Figure 2.

Wt1 is essential for epithelial cells characteristics maintenance and their normal proliferation. TUNEL assay of coelomic epitheliums (white lines) was detected in Wt1^R394W/R394W embryo and control embryo (A). A table outlining the number of TUNEL⁺ PGCs observed in control and Wt1^R394W/R394W E11.5 embryos (C). Proliferation of coelomic epitheliums (white lines) in Wt1^R394W/R394W embryos was examined, using BrdU incorporation assay (B). White arrows in B pointed to the BrdU⁺ coelomic epitheliums. Scale bars: 30 μm. Numbers in parentheses show the actual number of BrdU⁺ coelomic epitheliums over the total number of coelomic epitheliums counted for that embryo (D), and note that the BrdU labeling rate was significantly reduced in Wt1^R394W/R394W embryos (E). Four potential WT1 binding sites (GNGGGNG) in the cyclin E1 2.8-kb promoter were identified (F). TM4 cells were co-transfected with 0.2 μg cyclin E1-Luc (G), or 0.2 μg cyclin E1-Luc with different point mutants of the WT1A in modified PCB6+ vector (H), or the control PCB6+ vector. One group of mutants contained changes in the zinc finger region (R366C, H337Y and R394W), and the other group contained (F154S and S273A) changes outside that zinc finger region [37]. All of these assays were performed 36 hours after transfection. Fold activation was displayed by Firefly/Renilla ratio. Error bars represent SEM. *P <0.05 (by t-test). A significant elevation of mesenchymal markers vimentin (left) and a complete loss of the epithelial markers E-cadherin (right) and were detected by immunostaining assay (I). White arrows in I pointed to the signals. Scale bars: 30 μm.