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.

项目总结 选择性剪接为大规模扩展哺乳动物转录本的复杂性提供了一种手段 而且几乎所有的人类多外显子基因转录本都是交替剪接的。然而，与对 转录因子和转录基因表达程序在选择性剪接中的重要作用 发展项目在很大程度上仍未得到研究。使用全基因组细胞筛查我的实验室 发现了两个平行的上皮特异性剪接调节蛋白，ESRP1和ESRP2。ESRP 调节选择性剪接和选择性多聚腺苷作为广泛的 转录后网络。我们的研究表明，它们在上皮间充质中起着关键作用。 间质转化(EMT)和间质向上皮转化(MET)。然而，虽然我们细胞系中的数据是 与这一提议一致，支持这一论点的确凿证据需要在 活体遗传模型系统。为此，我们培育了具有条件和完全基因敲除的小鼠 Esrp1和Esrp2等位基因。利用这些小鼠，我们将研究以下假设：第一，我们 假设ESRP的表达是正常输尿管芽分支和集合管形成所必需的。 第二，我们提出后肾帽间充质细胞经历MET和生成肾脏的能力。 肾小管上皮细胞需要表达ESRP。我们进一步假设，ESRP的这些要求 涉及关键的ESRP靶向转录本，其协调的异构体开关是正常肾脏所必需的 构成和功能。我们将通过以下具体目标解决这些假设：1)定义 ESRPs在输尿管芽分枝和肾集合形成中的发育作用 系统。我们将有条件地去除ESRP在输尿管芽和与HoxB7杂交的衍生物中的表达 Cre小鼠。输尿管分支的潜在缺陷将在连续切片和3D重建后进行评估 成像。2)确定MET是否需要ESRP，以及 肾小管上皮细胞的发育。ESRP诱导在MET及后续过程中的作用 肾小球和肾小管上皮的形成将通过有条件的消融来确定。 SIX2-Cre小鼠品系中的间充质细胞。3)调查ESRP基因敲除的后果 在成人肾小管和集合管上皮细胞中进行ESRP的体内整体分析 使用高通量测序打靶转录本。我们假设在ESRP基因中表达的消融 成熟的肾上皮细胞会导致去分化和细胞损伤。除了确定 ESRP基因敲除的表型后果我们还将使用高通量测序技术 全面确定体内肾上皮剪接和聚腺苷酸化程序，这是由 埃斯普一家。ESRP调控靶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。