Molecular control of early urothelial differentiation in the murine ureter

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The urothelium, the inner epithelial lining of the mature urinary drainage system, serves as a physical barrier to prevent entry of toxic substances of the urine into the blood stream and to withstand pathogens from the external milieu. The mature tissue consists of three major cell types that are organized in radial layers of variable thickness. Bordering the lumen are large binucleated tightly-sealing superficial (S-)cells. Underneath are much smaller intermediate (I-)cells that serve as precursors. Finally, a layer of small but highly abundant cuboidal basal (B-)cells anchor the epithelium to the basal lamina. All these cell layers arise by an as yet poorly understood molecular and cellular program from a homogenous mono-layered epithelium during embryogenesis.Our genetic analysis in the mouse characterized a SHH-dependent FOXF1-BMP4 mesenchymal module as pivotal for proliferation, stratification and differentiation of the adjacent epithelial compartment in the ureter. We characterized the FGFR2 pathway as a second signaling input for epithelial stratification and I-/B-cell differentiation while retinoic acid (RA) signaling maintains I-cells and prevents their differentiation into B- and S-cells. The transcription factors delta NP63, PPARG and ELF5 were found as targets of these signaling activities in the ureteric epithelium. Here, we want to engage a combination of (tissue-specific) gene targeting and misexpression studies in vivo, pharmacological manipulation of ureter explant cultures, and small and large scale molecular assays to get a better understanding of how these signals and factors regulate early urothelial development in the mouse ureter. In a first work-program we wish to further characterize the individual and combinatorial function of the mesenchymal signals BMP4, FGFs and RA in initiating and maintaining urothelial differentiation by defining their phenotypic requirements, by characterizing their intracellular effector pathways (particularly SMAD and kinases) and by identifying their transcriptional target genes in the embryonic ureter.In a second work-program, we wish to decipher the individual contribution of the transcription factors delta NP63, PPARG and ELF5 to epithelial development in the ureter. For this, we will analyze the cellular and molecular changes resulting from conditional genetic loss and gain of these factors in the ureteric epithelium and its sublineages. Identification of direct target genes of these factors is aimed for by a combination of transcriptional profiling of mutant ureters and ChIP-Seq analysis. We expect from these studies decisive insights in the molecular control mechanism of induction and maintenance of urothelial differentiation in the ureter which may serve as a paradigm for other components of the urinary tract. From a clinical point of view, it may shed light on the etiology and possible molecular causes of congenital and acquired anomalies of the urothelial lining of the urinary tract.