Pbx-Directed Control of Cellular Behaviors that Drive Midface Morphogenesis

 描述(申请人提供)：唇腭裂(CL/P)是最常见的人类头面部出生缺陷(1/700出生)。小鼠为研究人类颅面形态发生及其异常提供了一个合适的模型，目前仍知之甚少。据报道，一种独特的细胞行为称为上皮间充质转化(EMT)，介导了雏鸡的头面部隆起融合。然而，其潜在的机制尚未确定，也尚不清楚EMT是否控制哺乳动物的上唇形态发生。我们的证据表明：1)在小鼠胚胎中，除了细胞凋亡外，隆起接缝处的上皮可塑性还介导组织重建和融合；2)这一过程依赖于PBX转录因子(TF)；3)PBX突变接缝处的上皮细胞失去了关键的EMT介体Snail1和核SMAD3/4的表达；以及4)PBX TF与Snail1和SMAD3潜在的调控元件结合，如面中部组织的染色质免疫沉淀(CHIP)所证明的那样。此外，对小鼠面部Pbx1结合区的全基因组序列分析确定了与上皮可塑性、EMT、细胞黏附和迁移相关的其他潜在的PBX靶基因。鉴于这些结果，我们假设，除了控制细胞凋亡外，PBX转录因子还可以作为缝隙上皮可塑性或EMT的新调节器。我们还假设，在小鼠面中部形态发生过程中，PBX转录因子同时控制密切相关的细胞行为，即细胞黏附和迁移。首先，我们将确定是局部上皮的可塑性还是完全的EMT介导了缝合处的组织重塑和隆起融合。为此，我们将使用我们的PBX缺陷小鼠品系来确定PBX在控制胚胎头部上皮可塑性方面的需求。然后，我们将研究PBX转录因子是否必要且足以驱动上皮可塑性或使用NMuMG上皮细胞完成EMT，NMuMG上皮细胞是EMT的经典模型。其次，我们将确定Snail1和Smad3在面中部形态发生过程中是否受PBX转录因子的调控。为了实现这一点，我们将在胚胎细胞中进行瞬时转基因，并在小鼠中进行瞬时转基因。第三，我们将描绘一个全面的调控网络，PBX导向的效应上皮细胞的可塑性，EMT，细胞黏附和迁移的小鼠面部形态发生。为此，我们将通过对野生型和PBX突变的头皮进行转录组分析，确定面中部组织上哪些Pbx1结合的增强子调控PBX靶基因。我们将在体内验证选定的PBX调控的新基因，优先考虑候选基因ZEB1和ZEB2以及Zpo1和Zpo2的最新发现，这些基因都是EMT、细胞黏附或迁移的效应者。这项研究将发现新的头面部顺式调控元件和PBX导向的网络，这些元件和网络驱动着对面部形态发生至关重要的相互连接的细胞行为，并揭示新的PBX靶基因用于CL/P的产前诊断。对头部发育中上皮行为的基本机制的研究将应用于其他依赖于相同细胞行为的过程，如肿瘤侵袭。

 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.