Hemogenic and aortic endothelium arise from a common hemogenic angioblast precursor and are specified by the Etv2 dosage

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 share a common precursor is controversial, and the molecular mechanisms governing their fate specification remain incompletely defined. Using a combination of fate mapping, time-lapse imaging, genetic manipulation, and single-cell RNA sequencing, here we show that HECs and cECs display strictly spatial separation in the DA where nearly all the endothelial cells in the floor and roof are HECs and cECs, respectively. We further show that HECs and cECs in the DA arise 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 by differentially regulating Fli1a, Notch, and Sclβ. Finally, we document that pan-endothelial overexpression of transcriptional factor runx1 is sufficient to promote HEC fate in the DA roof. Our study reveals the lineage origin of HECs and cECs in the DA and uncovers the molecular network controlling their fate specification.