Pbx, a Novel Regulator of EMT in Midface Morphogenesis

Pbx，中面部形态发生中 EMT 的新型调节器

• 批准号: 9121534
• 负责人: James Charles Hart
• 金额: $ 6.72万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-29 至 2017-09-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9121534
• 关键词: Adolescence; Adult; Affect; Age-Months; Anterior nares; Antibodies; Apoptosis; Behavior; Bilateral; Binding; Biology; Birth; Candidate Disease Gene; Cell Culture System; Cell Line; Cell Nucleus; Cell model; Cells; Cellular Morphology; Cephalic; Chromatin; Cleft Palate; Cleft lip with or without cleft palate; Congenital Abnormality; Cultured Cells; Data; Dental; Dentistry; Development; Developmental Process; Diagnostic; Differentiation and Growth; Electron Microscopy; Embryo; Embryonic Development; Epithelial; Epithelial Cells; Epithelium; Event; Exhibits; Face; Family; Future; Genes; Genetic; Genetic Markers; Growth; Health; Human; Immunofluorescence Immunologic; Individual; Knowledge; Laboratories; Lateral; Lentivirus Vector; Light; Lip structure; Live Birth; Long-Term Care; Maintenance; Mammals; Maps; Maxilla; Medial; Mediating; Mesenchymal; Modeling; Molecular; Molecular Genetics; Morphogenesis; Mus; Neoplasm Metastasis; Nose; Operative Surgical Procedures; Orthodontic; Orthodontics; Penetrance; Phenotype; Play; Population; Precipitation; Prevention; Procedures; Process; Psychology; Regulatory Element; Reporting; Role; Scanning Electron Microscopy; Societies; Specialist; Speech Pathology; Speech Therapy; System; Testing; Tissue Engineering; Transfection; Transforming Growth Factor beta; Transmission Electron Microscopy; Treatment/Psychosocial Effects; Tumor Cell Invasion; Vertebrates; Vimentin; craniofacial; craniofacial development; epithelial to mesenchymal transition; homeodomain; improved; in vivo; knock-down; lip morphogenesis; migration; molecular marker; mutant; novel; overexpression; palatal fusion; palate repair; prenatal; programs; repaired; research study; small hairpin RNA; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): As the most common craniofacial birth defect (1/500 to 1/1000 live births), cleft lip with or without cleft palate (CL/P) has a considerable impact on society. Treatment is intensive and prolonged, as surgical procedures are carried out from 3-6 months of age into adolescence. Dentistry, orthodontics and speech therapy continue into adulthood. Currently, our understanding of the cellular and molecular events perturbed in CL/P is inadequate. Epithelial to Mesenchymal Transition (EMT) is a fundamental cellular behavior believed to play a critical role in palatal fusion during embryonic craniofacial development. While in the chick EMT has also been demonstrated in the fusion of the facial processes that form the upper lip, it is debated whether it mediates this process in mammals. Because mice and humans share similar morphogenetic processes during craniofacial development, the mouse is a suitable model to study craniofacial morphogenesis and its abnormalities. The Pbx family of transcription factors (TFs) play critical roles in craniofacial development. Mouse embryos deficient for Pbx1 and Pbx2 (Pbx1/Pbx2) display CL/P with 100% penetrance and offer a new model for human CL/P. Here, mouse embryos with conditional loss of Pbx1 in the cephalic epithelium on a Pbx2-deficient background will be used to establish the cellular and molecular mechanisms underlying CL/P in vivo. Preliminary data highlight that: 1) Pbx1/Pbx2 loss causes persistence of the epithelial seams at the frontonasal processes, which do not fuse and yield CL/P; 2) Cells at the seams normally exhibit the mesenchymal marker vimentin, whereas in Pbx1/Pbx2 mutants they do not; 3) Pbx1 is upregulated during TGFb-mediated EMT in an epithelial cell culture system; and 4) Pbx1 over-expression in epithelial cells gives EMT phenotypes in culture. Given these results, it is hypothesized that Pbx1 acts as a novel regulator of EMT in midface morphogenesis. To test this hypothesis, Pbx1 requirements for EMT in lip morphogenesis/fusion will be dissected in vivo at the cellular (Aim1A) and molecular level (Aim1B). The effect of Pbx1 loss on cellular morphology and identity at the epithelial seam junction will be established by Electron Microscopy, Immunofluorescence with mesenchymal and epithelial markers, and genetic fate mapping of epithelial cells in mouse embryos. It will then be determined whether Snail1, a critical effector of EMT, is an in vivo target of Pbx1 at the seam junction by Chromatin Immuno Precipitation of embryonic midfaces with Pbx1 specific antibodies. Functionality of Pbx1 binding on Snail1 expression will be established by transient transfections in cultured cells. These studies will establish whether Pbx1 is required for EMT in vivo in the embryonic midface and if it executes this program by directly targeting Snail1. In parallel, Pbx1 requirements in a cellular system of EMT will be assessed using over-expression (Aim2A) and knock-down approaches (Aim2B) to uncover whether Pbx1 is sufficient and/or necessary to induce EMT in epithelial cells. Knowledge gained thorough these studies will improve prenatal diagnostics of CL/P and drive future pharmacological and tissue engineering approaches for repair.

