Fig. 5. Proximal epithelial structures are missing in DAPT-treated metanephroi. (A,E) E-cadherin (green), cytokeratin 8 (red) and Wt1 (blue, overlay of A on B and E on F with Canvas). Distal tubules (green only) formed in DAPT-treated metanephroi throughout the section (E, asterisk). (B,F): Wt1^HIGH (glomerular podocytes) and Wt1^LOW (induced/condensed mesenchyme and primitive epithelia) are detected in both DMSO- and DAPT-treated metanephroi. Only a few podocytes and well-defined glomeruli are found in the center of DAPT-treated metanephroi, and an increase in Wt1^LOW cells is also evident (F). (C,G) Detection of proximal tubules with Lotus tetragonolobus Lectin (LTL, green). Note the overall reduction in the number and the central location of proximal epithelia in DAPT-treated metanephroi. (D,H) Laminin 1 (red) was detectable in the basal laminae of tubules in both control and treated metanephroi; fewer renal tubules were detected in DAPT-treated cultures. (I,J) Proximal tubules are abundant in untreated metanephroi (cadherin 6, pink, inset in I) but are missing in DAPT-treated metanephroi (J). Distal tubules (E-cadherin, green) are present in both. (K,L) De novo formation of epithelia occurs in DAPT. Distal tubules (circled in K and L) but no podocytes (Wt1^HIGH, K) are present in 11.5 day metanephroi cultured for 6 days in DAPT. (Inset in L) Pax2 (red), expressed in induced mesenchyme, persists in newly formed epithelia at the periphery of DAPT treated cultures (stained with E-cadherin; green). A more central epithelial cluster loses staining of Pax2.