Exogenous Vegf-a₁₆₄ rescues the hematopoietic impairment of Wt1-null EBs. (A) Hematopoietic colony assay (Stem Cell Technologies) of disaggregated day 8 EBs following Vegf-a₁₆₄ treatment showing numbers of erythroid (BFU-E), myeloid (CFU-GM), and multipotent (CFU-mix) progenitor cells per 3 × 10⁵ EB cells. Addition of exogenous Vegf-a₁₆₄ significantly increases hematopoietic potential of Wt1-null EBs (total colony counts; P < .05). The data are the mean of 3 independent replicates, each conducted in triplicate. Bars display standard deviation; significance tested using analysis of variance. (B-C) Exogenous Vegf-a₁₆₄ blocks apoptosis in Wt1-null EBs. (B) Representative FACS analysis of annexin-V staining of cells within the earliest Ter119^+ve population of progenitor cells to emerge following 6 days of differentiation in (i) wild-type control, (ii) Wt1-null control, (iii) wild type + Vegf-a₁₆₄, and (iv) Wt1 null + Vegf-a₁₆₄. (C) Corresponding graph to (B) showing a significant reduction in apoptosis in Wt1-null EBs in response to Vegf-a₁₆₄ over 3 independent assays (P < .05). The numerical data are the mean of independent replicates, tested for significance using a paired Student t test; 2-tailed distribution. (D) WT1 controls the ratio of Vegf-a isoforms in hematopoietic progenitor cells. qRT-PCR analysis of wild-type and Wt1-null BL-CFCs for β-actin, Vegf-a₁₂₀, and pan–Vegf-a showing overrepresentation of Vegf-a₁₂₀ in Wt1-null BL-CFCs. (E) Vegf-a splicing assay. RT-PCR analysis of Vegf-a isoforms following transient transfection of Cos-7 cells with a genomic expression construct encompassing Vegf-a exons 4 to 8 (lanes 1-3) cotransfected with Wt1 expression constructs for the “-KTS” isoforms, Wt1(^−/−) and Wt1 (^+/−), lanes 1 and 2. Primers located in exons 4 and 8 amplify Vegf-a₁₂₀ (89 bp), Vegf-a₁₆₄ (221 bp), and Vegf-a₁₈₉ (293 bp). Nonspecific background bands are also present associated with an artifactual heteroduplex, as previously reported.¹⁵  Lower panels show Wt1 expression confirmed by RT-PCR and western blotting using monoclonal antibody 6F-H2 (DAKO).