Fig. 3

Fig. 3

Analysis of arterial and venous marker expression in SU5416-treated embryos at 24hpf after commencing treatment at multiple developmental time points. (A-H) Embryos were treated with either 0.1% DMSO or 20 μM SU5416 beginning at stages ranging from 15 somites to 23 somites and analyzed at 24 hpf by in situ hybridization. (A-D) Arterial marker cldn5b was severely downregulated when treated with SU5416 beginning at the 15-somite stage (16.5 hpf, B) as compared to DMSO treated controls (A). A similar phenotype was observed when treated at the 20-somite stage (19 hpf, C) but was less apparent when treated at the 23 somite stage (20.5 hpf, D). (E-H) Venous marker flt4 was downregulated upon SU5416 inhibition at the 15-somite stage (F) as compared to DMSO treated controls (E). Milder downregulation was observed when treatment was started at the 20-somite and 23-somite stages (G,H) and expansion of flt4 expression into the DA was observed. Arrows indicate DA and arrowheads indicate PCV. Lateral view of 24 hpf embryos, anterior to the left. Numbers of embryos showing the displayed phenotype are listed. (I) qPCR analysis of arterial (cldn5b, aqp8a), venous (flt4, stab1l, dab2, lyve1b, mrc1a) and pan-endothelial (cdh5, etv2) marker expression in SU5416-treated embryos. The treatment was started at the stages indicated and embryos were analyzed at 24 hpf. Note that arterial marker expression is greatly reduced at all stages indicated, while venous marker expression is significantly reduced at the early treatment stages (50%-epiboly and 5-somite), and only slightly reduced or unaffected (flt4, stab1l) or even expanded (dab2, 23-somite) at the later treatment stages. Pan-endothelial markers show strong reduction at early treatment stages and only slight or no reduction at the later treatment stages. * indicates p<0.05 and ** indicates p<0.01,±SEM is shown.