Fig. 6. Sema4d-induced inhibition of ureteric branching morphogenesis requires activation of the Rho-ROCK pathway. (A) Treatment with Sema4d leads to activation of RhoA in mice metanephroi cultured at E14.5. Lysates of kidneys treated with either Sema4d or mock medium for 5 hours were incubated with RBD-coupled sepharose and the amount of RhoA was analysed by western blotting with an anti-RhoA antibody. The amount of G13 was comparable in the two samples (internal control). (B-D) Typical examples of anti-calbindin-stained metanephroi cultured with either Sema4d or mock medium in the presence or absence of the ROCK inhibitor Y27632 (1 µM). In the presence of 1 µM Y27632, Sema4d did not attenuate ureteric branching. (E,F) Y27632 (3 µM) led to a hyperplasia and distortion of ureteric structures (arrowheads), to a lesser extent in Sema4d-treated kidneys than in mock-treated kidneys. (G-I) Higher magnification views of phalloidin-stained ureteric tips in Sema4d- or mock-treated kidneys cultured in the presence of 3 µM Y27632. The Y27632-induced distortion of apical wedge cells and actin bundles and the increase in tip lumen occurs to a lesser extent in Sema4d-treated kidneys in comparison with mock-treated kidneys. (J,K) Quantitative summary of the effects of Y27632 at either 1 µM or 3 µM on Sema4d- or mock-treated metanephroi. P<0.001 when compared with mock; ^*P<0.05 when compared with mock or Sema4d alone; ANOVA followed by post-hoc Fisher's test. Scale bars: 200 µm in B-F; 70 µm in G-I.