Fig. 1. In vitro colony formation from E11.5 metanephric mesenchyme. (A) Sheet-like colonies were formed on 3T3Wnt4, but not on 3T3lacZ. Arrows: fibroblast-like cells. (B) Colonies were not formed on 3T3Wnt4 with the addition of Fz-Fc chimeric protein. (C) RT-PCR analyses of genes expressed in metanephros and fully differentiated epithelia in glomeruli (podocyte), proximal and distal tubules, and the loop of Henle. Lane 1: E11.5 metanephric mesenchyme; 2: 3T3Wnt4 alone; 3: mesenchyme-derived cells cultured on 3T3Wnt4 at day 3; 4: at day 10; 5: at day 20; 6: 3T3lacZ alone; 7: mesenchyme-derived cells cultured on 3T3lacZ at day 3, 8: at day 10, 9: at day 20, 10: mesenchyme-derived cells at day 10 separated from 3T3Wnt4 feeder cells; 11: organ culture of E11.5 mesenchyme rudiments at day 10; 12: embryonic kidney (E17.5); 13: no RT reaction on mesenchyme-derived cells cultured on 3T3Wnt4 at day 10. (D-M) Immunocytochemistry of colonies for Pax2 (D-F), E-cadherin (G-I), Sall1 (J,K) and Aqp1 (L,M). (D-I) The expression of Pax2 and E-cadherin (red) was not detected at day 3 (D,G, respectively) but was observed at day 10 (E,H). (J,K) Sall1 expression (red) at day 10. (L,M) Aqp1 (red, proximal tubule marker) was expressed in some cells of the colony. Feeder cells have larger nuclei (DAPI, blue; arrows) than cells consisting of colonies. Control staining with rabbit (F,K,M) and mouse (I) IgGs. Mesenchyme of Sall1-GFP knock-in mice was used for J and K to visualize Sall1 expression using anti-GFP immunostaining, while EGFP transgenic mesenchyme was used for D-I,L,M. Scale bars: 50 µm.