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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要伴有或不伴有腭裂的唇裂（CL/P）是人类最常见的颅面出生缺陷。在胚胎发生过程中，中面部的内侧、外侧和上颌突出必须在称为 λ 的位置融合此前，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协调上皮溶解细胞凋亡和上皮间质转化；2) 在 PBX 基因缺陷的 CL/P 小鼠模型中上皮亚群不存在；3)仅去除了一半的上皮细胞这些过程促使我们的实验室进一步研究中面部是否存在新的亚群。在融合阶段，仅对小鼠中面部上皮进行单细胞 RNA 测序，在体内鉴定并验证了几个亚群，我发现其中一个亚群具有细胞周期停滞基因的独特转录组特征富集和细胞周期的耗竭基因转录本与小鼠和人类 CL/P 相关基因的进展表达一致我的体内分析发现该细胞亚群在时空上局限于该位点。 λ 融合，使我们将这些细胞命名为“λ 融合效应器”。与 CL/P 的发病机制有关，并且细胞周期停滞是一种尚未表征的机制与突出融合相关，以更好地了解这种上皮亚群和细胞周期的作用。在中面部融合过程中，我建议 1：评估和跟踪细胞周期进展以及 λ 融合中的停滞我会在野生型条件下上唇/初级腭形态发生和融合过程中的效应细胞。通过对小鼠胚胎进行 EdU/BrdU 双标记来评估体内亚群动态，我将使用 FUCCI 小鼠胚胎的外植体系统离体跟踪细胞周期阶段的变化 2：建立。上层时期λ融合效应细胞细胞周期停滞的分子机制我将分离和量化野生型条件下的唇/初级腭形态发生和融合。使用细胞计数方法从远端突出尖端检测效应细胞，并将识别 PBX1 基因靶标，重点关注细胞周期停滞基因，在 λ 融合效应细胞的分离亚群中与目标 3：确定在我们的 CL/P 小鼠模型中，胚胎是否存在复合损失。 Pbx1/2 表现出上皮 λ 融合效应子亚群细胞周期停滞的变化。使用 CL/P 量化和评估 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实现鉴于该亚群与人类 CL/P 的遗传联系，我希望该研究将为CL/P早期产前诊断、预防和治疗提供新靶点，并揭示细胞周期停滞机制对组织融合、修复和形态发生具有广泛影响。