Fig. 5. Detection of phosphorylated SMAD1/5/8 proteins (pSMAD) on tissue sections. The brown precipitate reveals pSMAD-positive nuclei. All sections are counterstained with Hoechst 33258 (light blue fluorescence) to reveal nuclei lacking pSMADs. All sections are sagittal along the primary embryonic axis with ventral to the left and dorsal to the right. Sections shown are representative of the results obtained by analysing serial sections of at least three embryos per genotype. (A,C,E) Wild-type mouse embryos at E11.0 (40 somites; A); E11.25 (47 somites; C) and E11.5 (50 somites, enlargement of tip region; E). (B,D,F) Grem1-deficient embryos at E11.0 (42 somites; B) and E11.25 (48 somites; D), an enlargement of the ureteric bud area in D is shown in F. The area within the red dashed line contains many picnotic nuclei indicative of the onset of cellular apoptosis (D,F). cm, condensing metanephric mesenchyme; mm, metanephric mesenchyme; sm, stromal mesenchyme; ub, ureteric bud; ue, ureteric epithelium (first branch); us, ureteric stalk; wd, Wolffian duct.