Hematopoiesis is significantly perturbed in Wt1-deficient EBs. (A) Representative FACS plots of disaggregated wild-type and Wt1-null^k1 EBs stained at (i) day 6 of differentiation for CD41/c-Kit (n = 7; P < .0005), (ii) day 6 for Ter119 (n = 5; P < .01), and (iii) day 8 for Mac-1 (n = 5; P < .0005), showing fewer hematopoietic progenitor cells, mature erythroid cells, and myeloid cells in Wt1-null EBs. The numerical data are the mean of independent replicates, tested for significance using Student t test; 2-tailed distribution assuming unequal variance. P values represent both Wt1-null clones vs wild type. (B) Hematopoietic colony assay (Stem Cell Technologies) of disaggregated day 8 EBs showing numbers of erythroid (BFU-E), myeloid (CFU-GM), and multipotent (CFU-mix) progenitor cells per 3 × 10⁵ EB cells. Both Wt1-null clones individually show a significant reduction in colony-forming potential (P < .005), including all individual colony types (P < .05), compared with wild type. The data are the mean of independent replicates, each conducted in triplicate. Bars display standard deviation; significance tested using analysis of variance (n [wild type vs K1] = 4; n [wild type vs K46] = 3). (C) Hematopoietic progenitor cells lacking Wt1 show elevated levels of apoptosis compared with wild type. Representative FACS plots for (i) annexin-V staining of cells gated for Ter119^+ve expression showing increased levels of apoptosis in Wt1-null EBs compared with wild type (n = 5; P < .02) and (ii) annexin-V staining of unfractionated, disaggregated wild-type and Wt1-null EBs showing that the increased apoptosis of hematopoietic progenitors is not a general feature of all EB cells lacking Wt1 (n = 5; P > .05). The numerical data are the mean of independent replicates, tested for significance using Student t test; 2-tailed distribution assuming unequal variance. P values represent both Wt1-null clones vs wild type. (D) FACS analysis of e15.5 fetal livers from chimeric embryos showing capacity of donor Wt1-null ES cells (GFP^+ve) to generate diverse hematopoietic cell types within chimeric fetal livers in similar proportions to the endogenous wild-type host (GFP^–ve): (i) Mac-1 (myeloid lineage), (ii) c-Kit (progenitor cells), (iii) Ter-119 (erythroid lineage), and (iv) B220 (lymphoid lineage). Data from the chimera with the highest contribution of GFP^+ve Wt1-null ES cells are shown.