Pbx, a Novel Regulator of EMT in Midface Morphogenesis

描述(申请人提供)：唇裂伴或不伴腭裂(CL/P)作为最常见的头面部出生缺陷(1/500至1/1000活产儿)，在社会上具有相当大的影响。治疗是密集和延长的，因为外科手术是从3-6个月到青春期进行的。牙科、正畸和语言治疗一直持续到成年。目前，我们对CL/P中扰动的细胞和分子事件的了解还不够充分。上皮向间充质转化(EMT)是一种基本的细胞行为，被认为在胚胎颅面发育过程中对腭部融合起关键作用。而当 在雏鸡的EMT中也已经证明了形成上嘴唇的面部突起的融合，但关于它是否在哺乳动物中参与了这一过程还存在争议。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转录因子家族在颅面发育过程中发挥着重要作用。Pbx1和Pbx2基因缺失的小鼠胚胎(Pbx1/Pbx2)表现出100%的CL/P外显率，为人类CL/P提供了一个新的模型。本文将利用Pbx2基因缺失的小鼠头上皮条件缺失Pbx1的小鼠胚胎来建立体内CL/P的细胞和分子机制。初步数据强调：1)Pbx1/Pbx2缺失导致额鼻突上皮缝持续存在，不融合而产生CL/P；2)缝隙细胞正常显示间充质标记vientin，而在Pbx1/Pbx2突变体中不显示；3)在TGFb介导的上皮细胞培养系统中，Pbx1表达上调；以及4)Pbx1在上皮细胞中过表达使培养中的EMT表型。根据这些结果，推测Pbx1在面中部形态发生中可能作为一种新的EMT调节因子。为了验证这一假设，将在体内从细胞(Aim1A)和分子水平(Aim1B)剖析Pbx1在嘴唇形态发生/融合中对EMT的需求。Pbx1缺失对上皮缝接合处细胞形态和特性的影响将通过电子显微镜、间质和上皮标记的免疫荧光以及小鼠胚胎上皮细胞的遗传命运图谱来确定。然后，通过用Pbx1特异性抗体对胚胎中面进行染色质免疫沉淀，确定EMT的关键效应者Snail1是否是缝合处Pbx1的体内靶点。Pbx1结合Snail1表达的功能将通过在培养细胞中瞬时转染来建立。这些研究将确定Pbx1是否是胚胎中面中部活体EMT所必需的，以及它是否通过直接靶向Snail1来执行这一程序。同时，Pbx1在EMT细胞系统中的需求将使用过度表达(Aim2A)和敲除方法(Aim2B)进行评估，以揭示Pbx1是否足以和/或必须诱导上皮细胞发生EMT。通过这些研究获得的知识将改善CL/P的产前诊断，并推动未来用于修复的药理学和组织工程学方法。

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.