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, Shh^c/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.

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