Cell cycle regulation in the cephalic epithelium as a driver of midface morphogenesis

头部上皮细胞周期调节作为中面部形态发生的驱动因素

• 批准号: 10605109
• 负责人: Brandon Hugh Chacon
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605109
• 关键词: Adult; Affect; Apoptosis; Behavioral Genetics; Bromodeoxyuridine; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cells; Cephalic; Cicatrix; Cleft Lip; Cleft Palate; Cleft lip with or without cleft palate; Congenital Abnormality; Cytometry; Dental; Development; Diagnostic; Dissection; Distal; Down-Regulation; Ectoderm; Effector Cell; Embryo; Embryonic Development; Epithelial; Epithelial Attachment; Epithelial Cells; Excision; Exhibits; Face; Failure; Flow Cytometry; Gene Expression; Gene Targeting; Genes; Genetic; Health; Heterogeneity; Human; Incentives; Individual; Infant; Label; Lateral; Lead; Link; Lip structure; Maxillary Prominence; Medial; Mesenchyme; Molecular; Molecular Profiling; Morphogenesis; Mus; Names; Nasal Prominence; Neural Crest; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Patients; Phenotype; Play; Population; Premaxillary palate; Prenatal Diagnosis; Prevention; Process; Research; Risk; Role; Site; Societies; Speech Pathology; Surface; System; Testing; Tissues; Transcript; United States; Up-Regulation; Validation; Wild Type Mouse; cell behavior; craniofacial; epithelial to mesenchymal transition; gene therapy; improved; in vivo; malformation; mouse model; mutant; novel; phase change; prenatal; prevent; repaired; single-cell RNA sequencing; tissue repair; transcriptomics

PROJECT SUMMARY Cleft lip with or without cleft palate (CL/P) is the most common craniofacial birth defect in humans. During embryogenesis, the medial, lateral, and maxillary prominences of the midface must fuse at a site called the λ junction to form the upper lip and primary palate. Previously, our lab discovered roles of the midface epithelium during fusion, including: 1) the coordination of epithelial dissolution via two discreet cell populations undergoing apoptosis and epithelial-to-mesenchymal transition; 2) in murine models of CL/P deficient in PBX genes these epithelial subpopulations are absent; and 3) only half of the epithelial cells removed were accounted for by these processes. This led our lab to investigate further the presence of novel subpopulations in the midface epithelium. In performing single cell RNA sequencing of mouse midface epithelium alone, during fusion stages, several subpopulations were identified and validated in vivo. I found that one subpopulation possessed a unique transcriptomic signature wherein enrichment for cell cycle arrest genes and depletion of cell cycle progression gene transcripts coincided with the expression of genes implicated in mouse and human CL/P pathogenesis. My in vivo analysis found this subpopulation of cells was spatio-temporally localized to the site of λ fusion, leading us to name these cells “λ fusion effectors.” I hypothesize that the λ fusion effectors are implicated in the pathogenesis of CL/P and that cell cycle arrest is a yet uncharacterized mechanism tied to prominence fusion. To better understand this epithelial subpopulation and the role that the cell cycle plays during midface fusion, I propose to 1: Assess and track cell cycle progression and arrest in λ fusion effector cells during upper lip/primary palate morphogenesis and fusion in wild type conditions. I will assess subpopulation dynamics in vivo by conducting EdU/BrdU dual labeling of mouse embryos and I will track cell cycle phase changes ex vivo by using an explant system from FUCCI mouse embryos. 2: Establish the molecular mechanisms underlying cell cycle arrest in λ fusion effector cells during upper lip/primary palate morphogenesis and fusion in wild type conditions. I will isolate and quantify λ fusion effector cells from the distal prominence tips using cytometry approaches and will identify PBX1 gene targets, with a focus on cell cycle arrest genes, in the isolated subpopulation of λ fusion effector cells versus neighboring λ epithelium. Aim 3: Determine if, in our mouse model of CL/P, embryos with compound loss of Pbx1/2 exhibit changes to cell cycle arrest in the epithelial λ fusion effector subpopulation. I will quantify and assess perturbations in the λ fusion effector cell subpopulation in Pbx1/2 mutants with CL/P. My research will investigate the molecular mechanism of cell cycle arrest during fusion of the λ junction and how it pertains to CL/P pathogenesis. Given the genetic links of this subpopulation to human CL/P, it is my hope that this study will provide novel targets for CL/P early prenatal diagnosis, prevention and treatment, and uncover mechanisms of cell cycle arrest with broad implications in tissue fusion, repair and morphogenesis.

项目总结 唇裂伴或不伴腭裂(CL/P)是人类头面部最常见的出生缺陷。在.期间 胚胎发生时，面中部的内侧、外侧和上颌突起必须在一个称为λ的位置融合。 形成上唇和主腭部的交界处。此前，我们的实验室发现了面中部上皮的作用 在融合过程中，包括：1)通过两个离散的细胞群经历 细胞凋亡与上皮向间充质转化；2)在PBX基因CL/P缺陷小鼠模型中 上皮亚群缺失；3)去除的上皮细胞中只有一半是由 这些过程。这使得我们的实验室进一步研究了面中部是否存在新的亚群 上皮组织。在仅对小鼠面中部上皮进行单细胞RNA测序时，在融合阶段， 在活体内鉴定和验证了几个亚群。我发现有一个亚群拥有一个 独特的转录转录特征，其中富含细胞周期停滞基因并耗尽细胞周期 进展基因转录本与小鼠和人类CL/P相关基因的表达一致 发病机制。我在体内的分析发现，这个细胞亚群在时空上定位于这个部位 我们将这些细胞命名为“λ融合效应器”。我假设λ融合效应器是 与CL/P的发病机制有关，细胞周期停滞是一种尚未确定的机制 与日冕融合有关。为了更好地了解这一上皮亚群以及细胞周期 在面中部融合中，我建议1：评估和跟踪λ融合中的细胞周期进展和停滞 野生型条件下上唇/初级腭部形态发生和融合过程中的效应细胞。这就做 通过对小鼠胚胎进行EDU/BrdU双重标记来评估体内亚群动态 使用Fucci小鼠胚胎的外植体系统在体外跟踪细胞周期的变化。2：建立 晚期λ融合效应细胞细胞周期停滞的分子机制 在野生型条件下唇部/初级腭部的形态发生和融合。我将分离并量化λ融合 使用细胞学方法从远端隆起尖端提取效应细胞，并将识别Pbx1基因靶点， 将重点放在细胞周期停滞基因上，在分离的λ融合效应细胞亚群中 邻近的λ上皮细胞。目的3：确定在我们的CL/P小鼠模型中，胚胎是否有复合性丢失 在上皮性λ融合效应亚群中，Pbx1/2显示细胞周期停滞的变化。这就做 量化和评估带有CL/P的PBX1/2突变体中λ融合效应细胞亚群的扰动 研究将探讨λ连接融合过程中细胞周期停滞的分子机制及其如何 与CL/P的发病机制有关。鉴于这个亚群与人类CL/P的遗传联系，我希望 本研究将为CL/P的产前早期诊断、防治和发现提供新的靶点 细胞周期停滞的机制在组织融合、修复和形态发生中具有广泛的意义。