Lymphoid potential of Gata3^−/− DN2 T-cell progenitors. (A) The graph shows limiting dilution and clonal frequency analysis of Gata3^−/− and Gata3^+/− DN2 cells (sorted from OP9Δ1 cultures) following replating on OP9 stroma in the presence of IL-7, Flt3L, and IL-2. Wells were scored for growth after 1 week. (B) Clones derived from single Gata3^−/− and Gata3^+/− DN2 cells after reculture on OP9 stroma with IL-2 (upper panels) or without IL-2 (lower panels) were analyzed for the expression of NK1.1 and CD19. (C) Gata3^−/− DN2 cells were recultured on OP9 stroma with IL-7, and NK1.1^- lymphocytes were analyzed for the expression of CD19 and IgM. The gate indicates the population that was sorted for analysis of transcripts (D). (D) RT-PCR analysis shows the expression of B- and T-cell transcription factors by B cells grown from sorted DN2 cells on OP9 stroma (DN2 > B, as shown in [C]) compared with sorted splenic B (B) and T (T) lymphocytes from a C57BL/6 control mouse. (E) Gata3^−/− T-cell precursors were retrovirally transduced with pMXI-GATA3-GFP, and single GFP^- or GFP⁺ DN2 cells were cultured on either OP9 (upper panels) or OP9Δ1 (lower panels) stroma with IL-7 and Flt3L. One week later, plates were scored for growth, and the colonies derived from a single DN2 were stained for NK1.1, CD3, or CD19 expression, as indicated.