Notch activity decreases during HSC maturation. (A) Expression of Hes1-GFP in endothelial cells (VC⁺CD45⁻CD43⁻; gate R1), pre-HSC type I (VC⁺CD45⁻CD43⁺; gate R2), and pre-HSC type II (VC⁺CD45⁺CD43⁺Sca1⁺; gate R3) defined by flow cytometry in the E11.5 Hes1-GFP⁺ AGM region. FMO GFP control (FMO control) was performed with wild-type cells. (B) Pre-HSCs type I are mainly Hes1-GFP⁺, whereas pre-HSCs type II reside in both the GFP⁻ and GFP⁺ fraction. VC⁺CD45⁻ (Pre-HSC type I) and VC⁺CD45⁺ (pre-HSC type II) cells were sorted from E10.5 and E11.5 AGM based on Hes1-GFP expression, coaggregated with OP9 cells and transplanted after culture (1 ee per recipient); n = 3. Levels of engraftment are plotted and number of repopulated vs total number of transplanted mice are shown in brackets (***P < .005, Mann-Whitney U test). (C) AGM dHSCs reside in both Hes1-GFP⁺ and Hes1-GFP⁻ populations. CD45⁺ cells were sorted from E12.5 AGM based on Hes1-GFP expression and directly transplanted into irradiated mice (4 ee per recipient); n = 2. (D) Expression of Hes1-GFP in E14.5 fetal liver dHSCs, phenotypically defined by Lin⁻cKit⁺Sca1⁺CD48⁻CD150⁺. Gray histogram: Hes1-GFP⁻ control. (E) Fetal liver (FL) HSCs reside in the GFP^−/low fraction. LSK populations were sorted based on Hes1-GFP expression from E13.5 and E14.5 fetal liver and directly transplanted into irradiated mice; (0.2 ee per recipient); n = 2. LSK, Lin⁻Sca⁺cKit⁺; LT-HSC, long-term HSC; SSC-A, side scatter.