Global programs of ESRP-regulated splicing in renal development and function

ESRP 调节剪接在肾脏发育和功能中的全球计划

• 批准号: 8545244
• 负责人: RUSS Paul CARSTENS
• 金额: $ 15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8545244
• 关键词: Ablation; Address; Adult; Alleles; Alternative Splicing; Behavior; Biological Models; Cell Differentiation process; Cell Line; Cell physiology; Cells; Cellular Morphology; Data; Defect; Development; Developmental Gene; Developmental Process; Disease; Duct (organ) structure; Ectopic Expression; Epithelial; Epithelial Cells; Epithelium; Exons; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Models; Health; Human; Image; In Vitro; Intercellular Junctions; Kidney; Kidney Glomerulus; Knock-out; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Mus; Nephrons; Organ; Organogenesis; Play; Polyadenylation; Process; Protein Isoforms; Protein Splicing; Proteins; RNA Splicing; Regulation; Renal function; Role; Set protein; Severities; Staging; System; Technology; Time; Transcript; Tubular formation; Variant; base; cell behavior; cell injury; cell type; epithelial to mesenchymal transition; genetic regulatory protein; genome-wide; in vivo; insight; migration; nephrogenesis; novel; progenitor; programs; transcription factor; urinary

PROJECT SUMMARY Alternative splicing provides a means of massively expanding the complexity of mammalian transcriptomes and nearly all human multi-exon gene transcripts are alternatively spliced. However, in contrast to studies of transcription factors and transcriptional gene expression programs, the role of alternative splicing in important developmental programs remains largely unstudied. Using a genome-wide cell-based screen my lab discovered two paralogous epithelial-specific splicing regulatory proteins, ESRP1 and ESRP2. The ESRPs regulate both alternative splicing as well as alternative polyadenylation as master regulators of an extensive post-transcriptional network. Our studies suggest that they play a key role in the epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). However, while our data in cell lines are consistent with this proposal, definitive evidence to support this contention requires analysis in more elegant in vivo genetic model systems. Towards this end we generated mice with conditional and complete knockout alleles for Esrp1 and Esrp2. Using these mice we will investigate the following hypotheses: First, we hypothesize that Esrp expression is required for proper ureteric bud branching and collecting duct formation. Second, we propose that the ability of metanephric cap mesenchyme cells to undergo MET and generate renal tubular epithelial cells requires Esrp expression. We further hypothesize that these requirements for ESRP involve critical ESRP target transcripts whose coordinated isoform switches are needed for proper kidney formation and function. We will address these hypotheses though the Following Specific Aims: 1) Define the developmental role of the ESRPs in ureteric bud branching and formation of the renal collecting system. We will conditionally ablate Esrp expression in the ureteric bud and derivatives in crosses with HoxB7 Cre mice. Potential defects in ureteric branching will be assessed after serial sectioning and 3D reconstructive imaging. 2) Determine whether the Esrps are required for the MET in cap mesenchyme and development of renal tubular epithelial cells. The role of Esrp induction during the MET and consequent formation of the glomerulus and renal tubular epithelium will be determined through conditional ablation in cap mesenchymal in breedings with Six2-Cre mouse lines. 3) Investigate the consequences of Esrp knockout in adult renal tubular and collecting duct epithelial cells and perform in vivo global profiling of ESRP target transcripts using high throughput sequencing. We hypothesize that ablation of Esrp expression in mature renal epithelial cells will induce dedifferentiation and cellular injury. In addition to determining the phenotypic consequences of Esrp knockout we will also use high throughput sequencing technologies to comprehensively determine the in vivo renal epithelial splicing and polyadenylation program that is enforced by the Esrps. The collective definition of Esrp regulated targets will reveal important gene transcripts and an extensive post-transcriptional network that function in renal organogenesis and function.

项目摘要 替代剪接提供了一种大规模扩大哺乳动物转录组复杂性的手段 几乎所有人类的多种外观基因转录本均被剪接。但是，与研究 转录因子和转录基因表达程序，替代剪接在重要的 发展计划在很大程度上没有研究。使用基于全基因组的细胞屏幕My Lab 发现了两个副上皮特异性剪接调节蛋白ESRP1和ESRP2。 ESRP 调节替代剪接以及替代聚腺苷酸化作为广泛的主调节剂 转录后网络。我们的研究表明，它们在上皮到间质的上皮中起着关键作用 过渡（EMT）和间质向上皮转变（MET）。但是，尽管我们在单元线中的数据是 与此提案一致，支持此争论的确切证据需要更优雅的分析 体内遗传模型系统。为此，我们用有条件而完整的淘汰赛产生了小鼠 ESRP1和ESRP2的等位基因。 使用这些小鼠，我们将研究以下假设：首先，我们 假设ESRP表达是正确的输尿管芽分支和收集管道形成所必需的。 其次，我们提出，元素盖间充质细胞经历MET并产生肾脏的能力 管状上皮细胞需要ESRP表达。我们进一步假设这些对ESRP的要求 涉及关键的ESRP目标成绩单，其适当的肾脏需要协调的同工型开关 形成和功能。我们将通过以下具体目的解决这些假设：1）定义 ESRP在输尿管芽分支和肾脏收集的形成中的发展作用 系统。我们将有条件地在带有hoxb7的十字架中的输尿管芽中燃烧ESRP的表达 CRE小鼠。 连续分段和3D重建性后，将评估输尿管分支中的潜在缺陷 成像。 2）确定盖盖间充质中Met的ESRP是否需要 肾小管上皮细胞的发展。 ESRP在MET中的作用和因此 肾小球和肾小管上皮的形成将通过帽中的条件消融确定 具有六个2-cre小鼠系的繁殖物中的间充质。 3）研究ESRP敲除的后果 在成年肾小管和收集导管上皮细胞中，并在ESRP的体内进行体内分析 使用高吞吐量测序的目标转录本。 我们假设ESRP表达在 成熟的肾上皮细胞将诱导去分化和细胞损伤。除了确定 ESRP敲除的表型后果我们还将使用高通量测序技术 全面确定由体内肾上皮剪接和聚丙基化程序。 ESRP。 ESRP规范目标的集体定义将揭示重要的基因转录本和 广泛的转录后网络，在肾脏器官发生和功能中发挥作用。