Pbx-Directed Control of Cellular Behaviors that Drive Midface Morphogenesis

项目总结 人类唇裂伴或不伴腭裂(CL/P)是最常见的头面部出生缺陷，由感染引起。 面部隆起的配对融合。在正常的形态发生过程中，额鼻和上颌前突- 神经融合在一个三向缝合处，即板状接合处(L)，形成上唇/主腭部。如果这句名言- 覆盖隆起的锂在L处持续存在，随后发生口裂，正如我们之前在小鼠身上报道的那样。 缺乏PBX转录因子(TF)的胚胎。我们描述了：1)日冕融合需要坐标-- 有两种截然不同的细胞行为的国家，分别是细胞凋亡和上皮向间充质转化(EMT)， 在L上皮内的两个离散的亚群中；2)这两个亚群具有不同的特征 分子特征；以及3)缺乏具有CL/P的PBX TFS的小鼠缺乏这两个上皮亚群。 我们在小鼠身上的初步证据表明：i)λ上皮是异质的，由6个主要的 单细胞RNA测序表明，日冕融合前的细胞亚群；ii) 我们称之为“L融合效应器”的亚群富含与人类相关的基因。 或小鼠口腔裂开，编码促凋亡因子的基因，以及细胞周期进展的抑制物； 3)“L融合效应”细胞位于日冕顶端；4)“L融合效应”细胞所占比例 与对照组相比，在具有CL/P的复合Pbx1/2突变胚胎中受到干扰。基于这些结果，我们 假设“L聚变效应”星团包含作为日冕聚变主要执行者的细胞；被叫停 在细胞周期中，随后的细胞变化的先决条件，如凋亡和EMT，以实现融合；以及 在CL/P小鼠模型中受到定量和/或定性的扰动。我们将通过 具体目标如下：1)测定上皮细胞“L融合效应器”的时空动力学 整个面中部突起融合的亚群。我们将确定“L融合效应器”是否属于-- TER是瞬变的还是融合后的持续存在，以及追踪器L融合效应器的祖先和后代 体内细胞跨越空间和发育时间。2)建立细胞的分子机制 “L融合效应”细胞在上唇/原腭部融合过程中的周期停滞。我们将在体内发现细胞 “L融合效应”细胞的周期动力学及PBX对细胞周期基因调控的研究 在这一簇中调节细胞周期控制。3)确定“λ聚变效应器”的摄动是否 Pbx1/2突变体中的簇在p63或BMPR1A缺陷的CL/P小鼠中被重写。我们将建立 在Pbx1/2突变体中，导致CL/P的L上皮的细胞和转录变化是否 在所有三种小鼠模型中概括，或者如果它们是不同的。这项研究将导致发现新的基因 CL/P的产前诊断和通过重新激活发育程序进行CL/P修复的开放策略 在口裂方面有缺陷的人。广泛地说，这项工作将促进机械研究，以测试其他 融合过程是通过细胞周期停滞来调节的。

PROJECT SUMMARY Human cleft lip with or without cleft palate (CL/P), the most common craniofacial birth defect, is caused by im- paired fusion of the facial prominences. During normal morphogenesis, the frontonasal and maxillary promi- nences fuse at a three-way seam, the lambdoidal junction (l), to form the upper lip/primary palate. If the epithe- lium that covers the prominences persists at the l, orofacial clefting ensues, as we previously reported in mouse embryos deficient for PBX transcription factors (TFs). We described that: 1) prominence fusion requires coordi- nation of two distinct cellular behaviors, apoptosis and epithelial-to-mesenchymal transition (EMT), respectively, in two discrete subpopulations within the l epithelium; 2) these two subpopulations are characterized by different molecular signatures; and 3) mice deficient for PBX TFs with CL/P lack both of these epithelial subpopulations. Our preliminary evidence in the mouse indicates that: I) the λ epithelium is heterogeneous and comprises 6 main cell subpopulations prior to prominence fusion, as demonstrated by single cell RNA sequencing; II) one of the subpopulations, that we termed “l fusion effectors”, is enriched for genes that have been associated with human or mouse orofacial clefting, for genes encoding pro-apoptotic factors, and for inhibitors of cell cycle progression; III) “l fusion effector” cells are located at the tip of the prominences; and IV) the proportion of “l fusion effectors” is perturbed in compound Pbx1/2 mutant embryos with CL/P compared to controls. Based on these results, we posit that the “l fusion effector” cluster comprises cells that are prime executors of prominence fusion; is halted in the cell cycle, a prerequisite for subsequent cellular changes, like apoptosis and EMT, to achieve fusion; and is quantitatively and/or qualitatively perturbed in mouse models of CL/P. We will test this hypothesis via the following Specific Aims: 1) Determine the spatiotemporal dynamics of the epithelial “l fusion effector” subpopulation throughout midface prominence fusion. We will establish whether the “l fusion effector” clus- ter is transient or if it persists after fusion, as well as track the progenitors and descendants of “l fusion effector” cells in vivo across space and developmental time. 2) Establish the molecular mechanisms underlying cell cycle arrest in “l fusion effector” cells during upper lip/primary palate fusion. We will uncover in vivo cell cycle dynamics of “l fusion effector” cells and assess whether PBX-dependent regulation of cell cycle genes mediates cell cycle control in this cluster. 3) Determine whether the perturbations of the “λ fusion effector” cluster in Pbx1/2 mutants are recapitulated in p63- or Bmpr1a-deficient mice with CL/P. We will establish whether the cellular and transcriptional changes of the l epithelium resulting in CL/P in Pbx1/2 mutants are recapitulated in all three mouse models, or if they are distinct. This research will lead to discover new genes for prenatal diagnostics of CL/P and open strategies for CL/P repair through reactivation of developmental programs that are defective in orofacial clefting. Broadly, this work will foster mechanistic studies testing whether other fusion processes are mediated by cell cycle arrest.