowheads indicate the location of runx1 expression in the floor of the DA. The numbers of embryos are shown in each panel as the number of embryos with phenotype/total number analyzed. Arterial EC, arterial endothelial cell.

See also Figure S4.