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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). Χ2 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.