Wnt4 has a direct effect on ETP and DN2 expansion. (A) Analysis of homing to the Wnt4-deficient and -sufficient adult thymus. The number and relative contribution of donor-derived (CD45.1⁺) Lin⁻ cells was determined 36 hours after injection. The histograms represent mean ± SEM from 7 mice per group. (B) Expression of chemokines and P-selectin in the Wnt4-deficient and -sufficient adult thymus. The histograms represent mean ± SEM from 4 to 5 mice per group. (C) Analysis of T-cell development in culture in the presence of GFP⁺ OP9-DW4 or OP9-DL1 stromal cells. GFP⁻ cells were analyzed by flow cytometry at indicated times and are expressed as number of cells/ETP seeded on day 0. The histogram represents mean ± SEM from a total of 8 replicates from 3 independent experiments. (D) Analysis of T-cell differentiation as defined by cKit and CD25 staining on day 5. The histogram on the left represents ETP and DN2 frequencies among GFP⁻ cells. The histogram in the middle represents the number of cells with ETP and DN2 phenotype on d5 per ETP seeded (mean ± SEM; n = 8 for wild type, n = 5 for Tcf7^−/−). Representative flow cytometry data gated on GFP⁻ cells are shown on the right. (E) Expression of selected genes associated with DN2-DN3 progression and early T-cell development. Histogram represents the relative mRNA levels from sorted GFP⁻ lymphocytes after 2 or 5 days of coculture (ratio OP9-DW4/OP9-DL1 from paired experiments; mean ± SEM; n = 3; *P < .05, **P < .01, ***P < .005).