Fig. 5

Fig. 5 miR-128 regulation of Notch (via jag1b) and Wnt (via csnk1a1) signalling in the EHT control nHSPC heterogeneity.

a,b, Confocal lateral view of IF zebrafish tail at 27 hpf of Tg(TCF:NLS-mCherry^ia5,kdrl:eGFP^zn1) (n = 20 (WT), 18 (128^Δ/Δ) and 19 (csnk1a1 g3′UTR) embryos) (a) and Tg(TP1:eGFP^um14,kdrl:mCherry^s896) (n = 18 (WT), 20 (128^Δ/Δ) and 21 (jag1b g3′UTR) embryos) (b), Wnt and Notch reported lines, respectively. Quantification of Wnt and Notch kdrl+ cells based on reporter intensity. Arrowheads represent kdrl+, TCF+ or TP1+ high (white) or low/negative (blue) cells. Cell quantification is reported in the ventral floor of the dorsal aorta (VDA) (three independent experiments; ordinary one-way ANOVA with Tukey’s multiple comparison). c, Confocal images of Tg(kdrl:mCherry^s896,cmyb:GFP^zf169) WT and g3′UTR mutants AGM at 32 hpf. Replicative nHSPCs are increased (kdrl⁺, cmyb⁺, EdU+, blue arrowhead) (n = 19 (WT), 17 (csnk1a1 g3′UTR) and 15 (jag1b g3′UTR) embryos) in the csnk1a1 g3′UTR while unchanged in jag1b g3′UTR. G2/M nHSPCs (kdrl⁺, cmyb⁺, pH3+, blue arrowhead) (n = 17 (WT), 19 (csnk1a1 g3′UTR), 16 (jag1b g3′UTR) embryos) are increased in the jag1b g3′UTR while unchanged in csnk1a1 g3′UTR (3 independent experiments; ordinary one-way ANOVA with Tukey’s multiple comparison). d, scRNA-seq analysis of kdrl+ cells experimental perturbation among genotype using MELD (Methods) assessed by the comparison between csnk1a1 g3′UTR versus WT, showing C8 as the most perturbed cluster. e, Violin plot of erythroid markers (gata1a and alas2, P < 0.0001) within C8.nHSPC pLEPs (Mann–Whitney test). f, MELD assessed by the comparison between jag1b g3′UTR versus WT, showing C8 and C5 as the most perturbed clusters (Methods). g, Violin plot of lymphoid markers (ikzf1, P = 0.0315 (C8) and 0.0099 (C5) and hmgb2a, P < 0.0001) within C8.nHSPC pLEPs and C5.nHSPC pLMPs (two-tailed Mann–Whitney test). All quantifications are represented with mean ± s.e.m. NS, not significant; P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. DA, dorsal aorta; PCV, posterior cardinal vein.