Figure 2.

Notch1 and TCF1 Fail to Prevent Inhibition of Early T Cell Lineage Differentiation by EBF1

(A) LSK (Lin⁻Sca1⁺c-kit⁺) cells were sorted from e14.5 Pax5^−/− fetal livers and transduced with control MigR1 or MigR-EBF1 virus. Viable GFP⁺ cells were sorted 24 hr after transduction onto pre-established OP9-DL4 cells with IL-7, FL, and SCF. Seven days later, cultures were stained and analyzed for absolute numbers of DAPI⁻CD45⁺GFP⁺Thy1.2⁺CD25⁺ cells. Data in right-most graph are means and SEMs from each group.

(B) e14.5 Ebf1^−/− fetal liver LSKs were isolated and cotransduced with MigY-EBF1 and MigR-ICN1 viruses and plated on OP9 stromal cells. A day later, cells were washed and replated on OP9s in fresh media supplemented with IL-7, FL, and SCF. On day 7, cultures were stained and analyzed for relative contribution of DAPI⁻CD45⁺ single-transduced (GFP⁺YFP⁻ or GFP⁻YFP⁺) or double-transduced (GFP⁺YFP⁺) cells that coexpressed Thy1.2 and CD25.

(C) e14.5 Ebf1^−/− fetal liver LSKs were isolated and cotransduced with MigY-EBF1 and TCF1-VEX viruses as in (B). Plots were gated on DAPI⁻CD45⁺ single- or double-transduced cells. All graphs show means ± SEMs of triplicate samples. Data are representative of three separate experiments. See also Figure S2.