Pbx-Directed Control of Cellular Behaviors that Drive Midface Morphogenesis

Pbx 定向控制驱动中面部形态发生的细胞行为

• 批准号: 10451656
• 负责人: Licia Selleri
• 金额: $ 57.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10451656
• 关键词: Antibodies; Apoptosis; Apoptotic; Behavioral Genetics; Bioinformatics; Biological Assay; Bromodeoxyuridine; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cells; Cephalic; Cleft Palate; Cleft lip with or without cleft palate; Congenital Abnormality; Development; Diagnostic; Effector Cell; Embryo; Epithelial; Epithelial Cells; Exhibits; Face; Fostering; Frontonasal Prominence; Genes; Genetic; Genetic Transcription; Grant; Histone H3; Human; Image; Immunofluorescence Immunologic; Impairment; In Situ Hybridization; Individual; Lip structure; Live Birth; Maxilla; Maxillary Prominence; Mediating; Mesenchyme; Microdissection; Molecular; Molecular Profiling; Morphogenesis; Mus; Neural Crest; Pathogenesis; Pathway interactions; Population; Pregnancy; Premaxillary palate; Process; Proliferating; Publishing; Quantitative Reverse Transcriptase PCR; Reporting; Research; Societies; Stains; Testing; Time; Tissues; Transcript; WNT5A gene; Work; base; cdc Genes; cell behavior; craniofacial; epithelial to mesenchymal transition; experimental study; in vivo; inhibitor; mouse model; mutant; orofacial cleft; palate repair; prenatal; progenitor; programs; repair strategy; single-cell RNA sequencing; spatiotemporal; tissue repair; transcription factor; transcriptome; wound healing

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.

项目摘要 人裂唇唇唇cle裂，有或没有left裂（Cl/p），这是最常见的颅面出生缺陷，是由IM-引起的 面部突出的配对融合。在正常形态发生期间，额骨和上颌偏向 Nences在三向接缝处熔断灯（l），形成上唇/原发性。如果是 覆盖阳性的lium持续存在于l，随之而来的是口面裂。 缺乏PBX转录因子（TFS）的胚胎。我们描述：1）突出融合需要坐标。 分别具有两种不同细胞行为的国家，分别 在L上皮内的两个离散亚群中； 2）这两个亚群以不同的特征 分子特征； 3）针对具有Cl/P的PBX TF的小鼠缺乏这两种上皮亚群。 我们在小鼠中的初步证据表明：i）λ上皮是异质的，构成6个主要 如单细胞RNA测序，在突出融合之前的细胞亚群。 ii）其中之一 我们称为“ l融合效应”的亚群，与与人有关的基因富含 对于编码促凋亡因子的基因以及细胞周期进展的抑制剂； iii）“ l融合效应子”细胞位于突出的尖端； iv）“ l融合效应子”的比例 与对照组相比，在具有Cl/P的化合物PBX1/2突变体胚胎中受到干扰。基于这些结果，我们 认为“ L Fusion效应子”群集组成的细胞是重大融合的主要执行者。被停止了 在细胞周期中，将随后的细胞变化（如细胞凋亡和EMT）实现融合的先决条件。和 在Cl/p的小鼠模型中进行定量和/或定性扰动。我们将通过 以下特定目的：1）确定上皮“ L融合效应器”的空间时间动力学 整个中间突出融合的亚群。我们将确定“ l融合效应子”是否clus- TER是瞬态的，或者在融合后持续存在，并且跟踪“ f融合效应器”的祖细胞和后代 细胞跨空间和发育时间。 2）建立细胞基础的分子机制 上唇/原发性粉状融合期间“ L融合效应器”细胞中的周期停滞。我们将在体内细胞中发现 “ L融合效应子”细胞的循环动力学，并评估PBX依赖性调节细胞周期基因 介导该群集中的细胞周期控制。 3）确定“λ融合效应器”的扰动是否 PBX1/2突变体中的簇在具有Cl/p的P63-或BMPR1A缺陷小鼠中概括。我们将建立 L上皮的细胞和转录变化是否导致PBX1/2突变体中Cl/p 在所有三种鼠标模型中概括，或者如果它们不同。这项研究将导致发现新基因 CL/P的产前诊断和通过重新激活发育计划的CL/P修复的开放策略 在口面上有缺陷。广义上，这项工作将培养机械研究，以测试其他是否 融合过程是通过细胞周期停滞介导的。