Project 1 - Molecular mechanisms controlling TEC dynamics and lineage hierarchies in the perinatal thymus

项目 1 - 围产期胸腺中控制 TEC 动态和谱系层次的分子机制

• 批准号: 10689287
• 负责人: Ellen R Richie
• 金额: $ 48.34万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10689287
• 关键词: Adolescent; Adult; Affinity; Age; Antigens; Atlases; Binding Proteins; Binding Sites; Bioinformatics; Biological Assay; Biometry; CDK4 gene; Cell Compartmentation; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Differentiation process; Cell Lineage; Cellular Structures; ChIP-seq; Characteristics; Chromatin; Coupled; Cyclin D1; Data; Development; Differentiation and Growth; Epigenetic Process; Epithelial Cell Proliferation; Event; Family member; Gene Expression Profile; Gene Expression Profiling; Gene Family; Generations; Genetic; Genetic Transcription; Goals; Growth; Homeostasis; Human; Image; In Vitro; Kidney; Knowledge; Link; Location; Maps; Mediating; Molecular; Mus; Newborn Infant; Organ Culture Techniques; Organizational Change; Pathway interactions; Perinatal; Process; Production; Proliferating; Proteomics; Regulatory T-Lymphocyte; Reporting; Resolution; Retinoblastoma; Self Tolerance; Series; Signal Pathway; Signal Transduction; T-Cell Development; T-Lymphocyte; T-cell receptor repertoire; Testing; Thymic Tissue; Thymic epithelial cell; Thymocyte Selection; Thymus Gland; Time; Tissues; Transgenes; Transplantation; autoreactivity; candidate identification; capsule; epithelial stem cell; experimental study; imaging approach; in vivo; in vivo evaluation; insight; keratin 5; multiplexed imaging; perinatal period; peripheral lymphoid organ; progenitor; programs; promoter; single-cell RNA sequencing; transcriptome sequencing

Project Summary/Abstract Thymic epithelial cells (TECs) provide growth and differentiation signals that are indispensable for the development of T cells with a diverse, yet self-tolerant TCR repertoire. The number of TECs increases exponentially in newborns as the thymus grows to support increased production of naïve T cells that are exported to colonize peripheral lymphoid organs. The perinatal period is a time of dynamic changes in TEC subset composition, differentiation, organization and function. Although the conversion of perinatal thymus growth to juvenile thymus homeostasis is well established, the specific changes that occur in the composition of transcriptionally distinct TEC subsets and identification of signaling pathways that initially promote and subsequently limit expansion and remodeling of the TEC compartment are poorly understood. Furthermore, the TEC progenitors in which such molecular re-programming events occur have not been defined. Our preliminary data suggest that the Cyclin D1-retinoblastoma (RB)-E2F pathway is a key molecular switch that modulates TEC proliferation during the perinatal to adult transition. We have shown that expansion of the perinatal TEC compartment can be maintained into adulthood by deleting Rb family members or by expressing a keratin 5 driven Cyclin D1 (K5.D1) transgene to inactivate RB function. Our overall hypothesis is that perinatal and juvenile TECs express distinct transcriptional profiles that coordinate TEC proliferation and differentiation, and that the Cyclin D1-RB-E2F pathway regulates both processes. We propose a series of experiments that will test this hypothesis and provide new insights into currently unresolved questions regarding the perinatal TEC compartment. These questions include: 1) what changes occur in the composition of TEC subsets during the perinatal to juvenile transition; 2) how do the transcriptional signatures of TEC subsets change across the transition; 3) what molecular mechanisms and pathways regulate these changes; 4) what are the identities and lineage hierarchies of TEC progenitors during the transition; and 5) do comparable cellular and molecular changes occur in human TECs. The overall goal of RP1 is to resolve these knowledge gaps. In Aim 1, we will use scRNA-seq to determine changes in the transcriptional profiles and advanced imaging to map localization of transcriptionally distinct TEC subsets during the perinatal to juvenile transition. Parallel scRNA-seq and imaging analyses will be performed on human TECs and thymus tissue. In Aim 2 we will perform in vitro and in vivo assays to directly test the differentiation potential of candidate TEC progenitors identified in Aim 1. In Aim 3 we will determine whether molecular pathways that regulate TEC proliferation and differentiation are coordinately linked by performing proteomic screens coupled with ChIP- seq analyses.

项目概要/摘要 胸腺上皮细胞 (TEC) 提供生长和分化信号，这些信号是细胞生长所必需的。 具有多样化且自我耐受的 TCR 库的 T 细胞的发育。 TEC 数量增加 随着胸腺的生长以支持输出的幼稚 T 细胞产量的增加，新生儿中的 T 细胞数量呈指数级增长 定植于外周淋巴器官。围产期是TEC子集动态变化的时期 组成、分化、组织和功能。尽管围产期胸腺生长转变为 幼年胸腺的稳态已得到很好的建立，其成分发生的具体变化 转录上不同的 TEC 子集和信号通路的鉴定最初促进和 随后对 TEC 隔间的限制扩展和改造知之甚少。此外， 发生此类分子重编程事件的 TEC 祖细胞尚未确定。我们的初步 数据表明 Cyclin D1-视网膜母细胞瘤 (RB)-E2F 通路是调节 TEC 的关键分子开关 围产期到成人过渡期间的增殖。我们已经证明围产期 TEC 的扩展 通过删除 Rb 家族成员或表达角蛋白 5 可以将隔室维持到成年 驱动 Cyclin D1 (K5.D1) 转基因使 RB 功能失活。我们的总体假设是围产期和 幼年 TEC 表达独特的转录谱，协调 TEC 增殖和分化，并且 Cyclin D1-RB-E2F 通路调节这两个过程。我们提出了一系列的实验来测试 这一假设并为目前尚未解决的有关围产期 TEC 的问题提供了新的见解 隔间。这些问题包括：1）在 围产期到青少年的过渡； 2）TEC子集的转录特征如何在整个过程中发生变化 过渡; 3）哪些分子机制和途径调节这些变化； 4) 身份是什么以及 过渡期间 TEC 祖细胞的谱系层次结构； 5）进行可比较的细胞和分子研究 人类 TEC 发生变化。 RP1 的总体目标是解决这些知识差距。在目标 1 中，我们将 使用 scRNA-seq 确定转录谱的变化并使用高级成像来绘制定位图 围产期到青少年过渡期间转录上不同的 TEC 子集。并行 scRNA 测序和 将对人类 TEC 和胸腺组织进行成像分析。在目标 2 中，我们将在体外和体内进行 体内测定直接测试目标 1 中鉴定的候选 TEC 祖细胞的分化潜力。 3 我们将确定调节 TEC 增殖和分化的分子途径是否 通过执行蛋白质组筛选和 ChIP-seq 分析来协调连接。