qRT-PCR analysis of the relative expression levels of TGF-β1, BMP-4 and Smads in embryonic bladders from E12.5 to neonatal day0.

Ten embryonic bladders per group were lysed to extract total RNA for qRT-PCR. Data were normalized to that of GAPDH and the relative expression levels are shown. Each experiment was repeated three times and the mean value was calculated as expression value for each stage of embryonic bladder development. A) BMP-responsive R-Smads. Smad1, Smad5 and Smad8 were expressed in all gestation stages. Smad1 expression was minimal in E12.5 and Smad8 expression was low in E18.5 and neonatal day0 (Mann-Whitney test, p<0.005)B) TGF-β responsive R-Smads. Smad2 and Smad3 showed relatively high expression in all gestation stages while Smad2 showed decreased expression at E18.5 and neonatal day0. (Mann-Whitney test, p<0.005)C) Common Smad. Smad4 showed the highest expression pattern from E12.5 to E16.5 and decreased in E18.5 and neonatal day0 (Mann-Whitney test, p<0.005) and D) Inhibitory Smads. Smad6 and Smad7. Smad6 showed higher expression from E12.5 to E16.5, and lowest expression levels in E18.5 and neonatal day0 (Mann-Whitney test, p<0.005), while Smad7 showed the highest expression level at E12.5 and decreased in the subsequent stages (Mann-Whitney test, p<0.005).