Activation of lineage competence in hemogenic endothelium precedes the formation of hematopoietic stem cell heterogeneity [Zebrafish.scATAC-seq]

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.