Fig. 5. Shh is required for mesenchymal cell proliferation. (A) The kidney volume/body weight of Shh mutant (Mu, n=4; 2.3±0.2 cm³/g) and wild-type (WT, n=3; 4.8±0.8 cm³/g) kidneys at the newborn stage. (B) The glomerular density of Shh mutant (Mu, 326.32±33.07) and wild-type (WT, 258.89±25.77) kidneys at the newborn stage. (C-E) Ureter sections from E14.5 wild-type (C) or mutant (D) kidneys were stained with anti-phospho-histone H3 antibodies (red), Dolichos bifloris agglutinin, which demarcates the surface of the epithelium (DBA, green), and a DNA dye (DAPI; blue). Mesenchymal cells within the broken line were counted for calculations in E. The mitotic index of the proximal ureter mesenchyme was 6.29±2.39% in Shh mutants (Mu) and 12.12±3.24% in wild-type (WT). The mitotic index of the distal ureter mesenchyme was 3.15±1.05% in Shh mutants and 6.5±1.08% in wild type. Scale bar: 50 µm in C,D. (F) Mesenchyme dissected from E12.5 ureter was cultured for 5 days without (control) or with proteins as indicated (see Materials and Methods), labeled with 10 µM BrdU for 11 hours and stained with anti-BrdU antibodies and the DNA dye DAPI. Proliferation index was calculated as the percentage of nuclei that incorporated BrdU. Control, 4.8%; SHH, 14.0%; Noggin, 8.4%; BMP4, 0%; BMP4+Noggin, 5.7%; SHH+Noggin, 12.6%.