描述（由申请人提供）：作为最常见的颅面出生缺陷（1/500至1/1000活产），带有或不带有left裂（Cl/p）的裂口对社会产生了相当大的影响。由于手术程序从3-6个月大至青春期进行，因此治疗是密集的，并且延长了。牙科，正畸和语音疗法一直持续到成年。当前，我们对Cl/P中扰动的细胞和分子事件的理解不足。间质转变（EMT）是一种基本的细胞行为，被认为在胚胎颅面发育过程中起着至关重要的作用。尽管 在小鸡EMT中，在形成上唇的面部过程的融合中也证明了这一点，是否在哺乳动物中介导了这一过程。由于小鼠和人类在颅面发育过程中具有相似的形态发生过程，因此小鼠是研究颅面形态发生及其异常的合适模型。 PBX转录因子（TFS）家族在颅面发育中起关键作用。缺乏PBX1和PBX2（PBX1/PBX2）的小鼠胚胎显示100％渗透率的CL/P，并为人类CL/p提供了新的模型。在这里，将使用PBX2缺陷型背景上的头皮上皮中有条件损失PBX1的小鼠胚胎来建立体内Cl/p下的细胞和分子机制。初步数据强调：1）PBX1/PBX2损失会导致上皮接缝在额叶过程中的持久性，而额叶过程不融合并产生Cl/p； 2）接缝处的细胞通常表现出间充质标记波形蛋白，而在PBX1/PBX2突变体中，它们没有； 3）在上皮细胞培养系统中TGFB介导的EMT期间，PBX1被上调； 4）上皮细胞中的PBX1过表达在培养中提供EMT表型。鉴于这些结果，可以假设PBX1在中表面形态发生中充当EMT的新调节剂。为了检验这一假设，将在细胞（AIM1A）和分子水平（AIM1B）的体内剖析唇形形态发生/融合中EMT的PBX1要求。 PBX1丧失对上皮接头的细胞形态和身份的影响将通过电子显微镜，具有间质和上皮标记物的免疫荧光以及小鼠胚胎中上皮细胞的遗传命运图的影响。然后，将确定EMT的关键效应子Snail1是否是通过具有PBX1特异性抗体的胚胎中间面的染色质免疫沉淀在接缝连接处PBX1的体内靶标。 PBX1结合在Snail1表达上的功能将通过培养细胞中的瞬时转染建立。这些研究将确定EMT在胚胎中心中的体内是否需要PBX1，并且是否通过直接靶向Snail1来执行该程序。同时，将使用过表达（AIM2A）和敲低方法（AIM2B）评估EMT细胞系统中的PBX1需求，以发现PBX1是否足够和/或必要以诱导上皮细胞中的EMT。知识获得了彻底的研究，这些研究将改善CL/P的产前诊断，并推动未来的药理和组织工程方法进行维修。