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

项目摘要/摘要 胸腺上皮细胞(TECs)提供生长和分化信号，这是胸腺细胞 发展具有多样性但自我耐受的TCR谱系的T细胞。TEC的数量增加 随着胸腺的生长以支持输出的幼稚T细胞的增加，新生儿的胸腺呈指数级增长 来定植外周淋巴器官。围产期是TEC亚群动态变化的时期 组成、分化、组织和功能。尽管围产期胸腺生长转换为 幼年胸腺的动态平衡是建立良好的，在组成上发生的具体变化 转录上不同的TEC亚群和识别最初促进和 因此，对TEC间隔部的极限扩张和重塑知之甚少。此外， 发生这种分子重编程事件的TEC前体还没有定义。我们的预赛 研究表明，Cyclin D1-视网膜母细胞瘤(Rb)-E2F通路是调控TEC的关键分子开关 在围产期向成人期过渡过程中的增殖。我们已经证明，围产期TEC的扩张 通过删除RB家族成员或通过表达角蛋白5可以将间隔保持到成年期 驱动Cyclin D_1(K5.D_1)转基因灭活Rb功能。我们的总体假设是围产期和 幼年TEC表达不同的转录谱，协调TEC的增殖和分化，以及 Cyclin D1-Rb-E2F通路调控这两个过程。我们提出了一系列实验，以检验 这一假说为目前尚未解决的有关围产期TEC的问题提供了新的见解 车厢。这些问题包括：1)TEC子集的组成在 围产期到青少年的转变；2)TEC亚群的转录签名是如何在不同时期变化的 转变；3)什么分子机制和途径调节这些变化；4)什么是身份和 过渡期间TEC祖细胞的谱系等级；以及5)具有可比性的细胞和分子 人类的TEC发生了变化。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来确定转录图谱的变化，并使用高级成像来定位地图 在围产期到幼年期的转变过程中转录上不同的TEC亚群。并行scRNA-seq和 成像分析将在人类TECs和胸腺组织上进行。在目标2中，我们将在体外和在 体内实验直接测试Aim 1中确定的候选TEC前体细胞的分化潜能 3我们将确定调控TEC增殖和分化的分子途径是否 通过进行蛋白质组筛选和芯片序列分析进行协调连接。

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.