The urinary outflow tract, including the renal pelvis, ureters, bladder and upper urethra, is lined by a specialized stratified epithelium called the urothelium, composed of a basal layer, an intermediate layer and a superficial layer consisting of umbrella cells. Umbrella cells are large multinucleated cells that are specialized for synthesis and trafficking of Uroplakins, a family of secreted proteins that assemble into the crystallin plaque lining the apical surface ofthe urothelium that generates a leak-proof barrier between the urine and the blood. The urothelium can be rendered dysfunctional by damage, or disease, including interstitial cystitis, inflammation and UTI, but at present there is no way to repair/regenerate this tissue. In addition the urothelium is thought to be a source of stem/progenitor cells that give rise to different forms of bladder cancer. One impediment to progress has been the paucity of markers distinguishing urothelial sub-types including progenitors We propose to generate a collection of genetic markers specific for urothelial sub-populations beginning with those that we have defined using transgenic and lineage-tagged mouse models expressing RFP or GFP. In addition to umbrella cells and basal cells, we have been able to define a novel cell type that resides in the intermediate layer that is a likely umbrella cell progenitor. Flow sorted cell populations will be genetically profiled by the Potter/Aronow labs using RNA-Seq, a technique that they have successfully used to expression profile kidney cell types. Once validated candidates are in hand, we will map their distribution in the LUT using in situ hybridization and immunohistochemistry. We will also map the distribution of existing candidates identified in GUDMAP1 as potentially urothelial-cell specific. We have been part of the GUDMAP1 consortium for the past 3 years, and during this time we have successfully generated LUT-focused expression data which has been incorporated into the existing alas. The relationships that we have developed with GUDMAP collaborators and experience with expression analysis in the LUT will be an advantage as we undertake the proposed studies.
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