Hematopoietic stem cells (HSCs) are generated from hemogenic endothelial cells (HECs) in the floor of the dorsal aorta (DA) via endothelial-to-hematopoietic transition (EHT). Yet whether HECs and conventional endothelial cells (cECs) in the DA shares a common precursor is controversial and the molecular mechanisms governing their fate specification remain incompletely defined. Here using a combination of fate mapping, time-lapse imaging, genetic manipulation, and single-cell RNA-seq, we show that before EHT, HECs and cECs display strict spatial separation in the DA where nearly all the ECs in the DA floor are HECs while all the ECs in the DA roof are cECs. We further document that HECs and cECs in the DA originate from a common hemogenic angioblast precursor, which differentiates into H...[ Read more ]

Hematopoietic stem cells (HSCs) are generated from hemogenic endothelial cells (HECs) in the floor of the dorsal aorta (DA) via endothelial-to-hematopoietic transition (EHT). Yet whether HECs and conventional endothelial cells (cECs) in the DA shares a common precursor is controversial and the molecular mechanisms governing their fate specification remain incompletely defined. Here using a combination of fate mapping, time-lapse imaging, genetic manipulation, and single-cell RNA-seq, we show that before EHT, HECs and cECs display strict spatial separation in the DA where nearly all the ECs in the DA floor are HECs while all the ECs in the DA roof are cECs. We further document that HECs and cECs in the DA originate from a common hemogenic angioblast precursor, which differentiates into HECs and cECs during axial migration prior to the DA formation. The specification of HECs and cECs from hemogenic angioblasts is governed by the Etv2 dosage through differentially activating Fli1a, Notch, and Sclβ. Finally, we showed that pan-endothelial overexpression of the HEC fate-specific transcriptional factor runx1 is sufficient to specify HEC fate in the DA roof. Our study answers the controversial lineage origin of HECs and cECs in the DA and uncovers the genetic and molecular network controlling their fate specification.