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Fig. 7. ß-catenin signaling triggers survival and proliferation of epithelial progenitors, but not tubulogenesis. (A) Introduction of stabilized ß-catenin (Ad-CTNNB_S37A) into epithelial progenitors marked by Pax2 (red) prevents apoptosis determined by immunostaining for activated caspase 3 (a-CASP3, blue) observed after 3 days of culture under control conditions (Ad-GFP only). This anti-apoptotic response is blocked by dominant-negative TCF (Ad-DN-TCF). (B) Proliferative activity measured by phospho-histone H3 staining (blue) is observed in Pax2-expressing (red) progenitors following infection with Ad-CTNNB_S37A, but not Ad-GFP (arrows). Note the presence of a small number of phospho-histone-positive cells in the Pax2-negative cell compartment in both conditions (arrowheads). (C) Only scattered Pax2-positive progenitors (red) are initially infected by Ad-CTNNB_S37A, as detected after 16 hours of culture following infection and staining for the HA epitope on the adenovirally coded protein (green). Infected cells display a strongly positive membrane pool of HA-CTNNB_S37A and a nuclear pool of variable intensity, which colocalizes with Pax2 (yellow areas). After 3 days of culture, HA-negative progenitors have largely disappeared suggesting positive selection of cells infected by Ad-CTNNB_S37A, which indicates that the effect of the adenovirus is cell-autonomous. (D) Metanephric mesenchymes infected with Ad-CTNNB_S37A develop into cell aggregates, but do not display evidence of tubulogenesis or segmentation. Conversely, tubular and glomerular-like structures are abundantly observed after treatment with a low dose of the Gsk3ß inhibitor BIO. (E) Aggregates induced by Ad-CTNNB_S37A stain negative for Cdh1 (green), a marker of polarized epithelial cells. By contrast, metanephric mesenchymes induced with the Gsk3ß inhibitors lithium chloride or BIO for the same time period display Cdh1-positive tubules. Note that treatment with BIO also induces glomerular-like structures marked by podocalyxin expression (Podxl, red). (F) As detected by real-time RT-PCR, markers of polarized epithelial cells are strongly induced after epithelial induction with BIO for 4 days (d4 BIO) or Lif for 7 days (d7 Lif) (time points of robust epithelial differentiation), but remain negative 4 days (d4) and 7 days (d7) after infection with Ad-CTNNB_S37A (S37A). Values are compared with freshly isolated metanephric mesenchymes (d0), expression levels of which were set to 1. ^*, P<0.05 versus metanephric mesenchyme. Gl, glomerular-like structure; Tb, tubule.

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