Extended Data Fig. 3: miR-128 regulates nHSPC heterogeneity prior EHT in zebrafish.
From: Haematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic endothelium
(a) Schematic representation of FACS isolation of zebrafish cells from 26 hpf Tg(kdrl:mCherrys896;cmyb:GFPzf169) nHSPCs (cmyb+, kdrl+), endothelia cells (ECs) (cmyb-,kdrl+) and non-ECs (cmyb-,kdrl-). RT-qPCR analysis of miR-128 (non-ECs, 5; ECs, 4; HSPCs, 5 independent pools of ~10 embryos) showing mature miR-128 expression enriched in ECs vs HSPCs or non-ECs (ordinary one-way ANOVA with Tukey’s multiple comparison). (b) Percentage of cells in scRNA-seq clusters expressing r3hdm1 and arpp21 in which the intronic miR-128 genes 1 and 2 are hosted, respectively. (c) Confocal images of zebrafish tail at 32 hpf in WT Tg(kdrl:GFPzn1) injected at one cell stage with Tol2 fli1a:miR-128-mCherry and gata2b:gal4 with Tol2 UAS:miR-128-mCherry (labeled as gata2b:miiR-128) (3 independent experiments). mCherry indicates successful miR-128 expression in the respective cells (arrowheads). (d) WISH analysis of runx1 (n = WT, 32; WT + fli1a, 27; 128Δ/Δ, 30; 128Δ/Δ + fli1a, 35; embryos) at 27 hpf with and without Tol2 fli1a:miR-128-mCherry injection at one cell stage (3 independent experiments; ordinary one-way ANOVA with Tukey’s multiple comparison). All quantification are represented with mean ± SEM. ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01,**** p ≤ 0.0001. Abbreviations: aorta gonad mesonephros (AGM).
