Generating molecular markers that selectively label urothelial sub-populations

描述(申请人提供)：尿流出道，包括肾盆、输尿管、膀胱和上尿路，由称为尿路上皮的特殊复层上皮构成，由基底层、中间层和由伞状细胞组成的浅层组成。伞状细胞是专门用于合成的大的多核细胞。 以及Uroplakins的运输，Uroplakins是一种分泌蛋白家族，聚集在尿路上皮顶端表面的晶体蛋白斑块中，在尿液和血液之间产生防泄漏的屏障。尿路上皮可能会因损伤或疾病，包括间质性膀胱炎、炎症和尿路感染而功能障碍，但目前还没有修复/再生这种组织的方法。此外，尿路上皮被认为是导致不同形式膀胱癌的干/祖细胞的来源。进展的一个障碍是缺乏区分包括祖细胞在内的尿路上皮亚型的标记。我们建议从我们使用表达RFP或GFP的转基因和谱系标记的小鼠模型定义的那些开始，产生一系列针对尿路上皮亚群的遗传标记。除了伞状细胞和基底层细胞外，我们已经能够定义一种新的细胞类型，它位于中间层，可能是伞状细胞的前体。波特/阿诺诺实验室将使用RNA-Seq对Flow分类的细胞群体进行遗传图谱分析，他们已经成功地使用了这项技术来表达肾脏细胞类型。一旦经过验证的候选人在手，我们将在 LUT采用原位杂交和免疫组织化学方法。我们还将绘制在GUDMAP1中确定的潜在尿路上皮细胞特异性的现有候选基因的分布。在过去的三年里，我们一直是GUDMAP1联盟的成员，在此期间，我们成功地生成了以LUT为重点的表达数据，该数据已被合并到现有的ALAS中。我们与GUDMAP合作者发展的关系以及在LUT中进行表达分析的经验将是我们开展拟议研究的优势。

DESCRIPTION (provided by applicant): 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.