UROPLAKIN mRNA IS NOT UROTHELIUM SPECIFIC AND CAN BE MANIPULATED BY HEDGEHOG INHIBITION IN AN EMBRYONIC ORGAN CULTURE SYSTEM

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Background.
Uroplakins are thought to be terminal markers of urothelium differentiation. Our study examines the expression and manipulation of uroplakin mRNA in mouse embryos and an embryonic organ culture (OCx) model.
Methods
mRNA was extracted from mouse embryo organs (including bladder, bowel, kidney) at different gestational ages using the RNeasy kit™. cDNA was reverse transcribed (RT) and Uroplakin (UP) Ia, Ib, 2, and 3a genes were amplified by the PCR, TA cloned, transformed, purified, and sequenced. Digoxigenin-labeled anti-sense cRNA probes were synthesized for each gene and used for RNA in-situ hybridization (ISH) of whole and sectioned embryos at different gestational ages.
Embryonic mouse bowel and bladders at e13.5 and e14.5 were harvested and cultured in serum supplemented DMEM with and without cyclopamine (10uM), an inhibitor of the hedgehog (Hh) pathway. OCx were fixed and processed at different time points for ISH.
Results
All plasmid inserts were found to have the correct sequence for their corresponding UP gene. cRNA probes showed specificity to mouse urothelium. Up1a and 1b mRNA was found in mouse embryo bladders and bowel by both RT-PCR (Figure 1) and ISH (Figure 2). Inhibition of the Hh pathway by cyclopamine leads to increased UP mRNA expression in both embryonic bowel and bladder (Figure 3)
Conclusion
Uroplakin mRNA is not urothelium specific and can be manipulated through inhibition of the Hh pathway in an OCx model. Our data suggests that the Hh pathway is important in urothelial differentiation thus providing further insight into a poorly understood area of embryogenesis.

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