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: 8915301
• 负责人: RUSS Paul CARSTENS
• 金额: $ 46.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-25 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8915301
• 关键词: Ablation; Accounting; 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; Harvest; Health; High-Throughput Nucleotide Sequencing; Human; Immunoprecipitation; Injection of therapeutic agent; Knock-out; Knockout Mice; Lead; Lip structure; Maintenance; Mesenchymal; Mesenchyme; Modeling; Molecular; Mus; Mutate; Palate; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Polyadenylation; Polyadenylation Pathway; Process; Processed Genes; Protein Isoforms; RNA; RNA Splicing; Regulation; Risk; Role; Signal Pathway; Signal Transduction; Testing; Time; Tissue-Specific Gene Expression; Transcript; Viral; cell type; craniofacial; crosslink; genetic regulatory protein; genome-wide; in vivo; innovation; mouse model; novel; oral cavity epithelium; orofacial; programs; receptor; tool; transcription factor; transcriptome sequencing; unpublished works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）：唇裂伴或不伴腭裂（CL/P）是人类患者中最常见的出生缺陷。与孤立的CP相反，CL/P在人类患者中更常见，但很少有CL/P的小鼠模型来定义引起CL/P的机制。已经确定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，但对这种串扰的基本分子事件知之甚少。虽然一些关键的转录因子和信号通路参与颅面发育已被确定，选择性剪接的作用在很大程度上是未开发的。我的实验室研究了成纤维细胞生长因子受体2（Fgfr 2）的选择性剪接，这是一种与颅面异常相关的基因。我们发现了两个旁系同源上皮细胞类型特异性剪接因子，Esrp 1和Esrp 2，这是需要表达的上皮Fgfr 2-IIIb亚型和更广泛的上皮程序的选择性剪接。我们产生的小鼠与敲除（KO）的Esrp基因和Esrp 1 KO小鼠的主要缺陷是100%的渗透CL/P。这些Esrp KO小鼠提供了一个神话般的新工具，以探测CL/P背后的机制，并确定新的基因和途径所需的正常面部发育。我们假设Esrp 1 KO导致口腔上皮细胞自主缺陷以及导致CL/P的基础间充质细胞非自主缺陷。我们进一步假设Esrp靶转录物编码上皮特异性蛋白质同种型，其对于维持上皮-间充质相互作用和唇和腭形成所需的信号传导事件至关重要。我们将通过以下目的全面定义外胚层中Esrp调节的靶点和导致Esrp 1 KO小鼠CL/P的机制：1）表征发育中的面部和腭中Esrp 1和Esrp 1/Esrp 2消融所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Esrp 1 KO导致CL/P的关键步骤和机制。2）确定发育中的外胚层和腭上皮细胞中Esrp调节的选择性剪接和多聚腺苷酸化的全局程序。我们将在Esrp 1flox/flox/Esrp 2-/- CKO胚胎中使用高通量测序（RNA-Seq）和剪接敏感的微阵列来定义外胚层和衍生物中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）来定义全基因组Esrp结合位点。我们将确定Esrp 1的全基因组结合位点，以确定体内Esrp调控的直接靶点。4)鉴定其损失导致CL/P的关键上皮特异性剪接形式。我们将使用Esrp调节的上皮同种型的病毒cDNA的羊膜内注射来测试胚胎发育期间CL/P表型的拯救。这些创新性研究有可能揭示CL/P的新候选基因，并确定这些疾病中出错的分子机制。