Extended Data Fig. 2: Single cell RNA sequencing analysis of miR-128^Δ/Δ EHT.

(a) Schematic representation of single cell RNA-sequencing (scRNAseq) strategy of kdrl+ tail endothelial cells (ECs) of WT and miR-128^Δ/Δ (128^Δ/Δ) at 26 hpf isolated by FACS from Tg(kdrl:GFP^zn1). (b) Heatmap showing the average gene expression of canonical vascular, hemogenic, blood stem cells and lineage gene markers in scRNAseq of 22,230 kdrl+ cells (WT + 128^Δ/Δ). Clusters selected in red correspond to the ones used for the re-analysis of specific EHT cells. (c) Gene Ontology (GO) analysis of reanalyzed 6096 kdrl + EHT cells. (d) Violin plots of kdrl, lyve1b and mrc1a expression in each cluster of WT cells (ordinary one-way ANOVA with Tukey’s multiple comparison). (e) Percentage of cells expressing gata2b, runx1 and cmyb in each cluster of WT cells. (f) UMAP of nHSPC clusters showing expression of defining HSPC markers, ptprc and lmo2. (g,h) Pseudotime analysis (g) and CellRank analysis (h) showing C8.nHSPC pLEPs and C5.nHSPC pLMPs are both terminal states of EHT cell clusters (Methods). (i) UMAP of WT and 128^Δ/Δ of EHT cluster kdrl+ cells with assigned identification. (j) Percentage of cells expressing gata2b in pre-EHT (C2 and C1) and hemEC (C4) clusters in WT and 128^Δ/Δ kdrl+ cells. (k) Percentage of cells in nHSPC clusters per genotype, showing the tendency of C3.nHSPCs to increase in 128^Δ/Δ compared to WT. (l) Quantification of ECs (kdrl+, cmyb⁻) edu+ (n = WT, 22; 128^Δ/Δ, 20 embryos) or pH3+ (n = WT, 18; 128^Δ/Δ, 24 embryos) counted in the same images as Fig. 2h (3 independent experiments, Two-tailed Mann-Whitney test). All Quantification are represented with mean ± SEM. ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Abbreviations: primed lympho-erythroid progenitors (pLEPs), primed lympho-myeloid progenitors (pLMPs), lymphatic progenitor (LP) and intersegmental vessels (ISV).