Fig. 4

(a) Quantitative RT–PCR for indicated genes, fold-change Notch reporter+ (NR+)/Notch reporter- (NR-) EC, relative to e1fa. Error bars, mean±s.e.m., N=5 replicates; one-sample Student’s T-test, **P≤0.01 and ***P≤0.001. (b–d) F0 mosaic transgenic zebrafish embryos (44 hpf) expressing GFP control (b) or GFP-tagged smad6b (c) from the vessel-specific fli promoter in the Tg(kdrl:mCherry) line. Arrows, GFP+ EC. (d) Arterial versus venous distribution of GFP+ EC quantified as percentage of total GFP+ EC on a per-embryo basis. Data bars, average per cent arterial and venous GFP+ EC, ±95% CI, representative of three independent experiments (n=18 fli:GFP and 30 fli:smad6b-GFP F0 embryos). ****P≤0.0001 by χ² analysis (1 degree of freedom). (e–h) Heat-shocked F1 embryos (heat shock at 26 hpf, analysed at 44–46 hpf) from Tg(fli:dCas9-EnR) and Tg(hsp70l:bmp2b;Tg(kdrl:GFP) crosses, injected with scrambled (scram) or with smad6b sgRNAs. Arrows, ectopic ISV sprouts. (g,h) have Z-planes with ectopic venous sprouts removed. (i) Quantification of arterial vascular defects (% segments with ectopic ISVs) in heat-shocked embryos of indicated genotypes, representative of two independent experiments (scram, n=4; smad6, n=6; scram/hsbmp, n=4; smad6/hsbmp, n=5). Error bars, mean±s.e.m.; *P≤0.05 and ****P≤0.0001 by one-way analysis of variance, with Tukey’s post-hoc test.