Wnt4-sufficiency in hematopoietic cells and extrathymic stromal cells does not rescue its absence in the thymic stroma. (A) Total thymic cellularity in Wnt4^+/+ and Wnt4^−/− grafts. (B) H&E-stained paraffin sections of Wnt4^+/+ and Wnt4^−/− thymic grafts. Original magnification ×40. (C) Numbers of thymocytes in Wnt4^+/+ and Wnt4^−/− grafts are depicted. (D) Representative flow cytometry data for cKit (CD117) and CD25 (gated on Lin^lo/neg cells for ETP, DN2, and DN3) and CD4/CD8 cell staining. The numbers within cytometry plots represent mean percentages of total. (E) Mice were injected intraperitoneally with 1 mg BrdU in PBS to provide a labeling pulse. Four hours later, thymic grafts were harvested, and BrdU uptake was detected with a BrdU flow kit (BD Biosciences Pharmingen). (F) Annexin V staining was performed to detect apoptotic T cells by flow cytometric analysis. (G) Representative flow cytometry data for Wnt4-overexpressing and control thymic aggregates (left panel). Graphs show the fold expansion of total CD45⁺ cells, CD25⁻cKit^hi (ETPs), and TECs in Wnt4 aggregates compared with controls (right panel). Histograms represent the mean ± SEM of 5-10 mice per group (*P < .05, **P < .01, ***P < .005).