Roles of Epithelial Splicing Regulatory Proteins in craniofacial development

描述（由申请人提供）：唇裂伴或不伴腭裂（CL/P）是人类患者中最普遍的出生缺陷之一。与分离的CP相比，CL/P在人类患者中更为常见，但很少有CL/P小鼠模型来确定引起CL/P的机制。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，但这种串扰背后的基本分子事件尚不清楚。虽然已经确定了颅面发育中涉及的一些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。我们发现了两个旁系上皮细胞类型特异性剪接因子Esrp1和Esrp2，它们是表达上皮细胞Fgfr2-IIIb亚型和更广泛的上皮细胞选择性剪接程序所必需的。我们产生了Esrp基因敲除（KO）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，并确定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潜在间质细胞非自主缺陷，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-间质相互作用和唇腭裂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esrp在外胚层调控的靶点以及导致Esrp1 KO小鼠CL/P的机制：1)描述在发育中的面部和腭裂中Esrp1和Esrp1/Esrp2消融导致的唇腭裂形成缺陷。我们将在两个不同的开发时间点有条件地消融Esrp1，以确定Esrp1 KO导致CL/P的关键步骤和机制。2)确定Esrp在发育中的外胚层和腭上皮细胞中调控的选择性剪接和聚腺苷化的全球程序。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剪接、聚腺酰化和基因表达的全基因组变化，这些变化发生在外胚层和衍生物中Esrp表达缺失。3)使用交联免疫沉淀结合高通量测序（CLIP-Seq）确定全基因组Esrp结合位点。我们将确定Esrp1的全基因组结合位点，以确定Esrp调控的体内直接靶点。4)鉴定缺失导致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来测试在胚胎发育过程中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。

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.