Fig. 3. Development of mouse metanephroi in whole-organ cultures and its modulation by Sema4d. (A-C) Phase-contrast images of a kidney rudiment isolated at E12 (A) and of the same kidney after 24 hours (B) and 48 hours (C) in culture. (D-F) Maximal projections of confocal images of whole cultured metanephroi showing the ureteric tree stained with an anti-calbindin antibody (green, D) and the condensing mesenchyme stained with an anti-WT1 antibody (red, E,F) at E12 (D) and after 24 hours (E) and 48 hours (F) in culture. UT, ureteric tip; CD, collecting duct; MM, metanephrogenic mesenchyme; CB, comma-shape bodies, two examples are indicated by the double arrow. (G) Illustration of the method used for quantifying ureteric branch points (bp) and ureteric tips (arrows, UT) in maximal confocal projections. (H,I) Typical examples of E12 metanephroi cultured for 24 hours in medium supplemented with concentrated medium of mock-transfected HEK293T cells (H) or Sema4d-AP-transfected HEK293T cells (I). The ureteric tree is labelled with an anti-calbindin antibody (green). (J) Western blot analysis demonstrating the efficacy of Sema4d in inducing RhoA activation in a GST-RBD-based assay in HEK293T cells transfected with plexin B1 (PlxnB1) and PDZRhoGEF (PRG). Anti-Myc-positive bands in western blotting serve as loading controls. (K) Sema4d treatment reduces the number of ureteric tips and branch points over mock treatment in sister cultures of metanephroi. (L) Sema4d treatment decreases the size of the developing kidney over mock treatment. Only sister kidneys cultured for 48 hours were taken for analysis. ^*P<0.001 in comparison with mock, Student's paired t-test. Scale bars: 200 µm in A-I.