Molecular control of early urothelial differentiation in the murine ureter

尿道上皮是成熟的尿液引流系统的内上皮衬里，其充当物理屏障以防止尿液中的有毒物质进入血流并抵抗来自外部环境的病原体。成熟组织由三种主要的细胞类型组成，它们以不同厚度的放射状层组织起来。与管腔相邻的是大的双核紧密封闭的表面（S-）细胞。下层是小得多的中间（I-）细胞，作为前体。最后，一层小但高度丰富的立方基底（B-）细胞将上皮固定在基底层上。所有这些细胞层的出现，但知之甚少的分子和细胞程序从一个同质的单层上皮在胚胎genetics.Our在小鼠的遗传分析的特点是一个SHH依赖FOXF 1-BMP 4间充质模块作为关键的增殖，分层和分化的相邻上皮细胞室在输尿管。我们将FGFR 2途径表征为上皮分层和I-/B-细胞分化的第二信号输入，而视黄酸（RA）信号维持I-细胞并阻止其分化为B-和S-细胞。发现转录因子Δ NP 63、PPARG和ELF 5是输尿管上皮中这些信号传导活动的靶点。在这里，我们希望结合体内（组织特异性）基因靶向和错误表达研究，输尿管外植体培养物的药理学操作，以及小规模和大规模的分子测定，以更好地了解这些信号和因子如何调节小鼠输尿管中的早期尿路上皮发育。在第一个工作计划中，我们希望进一步表征间充质信号BMP 4、FGF和RA在启动和维持尿路上皮分化中的单独和组合功能，通过定义它们的表型要求，通过表征它们的细胞内效应途径，（特别是SMAD和激酶）和鉴定它们在胚胎输尿管中的转录靶基因。我们希望解释转录因子Δ NP 63、PPARG和ELF 5对输尿管上皮发育的各自贡献。为此，我们将分析输尿管上皮及其亚系中这些因子的条件性遗传丢失和获得所导致的细胞和分子变化。这些因子的直接靶基因的鉴定旨在通过突变输尿管的转录谱分析和ChIP-Seq分析的组合进行。我们期望从这些研究中的决定性的见解，诱导和维持尿路上皮分化的分子控制机制，在输尿管，可作为一个范例，为其他组成部分的尿路。从临床的角度来看，它可能揭示的病因和可能的分子原因的先天性和后天性异常的尿路上皮衬里的尿路。

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.