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: 8800527
• 负责人: RUSS Paul CARSTENS
• 金额: $ 48.04万
• 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/8800527
• 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; 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 development; crosslink; crosslinking and immunoprecipitation sequencing; genetic regulatory protein; genome-wide; in vivo; innovation; mouse model; novel; oral cavity epithelium; orofacial; programs; public health relevance; receptor; tool; transcription factor; transcriptome sequencing; unpublished works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）：人类患者中最普遍的出生缺陷之一。与孤立的CP相反，Cl/P在人类患者中更为常见，但是CL/P的小鼠模型很少用于定义引起Cl/p的机制。众所周知，上皮细胞和间质细胞之间的串扰是面部和口感的形成，但是该串扰的基本分子事件知之甚少。尽管已经确定了许多关键的转录因子和涉及颅面发育中的信号通路，但替代剪接的作用在很大程度上尚未探索。我的实验室研究了与颅面异常相关的基因成纤维细胞成年因子受体2（FGFR2）的替代剪接。我们发现了两个寄生态上皮细胞特异性剪接因子ESRP1和ESRP2，这是表达上皮FGFR2-IIIB同工型和更广泛的替代拼接上皮上皮程序所必需的。我们用ESRP基因的基因敲除（KO）产生小鼠，而ESRP1 KO小鼠的主要缺陷为100％渗透液CL/p。这些ESRP KO小鼠提供了一种神话般的新工具，可以探测Cl/P背后的机制，并确定正常面部发育所需的新基因和途径。我们假设ESRP1 KO会导致口腔上皮的细胞自主缺陷以及导致Cl/p的下层间充质中的细胞非自主缺陷。我们进一步假设ESRP目标转录本编码上皮特异性蛋白质同工型，这些蛋白质同工型对于维持上皮 - 间质相互作用和嘴唇形成所需的信号事件至关重要。我们将通过以下目的全面定义了外胚层中ESRP调节的靶标，以及导致ESRP1 KO小鼠中Cl/p的机制：1）表征唇部和pape形成中的缺陷，而ESRP1和ESRP1和ESRP1/ESRP2在发育中的面部和口感中发生了。我们将有条件地在开发中的两个不同时间点上烧掉ESRP，以定义ESRP1 KO导致Cl/p的关键步骤和机制。 2）确定ESRP调节的替代剪接和多腺苷酸化的全局程序，以发展出外胚层和palatal上皮细胞。我们将使用高吞吐量测序（RNA-SEQ）和ESRP1FLOX/FLOX/ESRP2 - / - CKO胚胎中的敏感微阵列来定义剪接，聚腺苷酸化和基因表达的全基因组的变化，这些变化是伴有Ectoderm和derivatient和derivatient ectoderp s ectrp表达的基因表达。 3）使用交联的免疫沉淀与高吞吐量测序（夹子seq）定义全基因组ESRP结合位点。我们将确定ESRP1全基因组结合位点，以定义ESRP调控的体内直接靶标。 4）确定损失导致Cl/p的关键上皮剪接形式。我们将使用ESRP调节的上皮同工型的病毒cDNA的散热性注射，以测试胚胎发育过程中CL/P表型的营救。这些创新的研究有可能揭示CL/P的新候选基因，并定义这些疾病中出现问题的分子机制。