Pbx, a Novel Regulator of EMT in Midface Morphogenesis

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），唇腭裂（CL/P）对社会有相当大的影响。治疗是密集和长期的，因为外科手术是从3 - 6个月大到青春期进行的。牙科、矫正术和语言治疗一直持续到成年。目前，我们对CL/P中受干扰的细胞和分子事件的理解是不够的。上皮细胞向间充质细胞转化（EMT）是一种基本的细胞行为，被认为在胚胎颅面发育过程中腭融合中起关键作用。而 在鸡的EMT中，也已经在形成上唇的面部突起的融合中得到证实，但在哺乳动物中，它是否介导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。转录因子Pbx家族在颅面发育中起着重要作用。小鼠胚胎Pbx1和Pbx2（Pbx1/Pbx2）缺陷显示CL/P与100%的突变率，并提供了一个新的模型，为人类CL/P。在这里，小鼠胚胎与条件损失的Pbx1在头部上皮的Pbx2缺陷的背景将被用来建立CL/P在体内的细胞和分子机制。初步数据显示：1）Pbx1/Pbx2缺失导致额鼻突处上皮接缝的持续存在，其不融合并产生CL/P; 2）接缝处的细胞通常表现出间充质标志物波形蛋白，而在Pbx1/Pbx2突变体中它们不表现出; 3）在上皮细胞培养系统中，Pbx1在TGF β介导的EMT期间上调;以及4）Pbx1在上皮细胞中的过表达在培养物中产生EMT表型。鉴于这些结果，我们假设Pbx1在面中部形态发生中作为EMT的一种新的调节因子。为了验证这一假设，Pbx1的EMT在唇形态发生/融合的要求将在体内解剖在细胞（Aim1A）和分子水平（Aim1B）。将通过电子显微镜检查、间充质和上皮标志物的免疫荧光以及小鼠胚胎上皮细胞的遗传命运作图来确定Pbx 1缺失对上皮接缝连接处细胞形态和身份的影响。然后将通过用Pbx 1特异性抗体对胚胎中面进行染色质免疫沉淀来确定Snail 1（EMT的关键效应子）是否是Pbx 1在接缝连接处的体内靶点。Pbx 1结合Snail 1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）来评估EMT的细胞系统中的Pbx1需求，以揭示Pbx1是否足以和/或必需诱导上皮细胞中的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。

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.