(A) New developmental trajectory from endothelium to pre-HSCs. An intermediate pre-HE stage generates HE cells through the upregulation of RUNX1. An initial wave of LMPs followed by a second wave of pre-HSCs contribute to hematopoietic clusters at E10.5 and E11.5, respectively. (B) The ligand-receptor couple ADM-RAMP2 and the secreted factor SVEP1 regulate HSPCs production in vivo. (C) Schemes of the embryo species and samples used for Tomo-seq. The red areas mark the ventral aortic region of emergence of clusters and pre-HSCs.