Fig. 2 (a,b) Lateral view of vessel-specific (a) or Notch-activated (b) GFP expression in 28 hpf fish embryos. Scale bar, 50 μm. (c,d) Depth-encoded lateral view of44 hpf fish embryos heat-shocked at 26 hpf. Scale bar, 50 μm. (c) Tg(hsp70l:bmp2b) embryos overexpress Bmp2b; (d) Tg(hsp70l:bmp2b);Tg(UAS:NICD); Tg(hsp70l:gal4) overexpress both Bmp2b and NICD. (e) Quantification of ectopic venous sprouts, representative of two independent experiments. Data points, individual embryos (n=12 WT, 11 Tg(hsp70l:Gal4);Tg(UAS:NICD), 9 Tg(hsp70l:bmp2b), 13 compound transgenics). Error bars, mean±95% CI. ****P≤0.0001 by one-way analysis of variance (ANOVA), with Tukey’s post-hoc test. (f–k) Depth-encoded compressed z-stack lateral views of 44 hpf embryos heat shocked at 26 hpf and treated with vehicle (f–h) or DAPT (i–k) from 10 hpf, representative of three independent experiments. (f,i) WT embryos; (g,j) Tg(hsp70l:bmp2b) embryos with ectopic venous z-slices removed to visualize intersegmental arteries; (h,k) Tg(hsp70l:bmp2b) embryos. (l) quantification of ectopic arterial sprouts. Data points, individual embryos (n=27 WT/dimethyl sulfoxide (DMSO), 26 Bmp2b/DMSO, 23 WT/DAPT and 26 Bmp2b/DAPT). Error bars, mean±95% CI. *P≤0.05, **P≤0.01 and ****P≤0.0001 by Kruskal–Wallis with Dunn’s post-hoc test. DA, dorsal aorta; DV, dorsal vein; ISV, intersegmental vessel; VV, ventral vein. (m) BMP6 twofold dose–response curve (indicated on x axis) in HUVEC after Notch activation (Dll4-Fc, red line) versus control (IgG-Fc, green line), representative of two independent experiments. Data are four-parameter best-fit curves (solid lines) ±95% confidence bands (filled areas). *P≤0.05 by nonlinear regression. Quantification (n) and panels (o) of nuclear pSMAD1/5 expression in individual HUVEC with indicated conditions, representative of two independent experiments. Yellow arrow, EC expressing FLAG-NICD. Scale bar, 10 μm. Error bars, mean±s.e.m. *P≤0.05 and ****P=0.0001 by one-way ANOVA with Tukey’s post-hoc test. |
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 χ^{2} 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. |
Fig. S4 SMAD6 mediates Notch-dependent suppression of BMP signaling and angiogenesis. (a-c) CRISPRi phenotypes with sgRNA manipulations (Scale bar, 100 μm). (a) WT control injected with 50pg dCas9-EnR mRNA, (b) dCas9-EnR + 50pg bmp6 sgRNA (predicted loss-of-function for BMP signaling), (c) dCas9-EnR + 50pg smad6b sgRNA (predicted gain-of-function for BMP signaling); two different sgRNAs produced the ventralized phenotype. (d) Quantification of early embryonic phenotypes of indicated injections. (#embryos: dCAS+scram=36; dCAS+smad6=57; dCas=48; scram=55; smad6=76; uninjected=101). Χ^{2} analysis; *, p≤0.05; ***, p≤0.001; NS, not significant. (e) qRT-PCR of whole embryos for indicated mRNAs targeted with two unique sgRNAs each. Data points, replicated experiments using 25 embryos per condition, displayed as a log-conversion of the ΔΔCT vs. controls. (f-g) WT (f) or dCas9-EnR+ (with a linked myl7:GFP transgene - inset) (g) F1 embryos from Tg(fli:dCas9-EnR) and Tg(hsp70l:bmp2b) crosses were indistinguishable by gross morphology and developmental staging. Scale bar, 100 μm. (h) RT-PCR with dCas9-EnR primers from 24hpf mRNA from WT (left) or Tg(fli:dCas9-EnR) (Tg, right) embryos. dCas9-EnR PCR product is at expected size (~1.2kb) (Supplementary Fig. 5e). All embryos are at 24 hpf. (i-l) Heat-shocked F1 embryos (heat shock at 26 hpf, analyze at 44-46 hpf) from Tg(fli:dCas9-EnR) and Tg(hsp70l:bmp2b;Tg(kdrl:GFP) crosses, uninjected or injected with smad6b sgRNAs. Arrows, ectopic ISV sprouts. Panels k and l have Z planes with ectopic venous sprouts removed. (m) Quantification of arterial vascular defects (% segments with ectopic ISVs) in heat-shocked embryos of indicated genotypes, representative of 2 independent experiments, N values on graph. Error bars, mean +/- SEM; *, p≤0.05; ****, p ≤ 0.0001 by 1-way ANOVA, with Tukey’s post-hoc. |
Acknowledgments: |
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ZFIN wishes to thank the journal Nature communications for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Nat. Commun. |