Extrathymic T-lineage progenitors are Notch dependent. B6 mice were reconstituted with Ly5^SJL BM cells after transduction with a retrovirus expressing green fluorescent protein (GFP) (MigR1), the pan-Notch inhibitor DNMAML1, or the Notch modulator Deltex1 (MigDtx1). All retroviruses expressed a GFP tag. Spleen cells were analyzed 2 weeks after BMT. Data are representative of 2 experiments with at least 4 mice/group. (A) GFP expression after infusion of MigR1-transduced progenitors. Donor-derived Lin⁺ Thy1.2^– cells were used as an internal control for the efficiency of retroviral transduction. The level of GFP expression was comparable in this population and in SpT and Thy1.2^–CD44⁺ cells as defined in Figure 2. (B) GFP expression after infusion of DNMAML1-transduced progenitors was absent from the SpT population, indicating that its generation was Notch dependent. GFP expression was modestly reduced among Thy1.2^–CD44⁺ cells. (C) GFP expression after infusion of Dtx1-transduced progenitors was reduced but not abolished in SpT and Thy1.2^–CD44⁺ cells. (D) Graphical representation of the results shown in Figure 5B-C. The percentage of transduction in the Lin⁺Thy1.2^– population (internal control) is normalized to 100%. The percentage of GFP⁺ cells in the SpT and Thy1.2^–CD44⁺ populations is shown as a fraction of the internal control. Data are shown as mean ± SEM. * indicates a significant difference as compared with the internal control (P < .01, Student t test). (E) Relative expression of Notch1, Notch2, Notch3, Hes1, and Ptcra mRNA at day 14 after BMT in sorted MigR1 or MigDtx1-transduced SpT cells, showing that Dtx1 reduces the intensity of Notch signaling and decreases Notch1 but not Notch2 mRNA levels. Data are shown in arbitrary units after normalization for Hprt1 mRNA. Each triangle shows the mRNA level measured in triplicate in SpT cells from a pool of 2 recipient spleens (6 recipients in each group).