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：
9174502
负责人：
Licia Selleri
金额：
$ 32.08万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
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 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9174502
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（由适用提供）：嘴唇和/或pa裂（Cl/p）是最常见的人类颅面先天缺陷（1/700出生）。该小鼠提供了研究人类颅面形态发生及其异常的合适模型，这些模型仍然很少理解。据报道，一种称为上皮间充质转变（EMT）的独特细胞行为介导了雏鸡的颅面突出融合。但是，尚未确定潜在的机制，并且尚不清楚EMT是否控制哺乳动物中的上唇形态发生。我们的证据表明：1）在小鼠胚胎中，除凋亡外，在突出缝上的上皮可塑性还介导组织的重塑和融合； 2）此过程取决于PBX转录因子（TFS）； 3）PBX突变体接缝处的上皮细胞失去了蜗牛1和核Smad3/4的表达，即关键EMT介质； 4）PBX TFS与Snail1和Smad3潜在调节元件结合，如中心组织上的染色质免疫沉淀（CHIP）所证明。此外，鼠面部的PBX1结合区域的全基因组序列分析确定了与上皮可塑性，EMT，细胞粘合剂和迁移相关的其他潜在PBX靶基因。鉴于这些结果，我们肯定的是，除了控制凋亡外，PBX TFS是上皮可塑性或接缝处的EMT的新型调节剂。我们还阳性PBX TF在小鼠中表面形态发生中，PBX TF伴随控制密切相关的细胞行为，即细胞粘附和迁移。首先，我们将确定局部上皮可塑性或完整的EMT是否介导了接缝处的组织重塑和突出融合。为此，我们将使用我们的PBX缺陷小鼠系列来定义PBX在胚胎头上控制上皮可塑性中的要求。然后，我们将研究PBX TF是否需要，并且还足以使用Nmumg上皮细胞（一种经典的EMT模型）驱动上皮可塑性或完整的EMT。其次，我们将确定Snail1和Smad3是否受到中脸形态发生的PBX TF调节。为此，我们将在胚胎细胞中进行瞬时转染和小鼠中的瞬时转基因。第三，我们将描述一个综合的调节网络，这些网络是上皮可塑性，EMT，细胞粘合剂和鼠面临形态发生的迁移的PBX指导效应。为此，我们将通过野生型和PBX突变的头顶上皮细胞上的转录组分析来确定哪些PBX1结合的增强子在中间组织上通过ChIP-Seq鉴定出PBX靶基因。我们将在体内验证选定的新型PBX调节基因，并基于当前对Zeb1＆Zeb2和Zpo1＆ZPO2的候选结果的发现，所有这些都是EMT的影响，细胞粘附或迁移的作用。该重新搜索将确定新型的颅面顺式调节元件和PBX定向网络驱动相互联系的细胞行为，这对于面部形态发生至关重要，并揭示了新的PBX靶基因以用于CL/P的纳塔尔前诊断。研究头部发育中上皮行为的基本机制将对其他依赖相同细胞行为（例如肿瘤侵袭）的过程应用。