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: 8892814
• 负责人: James Charles Hart
• 金额: $ 6.5万
• 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/8892814
• 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; 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（申请人提供）：作为最常见的颅面出生缺陷（1/500至1/1000活产），唇裂伴或不伴腭裂（CL/P）对社会影响相当大。治疗强度大且时间长，因为外科手术从 3-6 个月大到青春期进行。牙科、牙齿矫正和言语治疗一直持续到成年。目前，我们对 CL/P 中受到干扰的细胞和分子事件的理解还不够。上皮向间质转化（EMT）是一种基本的细胞行为，被认为在胚胎颅面发育过程中的腭融合中发挥着关键作用。尽管 在小鸡中，EMT 也被证明存在于形成上唇的面部过程的融合中，但它是否在哺乳动物中介导这一过程存在争议。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 (TF) 家族在颅面发育中发挥着关键作用。缺乏 Pbx1 和 Pbx2 (Pbx1/Pbx2) 的小鼠胚胎显示出 100% 外显率的 CL/P，并为人类 CL/P 提供了新模型。在这里，在 Pbx2 缺陷背景下头颅上皮中 Pbx1 有条件丢失的小鼠胚胎将用于建立体内 CL/P 的细胞和分子机制。初步数据强调：1）Pbx1/Pbx2 丢失导致额鼻突上皮接缝持续存在，不会融合并产生 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 突变体中则没有； 3) 在上皮细胞培养系统中，TGFb介导的EMT过程中Pbx1上调； 4) 上皮细胞中 Pbx1 过度表达在培养物中产生 EMT 表型。鉴于这些结果，假设 Pbx1 在中面部形态发生中充当 EMT 的新型调节剂。为了检验这一假设，我们将在体内细胞水平 (Aim1A) 和分子水平 (Aim1B) 上剖析唇形态发生/融合中 Pbx1 对 EMT 的要求。 Pbx1 缺失对上皮缝连接处细胞形态和身份的影响将通过电子显微镜、间充质和上皮标记的免疫荧光以及小鼠胚胎中上皮细胞的遗传命运图谱来确定。然后通过使用 Pbx1 特异性抗体对胚胎中面进行染色质免疫沉淀，确定 Snail1（EMT 的关键效应子）是否是接缝处 Pbx1 的体内靶标。 Pbx1 结合 Snail1 表达的功能将通过培养细胞中的瞬时转染来确定。这些研究将确定 Pbx1 是否是胚胎中面部体内 EMT 所必需的，以及它是否通过直接靶向 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，以揭示 Pbx1 是否足以和/或必要以诱导上皮细胞中的 EMT。通过这些研究获得的知识将改善 CL/P 的产前诊断，并推动未来的药理学和组织工程修复方法。