Differential Notch and Wnt activity in lineage-biased MPP subsets. (A) Quantitative RT-PCR analysis of canonical Notch, TGFβ, Hedgehog (Hh), and Wnt pathway genes in HSPCs (n = 3, in one experiment). Results are expressed as log₂ fold expression relative to HSCs (set to 0). (B) SABiosciences PCR array of Notch and Wnt pathway-related genes in HSPCs (n = 3, in two independent experiments). Results are expressed as log₂ mean fold expression relative to HSCs (set to 0). (C) Schematic of the ex vivo stimulation of Hes1-Gfp HSPCs by plate-bound DLL1 ligand, representative FACS plots and quantification of Notch activity (n = 6 for HSCs, n = 5 for MPP3 and MPP4, in four independent experiments). Results are expressed as fold changes in GFP mean fluorescence intensity (MFI) relative to respective unstimulated population (set to 1). S, stimulated; US, unstimulated. (D) Schematic of the NICD IF analysis and percentage of nuclear NICD–positive (nuc. NICD⁺) cells in HSPCs (n = 3, in two independent experiments). (E) Schematic of the IF analysis with representative images and percentage of nuclear β-catenin–positive (nuc. β-cat⁺) cells in HSPCs (n = 5 for HSC and MPP4, n = 4 for MPP3, in five independent experiments). Scale bar, 10 µm. (F) Model depicting differential Notch and Wnt activity in HSPCs at steady state. Results are expressed as mean ± SD except in B; unpaired Student’s t test was used. *, P ≤ 0.05; **/°°, P ≤ 0.01; ***/°°°, P ≤ 0.001; *, versus HSC levels; °, in between MPPs.