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Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney

Department of Molecular and Cellular Biology, 16 Divinity Avenue, Harvard University, Cambridge MA 02138, USA

View larger version (126K): [in a new window]   Fig. 1. Expression pattern of Shh, Ihh, Ptch and Bmp4 in the prenatal and neonatal kidney and ureter. In situ hybridization with probes as indicated to stages as indicated. (A-D) Parasagittal sections of E14.5 kidney and ureter. Scale bar: 200 µm. (E-H) Coronal sections of the newborn kidney. Arrow in H indicates signals in glomeruli. Scale bar: 500 µm. (I-L) Cross-sections of the newborn ureter. Scale bar: 100 µm.

View larger version (39K): [in a new window]   Fig. 2. HoxB7/Cre reporter analysis. HoxB7/Cre mice were crossed to either Rosa (A-F) or Z/AP (G-I) reporter mice and subjected to histological staining to visualize ß-galactosidase activity (A-F; G-I, red, lacZ) or alkaline phosphatase (I, blue, AP) activity. Ages of embryos are indicated. (A-D,G-I) Whole-mount views; (E-F) sections. (A) Mesonephric duct (arrow). (B) Mesonephric duct and onset of ureteric bud invasion into the metanephric mesenchyme (arrow; low levels of HoxB7/Cre activity were also detected in the dorsal root ganglia (arrowhead) and the spinal cord). (C) The ureteric bud has branched once in the metanephric mesenchyme (arrow). (D) The ureteric bud has undergone several branches, the ureter (arrow) and the Wolffian duct (arrowhead) are evident. (E,F) Sections of different regions of a E12.5 kidney. (G-I) The ß-galactosidase activity was high in ureteric bud derivatives of the Z/AP kidney in the absence of HoxB7/Cre (G), but was completely removed in the presence of HoxB7/Cre (H). Alkaline phosphatase activity was detected in all ureteric bud branches (I).

View larger version (45K): [in a new window]   Fig. 3. Conditional removal of Shh activity from the urothelium with HoxB7/Cre results in hypoplasia, hydroureter and hydronephrosis. (A,B) Whole-mount view of the newborn kidney and ureter of wild-type (WT) and HoxB7/Cre, Shhc/n mice. (C-J) Hematoxylin and Eosin staining of coronal sections (C,D) and parasagittal sections (G,H) of the kidney (C,D,G,H) and cross-sections of the ureter (E,F,I,J) at stages indicated. Scale bars: 500 µm in C,D; 100 µm in E,F,I,J; 1 mm in G,H.

View larger version (37K): [in a new window]   Fig. 4. Ptch and Bmp4 expression in mutant kidneys. (A-H) In situ hybridization of Ptch and Bmp4 probes to E14.5 kidney (A,B,E,F) and ureter (C,D,G,H). The insets show Bmp4 expression in glomeruli. Scale bars: 100 µm in A,B,E,F; 50 µm in C,D,G,H. (I) RT-PCR of Bmp4 transcripts from E12.5 mesenchymal cells dissected from the ureter (lane 1) and those cultured without (lane 2) and with (lanes 3 and 4) the addition of N-SHH protein at concentrations indicated. PCR with ß-actin primers (ß-actin) indicated equal cDNA inputs in all lanes.

View larger version (28K): [in a new window]   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%.

View larger version (54K): [in a new window]   Fig. 6. Smooth muscle differentiation in wild-type kidneys and ureter. Hematoxylin and Eosin (H&E; A,C,E,G,I,K) and immunohistochemical staining for smooth muscle -actin (SM -actin, brown; B,D,F,H,J,L) in the proximal (A,B,E,F,I,J) and distal (C,D,G,H,K,L) ureter at the indicated stages. Arrows in B indicate scattered cells that produce SM -actin. Scale bar: 50 µm.

View larger version (61K): [in a new window]   Fig. 7. Smooth muscle differentiation in the wild-type (A,C,E,G) and Shh mutant (B,D,F,H) kidneys. Immunohistochemical staining for smooth muscle -actin (brown) in the cross-sections of the proximal (A,B,E,F) and distal (C,D,G,H) ureters at the stages indicated. NB, newborn. Scale bar: 50 µm in A-D; 100 µm in E-H.

View larger version (73K): [in a new window]   Fig. 8. Mesenchymal cells between smooth muscle and the epithelium in the ureter are absent in Shh mutants. (A,B) Newborn kidneys and ureters stained with anti-smooth muscle -actin antibody (red), Dolichos bifloris agglutinin (DBA), which stains the cell surface and demarcates the urothelium (green), and the DNA dye DAPI (blue). Note that one to two layers of mesenchymal cell nuclei immediately subjacent to the ureteral epithelium (the boundary marked by DBA staining) in the ureter of wild type are not surrounded by anti-smooth muscle -actin antibody staining (A; inset, arrow), suggesting that these cells do not express smooth muscle -actin. By contrast, in the mutants (B; inset), those cells recognized by the anti-smooth muscle -actin antibody lie immediately adjacent to the urothelium (the boundary marked by DBA staining). Thus, the smooth muscle -actin-negative mesenchymal cell population appears to be absent in HoxB7/Cre, Shhc/n kidneys. (C,D) Ptch-lacZ+/- mice at the newborn stage stained for ß-galactosidase (black), smooth muscle -actin (red) and Dolichos bifloris agglutinin (DBA, green) indicates that those cells expressing highest level of Ptch-lacZ do not produce SMA. Arrow in D indicates such cells. Scale bar: 50µm. (E) Shh adversely affects smooth muscle differentiation in a dose-dependent manner. RT-PCR of SM -actin (SM -actin) transcripts from E12.5 mesenchymal cells dissected from the ureter (lane 1) and those cultured without (lane 2) and with (lanes 3 and 4) the addition of N-SHH protein at concentrations indicated. PCR with ß-actin primers (ß-actin) indicated equal cDNA inputs in all lanes.

View larger version (44K): [in a new window]   Fig. 9. A model for the functions of Shh in ureteral mesenchymal cell proliferation and differentiation. Shh produced in the ureteral epithelium promotes proliferation and inhibits smooth muscle differentiation of ureteral mesenchymal cells. It also induces Bmp4 expression in these cells. SM, smooth muscle.