Expression of Notch receptors in HSC lineage in AGM region. (A) Expression levels of Notch receptors assessed by qRT-PCR in endothelial cells (VC⁺CD45⁻CD43⁻), pre-HSC type I (VC⁺CD45⁻CD43⁺) and pre-HSC type II (VC⁺CD45⁺) sorted from the E11.5 AGM region (n = 3). Data are mean ± standard error of the mean (s.e.m.). *P < .05, **P < .01. (B) Expression of Notch1 and Notch2 in HSC lineage. FACS analysis representing Notch1 or Notch2 presence at the surface of endothelial cells (VC⁺CD45⁻CD41⁻), pre-HSC type I (VC⁺CD45⁻CD41⁺) and pre-HSCs type II (VC⁺CD45⁺) in the E11.5 AGM region (n = 3). The graphs on the right indicate mean fluorescence intensity (MFI) ratios between Notch1 or Notch2 and their respective FMO controls during the endothelial-to-pre-HSC transition in 3 independent experiments. Data are mean ± s.e.m. *P < .05. (C) All functional pre-HSCs express Notch1. E11.5 AGM cells were sorted based on Notch1 expression and 2 populations (Notch1⁺ and Notch1⁻) were coaggregated with OP9 cells and cultured for 5 days before transplantation into irradiated mice in order to functionally assess the presence of pre-HSCs (0.5 ee per recipient). n = 2; *P < .05, Mann-Whitney U test. (D) Notch2 is expressed in functional pre-HSC type II, but not in pre-HSC type I. E11.5 VC⁺CD45⁻ cells (type I) and VC⁺CD45⁺ (type II) were sorted based on Notch2 expression level and coaggregated with OP9. After 5 days of culture, they were injected into irradiated recipients (pre-HSC type I: 1 ee per recipient; pre-HSC type II: 0.1 ee per recipient); n = 2; ***P < .005, Mann-Whitney U test. ns, nonsignificant, t test.