FIGURE 4.

Deletion of Gpr54 down-regulates Bmp7 expression and Smad1 phosphorylation in developing kidney. A, Bmp7 mRNA expression was decreased in E12.5 Gpr54^−/− kidneys. Total RNA was isolated from E12.5 wild type (WT) and Gpr54^−/− kidneys, and key genes involved in kidney development were examined by RT-PCR. GDNF, glial cell-derived neurotrophic factor. B, real-time PCR analysis of Bmp7 mRNA expression in E12.5, E17.5, and P0 wild type and Gpr54^−/− kidneys is shown. Decreased Bmp7 mRNA expressions were observed in E12.5, E17.5, and P0 Gpr54^−/− kidneys compared with wild type kidneys. C-H, Gpr54 deletion leads to decreased Bmp7 protein expression in E12.5, E17.5, and P0 kidneys. Immunohistochemistry staining was performed in E12.5, E17.5, and P0 wild type and Gpr54^−/− kidneys with anti-Bmp7 antibody. C and D, detectable Bmp7 immunostaining was found in ureteric buds (UB), metanephric mesenchymal cells (CM), and the epithelial cells of collecting buds (CD) in E12.5 wild type kidneys but not in E12.5 Gpr54^−/− kidneys. E and F, Bmp7 is expressed in the epithelial cells of proximal and distal tubules (PT and TU) and collecting ducts (CD) in E17.5 wild type kidneys. GL, glomeruli. Bmp7 immunostaining was significantly decreased in Gpr54^−/− kidneys. G and H, Bmp7 is expressed in the epithelial cells of proximal and distal tubules (TU) and collecting ducts (CD) in P0 wild type and Gpr54^−/− kidneys. I–L, immunostaining of phosphorylated Smad1 (p-Smad1) in E17.5 and P0 kidney is shown. p-Smad1 was decreased in the epithelial cells of proximal and distal tubules (TU) and the collecting ducts (CD) in E17.5 Gpr54^−/− kidneys (J and L) compared with wild type kidneys (I and K). Scare bar = 10 μm. Arrows show detectable Bmp7 or p-Smad1 immunostaining.