Pbx-Directed Control of Cellular Behaviors that Drive Midface Morphogenesis

 描述（由申请人提供）：唇腭裂（CL/P）是最常见的人类颅面出生缺陷（1/700出生）。小鼠提供了一个合适的模型来研究人类颅面形态发生及其异常，这仍然知之甚少。据报道，一个独特的细胞行为称为上皮间充质转化（EMT）介导的颅面隆起融合在鸡。然而，潜在的机制尚未确定，它仍然是未知的EMT是否控制哺乳动物上唇形态发生。我们的证据表明：1）在小鼠胚胎中，除了细胞凋亡，突起接缝处的上皮可塑性介导组织重塑和融合; 2）该过程依赖于Pbx转录因子（TF）; 3）Pbx突变接缝处的上皮细胞失去Snail 1和核Smad 3/4（关键EMT介质）的表达; 4）Pbx转录因子与Snail 1和Smad 3潜在的调控元件结合，如面中部组织的染色质免疫沉淀（ChIP）所示。此外，全基因组序列分析的Pbx 1结合区域在小鼠的脸确定其他潜在的Pbx靶基因与上皮可塑性，EMT，细胞粘附和迁移。鉴于这些结果，我们认为，除了控制细胞凋亡，Pbx TF作为上皮可塑性或EMT在接缝的新的调节剂。我们还证实，Pbx TF伴随控制密切相关的细胞行为，即细胞粘附和迁移，在小鼠面中部形态发生。首先，我们将确定是否局部上皮可塑性或完整的EMT介导的组织重塑和突出融合的接缝。为此，我们将使用我们的Pbx缺陷小鼠系定义Pbx在胚胎头部上皮可塑性控制中的要求。然后，我们将研究Pbx TF是否是必要的，也足以驱动上皮可塑性或使用NMuMG上皮细胞完成EMT，EMT的经典模型。其次，我们将确定Snail 1和Smad 3是否受Pbx TF在中面形态发生中的调控。为了实现这一目标，我们将在胚胎细胞中进行瞬时转染，并在小鼠中进行瞬时转基因。第三，我们将描绘小鼠面部形态发生中上皮可塑性、EMT、细胞粘附和迁移的Pbx定向效应器的全面调节网络。为此，我们将通过对野生型和Pbx突变型头上皮的转录组测定，确定哪些Pbx 1结合增强子通过ChIP-Seq在面中部组织上鉴定，从而调节Pbx靶基因。我们将在体内验证选定的新型Pbx调控基因，优先考虑候选人Zeb 1和Zeb 2以及Zpo 1和Zpo 2的当前发现，所有这些都是EMT，细胞粘附或迁移的效应子。这项研究将确定新型颅面顺式调节元件和驱动相互关联的细胞行为的Pbx定向网络，这些行为对面部形态发生至关重要，并揭示用于纳塔尔诊断CL/P的新Pbx靶基因。对头部发育中上皮行为的基本机制的研究将应用于依赖于相同细胞行为的其他过程，例如，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.