Pbx-Directed Control of Cellular Behaviors that Drive Midface Morphogenesis

Pbx 定向控制驱动中面部形态发生的细胞行为

基本信息

批准号：
8964584
负责人：
Licia Selleri
金额：
$ 10.3万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2015-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8964584
关键词：
Adhesions Adult Apoptosis Automobile Driving Behavior Binding Biological Assay Biological Process Birth Boxing Candidate Disease Gene Caring Cell Adhesion Cell Culture System Cell-Cell Adhesion Cells Cephalic Chromatin Cleft Lip Cleft Palate Cleft lip with or without cleft palate Complement Congenital Abnormality Development Developmental Process Diagnostic Elements Embryo Enhancers Epithelial Epithelial Cells Epithelium Face Frontonasal Prominence Gene Expression Profile Gene Targeting Gene Transfer Techniques Genes Genetic Growth Head Human Immigration Immunofluorescence Immunologic Individual Investigation Knowledge Lateral Lead Mammals Maps Maxillary Prominence Medial Mediating Mediator of activation protein Mesenchymal Modeling Molecular Profiling Morphogenesis Mus Nasal Prominence Nuclear Nucleic Acid Regulatory Sequences Palate Personal Communication Plastics Process Proteins Regulatory Element Regulatory Pathway Reporting Research Sequence Analysis Societies Structure System Testing Tissues Transfection Tumor Cell Invasion Validation Zinc Fingers base blastomere structure cell behavior cell motility chromatin immunoprecipitation craniofacial driving behavior embryonic cell culture epithelial to mesenchymal transition genome sequencing homeodomain in vivo lip morphogenesis migration mutant novel prenatal prevent public health relevance repaired social transcription factor

项目摘要

DESCRIPTION (provided by applicant): Clefts of the lip and/or palate (CL/P) are the most common human craniofacial birth defect (1/700 births). The mouse offers a suitable model to study human craniofacial morphogenesis and its abnormalities, which remain poorly understood. It was reported that a unique cellular behavior known as Epithelial Mesenchymal Transition (EMT) mediates craniofacial prominence fusion in the chick. However, the underlying mechanisms were not determined and it remains unknown whether EMT controls upper lip morphogenesis in mammals. Our evidence indicates that: 1) in the mouse embryo, in addition to apoptosis, epithelial plasticity at the prominence seam mediates tissue remodeling and fusion; 2) this process is dependent upon Pbx transcription factors (TFs); 3) epithelial cells at the Pbx mutant seam lose expression of Snail1 and nuclear Smad3/4, critical EMT mediators; and 4) Pbx TFs bind to Snail1 and Smad3 potential regulatory elements, as demonstrated by Chromatin immunoprecipitation (ChIP) on midface tissue. Moreover, whole-genome sequence analysis of Pbx1-bound regions in the murine face identifies additional potential Pbx target genes associated with epithelial plasticity, EMT, cell adhesion, and migration. In view of these results, we posit that, in addition to controlling apoptosis, Pbx TFs act as novel regulators of epithelial plasticity or EMT at the seam. We also posit that Pbx TFs concomitantly control closely related cellular behaviors, namely cell adhesion and migration, in mouse midface morphogenesis. First, we will determine whether local epithelial plasticity or complete EMT mediates tissue remodeling and prominence fusion at the seam. To this end, we will define the requirement for Pbx in the control of epithelial plasticity in the embryonic head using our Pbx-deficient mouse lines. Then we will investigate whether Pbx TFs are necessary and also sufficient to drive epithelial plasticity or complete EMT using NMuMG epithelial cells, a classic model of EMT. Second, we will establish whether Snail1 and Smad3 are regulated by Pbx TFs in midface morphogenesis. To achieve this, we will perform transient transfections in embryonic cells and transient transgenesis in the mouse. Third, we will delineate a comprehensive regulatory network of Pbx-directed effectors of epithelial plasticity, EMT, cell adhesion and migration in murine face morphogenesis. To this end, we will determine which Pbx1-bound enhancers identified by ChIP-Seq on midface tissue regulate Pbx target genes by transcriptome assays on wild type and Pbx-mutant cephalic epithelium. We will validate selected novel Pbx-regulated genes in vivo, with priority based on current findings for candidates Zeb1 & Zeb2 and Zpo1 & Zpo2, all of which are effectors of EMT, cell adhesion, or migration. This re- search will identify novel craniofacial cis-regulatory elements and Pbx-directed networks driving interconnected cellular behaviors that are critical for face morphogenesis, as well as revealing new Pbx target genes for pre- natal diagnostics of CL/P. Investigations on basic mechanisms underlying epithelial behaviors in head development will have applications to other processes that rely on the same cell behaviors, e.g., tumor invasion.

描述（由申请人提供）：唇裂和/或腭裂（CL/P）是最常见的人类颅面部出生缺陷（1/700 出生），小鼠为研究人类颅面部形态发生及其异常提供了合适的模型。据报道，一种称为上皮间质转化（EMT）的独特细胞行为介导颅面突出融合。然而，潜在的机制尚未确定，并且仍不清楚 EMT 是否控制哺乳动物的上唇形态发生。我们的证据表明：1) 在小鼠胚胎中，除了细胞凋亡之外，突出缝处的上皮可塑性还介导组织重塑和发育。融合；2) 该过程依赖于 Pbx 转录因子 (TF)；3) Pbx 突变接缝处的上皮细胞失去 Snail1 和核 Smad3/4 的表达，这一点至关重要EMT 介质；4) Pbx TF 与 Snail1 和 Smad3 潜在调控元件结合，如中面部组织染色质免疫沉淀 (ChIP) 所示。此外，对小鼠面部 Pbx1 结合区域的全基因组序列分析发现了其他潜在的 Pbx 靶点。与上皮可塑性、EMT、细胞粘附和迁移相关的基因鉴于这些结果，我们认为，除了控制细胞凋亡之外，Pbx TF 作为接缝处上皮可塑性或 EMT 的新型调节剂，我们还假设 Pbx TF 同时控制小鼠中面部形态发生中密切相关的细胞行为，即细胞粘附和迁移。介导接缝处的组织重塑和突出融合为此，我们将定义 Pbx 在控制上皮可塑性方面的要求。然后我们将研究 Pbx TF 是否是必要且足以驱动上皮可塑性或使用 NMuMG 上皮细胞（EMT 的经典模型）完成 EMT。为了实现这一点，我们将在胚胎细胞中进行瞬时转染，并在小鼠中进行瞬时转基因。描绘小鼠面部形态发生中上皮可塑性、EMT、细胞粘附和迁移的 Pbx 定向效应器的综合调控网络为此，我们将确定 ChIP-Seq 在中面部组织上鉴定的哪些 Pbx1 结合增强子通过调节 Pbx 靶基因。我们将优先根据当前的发现，在体内验证选定的新型 Pbx 调节基因。对于候选者 Zeb1 和 Zeb2 以及 Zpo1 和 Zpo2，它们都是 EMT、细胞粘附或迁移的效应器。这项研究将确定新的颅面顺式调节元件和 Pbx 导向的网络，驱动对面部至关重要的互连细胞行为。形态发生，以及揭示用于 CL/P 产前诊断的新 Pbx 靶基因。对头部发育中上皮行为的基本机制的研究将应用于其他领域。依赖相同细胞行为的过程，例如肿瘤侵袭。