Activation of lineage competence in hemogenic endothelium precedes the formation of hematopoietic stem cell heterogeneity. Activation of lineage competence in hemogenic endothelium precedes the formation of hematopoietic stem cell heterogeneity

Using a combination of single-cell multi-omics, lineage tracing and functional assays, we show that embryonic HSPCs are originated from heterogeneous hemogenic endothelial cells (HECs) during zebrafish embryogenesis. Overall design: Hematopoietic stem and progenitor cells (HSPCs) are considered as a heterogeneous population, but where and how HSPC heterogeneity occurs remain unclear. Here, we performed scRNA-seq and scATAC-seq with zebrafish 36 hpf VDA-derived kdrl+runx1-, kdrl+runx1+, and kdrl-runx1+ cells. To determine the transcriptional signatures of spi2+ lineages in zebrafish, we performed STRT-seq with spi2: Gal4;UAS:GFP+ kdrl:mCherry+ HECs and spi2: Gal4;UAS:GFP+ kdrl:mCherry- hematopoietic cells at 36 hpf. To investigate the underlying molecular mechanism upon spi2 deficiency, we performed scRNA-seq with the sorted ECs (kdrl+runx1-), HECs (kdrl+runx1+) and hematopoietic cells (kdrl-runx1+) from spi2 morphants at 36 hpf. To determine whether spi2 can directly modulate transcriptional programs in EC/HEC, we examined genome-wide spi2 binding by cut-tag assay in fli1a-flag-spi2-EGFP+ cells sorted from trunk region of Tg (fli1a-flag-spi2-GFP) embryos at 36 hpf.