Roles of Epithelial Splicing Regulatory Proteins in craniofacial development

上皮剪接调节蛋白在颅面发育中的作用

• 批准号: 9267966
• 负责人: RUSS Paul CARSTENS
• 金额: $ 58.01万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9267966
• 关键词: Ablation; Alternative Splicing; Binding Sites; Birth; Candidate Disease Gene; Cells; Cleft Palate; Cleft lip with or without cleft palate; Complementary DNA; Congenital Abnormality; Congenital abnormal Synostosis; Coupled; Craniofacial Abnormalities; Defect; Development; Disease; Ectoderm; Embryo; Embryonic Development; Epithelial; Epithelial Cells; Epithelium; Event; Face; Fibroblast Growth Factor Receptor 2; Fibroblasts; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Harvest; High-Throughput Nucleotide Sequencing; Human; Immunoprecipitation; Injection of therapeutic agent; Knock-out; Knockout Mice; Lead; Lip structure; Maintenance; Mesenchymal; Mesenchyme; Messenger RNA; Modeling; Molecular; Mus; Mutate; Palate; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Polyadenylation; Process; Protein Isoforms; RNA; RNA Splicing; Regulation; Risk; Role; Signal Pathway; Signal Transduction; Testing; Time; Tissue-Specific Gene Expression; Transcript; Viral; cell type; craniofacial development; crosslink; crosslinking and immunoprecipitation sequencing; genetic regulatory protein; genome-wide; in vivo; innovation; mouse model; novel; oral cavity epithelium; orofacial cleft; programs; public health relevance; receptor; tool; transcription factor; transcriptome sequencing; unpublished works

DESCRIPTION (provided by applicant): Cleft lip with or without cleft palate (CL/P) is among the most highly prevalent birth defects in human patients. In contrast to isolated CP, CL/P is more common in human patients and yet there are few mouse models for CL/P to define mechanisms that give rise to CL/P. It is well established that crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, yet the basic molecular events underlying this crosstalk are poorly understood. While a number of key transcription factors and signaling pathways involved in craniofacial development have been identified, the role of alternative splicing is largely unexplored. My lab studied alternative splicing of fibroblast growt factor receptor 2 (Fgfr2), a gene associated with craniofacial abnormalities. We discovered two paralogous epithelial-cell-type-specific splicing factors, Esrp1 and Esrp2, which are required for expression of the epithelial Fgfr2-IIIb isoform and a broader epithelial program of alternative splicing. We generated mice with knockout (KO) of the Esrp genes and the primary defect in Esrp1 KO mice is 100% penetrant CL/P. These Esrp KO mice provide a fabulous new tool to probe the mechanisms behind CL/P and to identify novel genes and pathways required for normal facial development. We hypothesize that Esrp1 KO causes cell autonomous defects in oral epithelium as well as cell- non-autonomous defects in underlying mesenchyme that lead to CL/P. We further hypothesize that Esrp target transcripts encode epithelial-specific protein isoforms that are essential for maintenance of epithelial- mesenchymal interactions and signaling events that are required for lip and palate formation. We will comprehensively define Esrp-regulated targets in ectoderm and the mechanisms leading to CL/P in Esrp1 KO mice through the following aims: 1) Characterize the defects in lip and palate formation that occur with ablation of Esrp1 and Esrp1/Esrp2 in the developing face and palate. We will conditionally ablate the Esrps at two distinct time points in development to define the key steps and mechanisms by which Esrp1 KO leads to CL/P. 2) Identify global programs of Esrp regulated alternative splicing and polyadenylation in developing ectoderm and palatal epithelial cells. We will use high throughput sequencing (RNA-Seq) and splicing sensitive microarrays in Esrp1flox/flox/Esrp2-/- CKO embryos to define genome-wide changes in splicing, polyadenylation, and gene expression that occur with loss of Esrp expression in ectoderm and derivative. 3) Define genome-wide Esrp binding sites using crosslinking immunoprecipitation coupled with high throughput sequencing (CLIP-Seq). We will identify genome-wide binding sites for Esrp1 to define in vivo direct targets of Esrp regulation. 4) Identify key epithelial-speific splice forms whose loss leads to CL/P. We will use intra-amniotic injections of viral cDNAs of Esrp regulated epithelial isoforms to test for rescue of CL/P phenotypes during embryonic development. These innovative studies have the potential to unveil new candidate genes for CL/P as well as to define molecular mechanisms that go awry in these disorders.

描述（由申请人提供）：伴有或不伴有腭裂的唇裂（CL/P）是人类患者中最常见的出生缺陷之一。与孤立性 CP 相比，CL/P 在人类患者中更为常见，但很少有 CL/P 小鼠模型来定义产生 CL/P 的机制。众所周知，上皮细胞和间充质细胞之间的串扰是面部和腭形成的基础，但人们对这种串扰背后的基本分子事件知之甚少。虽然已经确定了与颅面发育有关的许多关键转录因子和信号通路，但选择性剪接的作用在很大程度上尚未被探索。我的实验室研究了成纤维细胞生长因子受体 2 (Fgfr2) 的选择性剪接，这是一种与颅面异常相关的基因。我们发现了两种旁系同源上皮细胞类型特异性剪接因子 Esrp1 和 Esrp2，它们是上皮 Fgfr2-IIIb 亚型表达和更广泛的上皮选择性剪接程序所必需的。我们生成了 Esrp 基因敲除 (KO) 的小鼠，Esrp1 KO 小鼠的主要缺陷是 100% 渗透 CL/P。这些 Esrp KO 小鼠提供了一个极好的新工具来探索 CL/P 背后的机制并识别正常面部发育所需的新基因和途径。我们假设 Esrp1 KO 导致口腔上皮细胞自主缺陷以及底层间充质细胞非自主缺陷，从而导致 CL/P。我们进一步假设 Esrp 靶转录物编码上皮特异性蛋白亚型，这些亚型对于维持上皮-间质相互作用以及唇和腭形成所需的信号事件至关重要。我们将通过以下目标全面定义外胚层中 Esrp 调节的靶点以及导致 Esrp1 KO 小鼠 CL/P 的机制：1）表征发育中面部和腭中 Esrp1 和 Esrp1/Esrp2 消融时发生的唇和腭形成缺陷。我们将在发育过程中的两个不同时间点有条件地消融 Esrps，以确定 Esrp1 KO 导致 CL/P 的关键步骤和机制。 2) 确定 Esrp 在发育外胚层和腭上皮细胞中调节选择性剪接和聚腺苷酸化的全局程序。我们将在 Esrp1flox/flox/Esrp2-/- CKO 胚胎中使用高通量测序 (RNA-Seq) 和剪接敏感微阵列来定义外胚层及其衍生物中 Esrp 表达丧失时发生的剪接、多聚腺苷酸化和基因表达的全基因组变化。 3) 使用交联免疫沉淀结合高通量测序 (CLIP-Seq) 定义全基因组 Esrp 结合位点。我们将确定 Esrp1 的全基因组结合位点，以定义 Esrp 调节的体内直接目标。 4) 确定其丢失导致 CL/P 的关键上皮特异性剪接形式。我们将使用羊膜内注射 Esrp 调节的上皮亚型的病毒 cDNA 来测试胚胎发育过程中 CL/P 表型的拯救。这些创新研究有可能揭示 CL/P 的新候选基因，并确定这些疾病中出错的分子机制。