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

项目摘要 选择性剪接提供了一种大规模扩展哺乳动物转录组复杂性的手段 并且几乎所有的人多外显子基因转录物都是选择性剪接的。然而，与研究相比， 转录因子和转录基因的表达程序，选择性剪接的作用，在重要的 发展计划在很大程度上尚未得到研究。我的实验室使用全基因组细胞筛选 发现了两种旁系同源的上皮特异性剪接调节蛋白ESRP 1和ESRP 2。ESRP 调节可变剪接以及可变多聚腺苷酸化作为广泛的 转录后网络我们的研究表明，它们在上皮细胞向间质细胞转化中起着关键作用。 间质细胞向上皮细胞转化（MET）。然而，虽然我们在细胞系中的数据 与这一建议相一致，支持这一论点的明确证据需要在更优雅的分析中进行分析。 体内遗传模型系统。为了达到这个目的，我们产生了条件性和完全敲除的小鼠， Esrp1和Esrp2的等位基因。 利用这些小鼠，我们将研究以下假设：首先，我们 推测Esrp表达是正确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并产生肾功能的能力， 肾小管上皮细胞需要Esrp表达。我们进一步假设ESRP的这些要求 涉及关键的ESRP靶向转录物，其协调的同种型开关是正常肾脏所必需的， 形成和功能。我们将通过以下具体目标来解决这些假设：1）定义 ESRPs在输尿管芽分支和肾集合形成中的发育作用 系统我们将在与HoxB7杂交中有条件地消除输尿管芽和衍生物中的Esrp表达。 克雷老鼠。 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 显像2)确定帽间充质中MET是否需要ESrps， 肾小管上皮细胞的发育。Esrp诱导在MET过程中的作用及其后果 肾小球和肾小管上皮的形成将通过CAP中的条件消融来确定。 用Six2-Cre小鼠系在乳腺间充质中进行。3)研究Esrp敲除的后果 在成人肾小管和集合管上皮细胞中进行ESRP的体内总体分析 使用高通量测序靶向转录物。 我们假设，在胰腺癌中， 成熟的肾上皮细胞将诱导去分化和细胞损伤。除了确定 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多聚腺苷酸化程序， ESRP。Esrp调控靶点的集体定义将揭示重要的基因转录本和一个新的基因表达。 广泛的转录后网络，在肾器官发生和功能中发挥作用。

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.