Forced Notch activity blocks pre-HSC type I maturation. (A-B) Forced activation of Notch activity elevates Hes1-GFP expression in pre-HSCs type I and type II. Hes1-GFP⁺ pre-HSCs type I (lin⁻VC⁺CD45⁻CD43⁺cKit⁺) and type II (lin⁻VC⁺CD45⁺Sca1⁺cKit⁺) were sorted from E11.5 AGM and coaggregated either with OP9-WT (left column) or OP9-expressing DLL1 upon doxycycline addition (right columns). After 1 day of culture, VC⁺CD45⁺ cells (dot plots) derived from pre-HSCs type I and type II were analyzed for Hes1-GFP expression (black histograms) and compared with Hes1-GFP⁻ control cells (gray histograms). The data are representative of 2 independent experiments. (C) Forced Notch activity prevents pre-HSC type I maturation. Hes1-GFP⁺lin⁻VC⁺CD45⁻CD43⁺cKit⁺ pre-HSCs type I and Hes1-GFP⁺lin⁻VC⁺CD45⁺Sca1⁺cKit⁺ pre-HSCs type II were sorted from E11.5 AGM regions and reaggregated with OP9-Dll1. The coaggregates were cultured with cytokines with or without doxycycline for 5 days before transplantation (0.1 ee per recipient); n = 2; **P < .01, Mann-Whitney test.