Harnessing intrinsic cell clocks to control growth & regeneration

利用内在细胞时钟来控制生长

• 批准号: 10460131
• 负责人: Megan M Sperry
• 金额: $ 6万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460131
• 关键词: ARNTL gene; Address; Affect; Algorithms; Amputation; Arrhythmia; Biological; CRISPR/Cas technology; Cartilage; Cell Communication; Cell Reprogramming; Cells; Circadian Rhythms; Clinical; Cluster Analysis; Communication; Complex; Cues; DNA; Development; Developmental Biology; Disease; Enhancers; Epidermis; Epigenetic Process; Epithelium; Exhibits; Failure; Frequencies; Gene Expression; Genes; Genetic; Genetic Medicine; Goals; Growth; Human; Individual; Injury; Intestines; Knowledge; Larva; Learning; Maintenance; Mammals; Measures; Modeling; Molecular; Monitor; Morphology; Natural regeneration; Nature; Outcome; Output; Pacemakers; Patients; Pattern; Population; Process; Proliferating; Property; Regenerative Medicine; Regenerative capacity; Regenerative response; Regulation; Reporter; Reporter Genes; Role; Sampling; Speed; Structure; System; Tadpoles; Tail; Techniques; Therapeutic; Time; Tissue Transplantation; Tissues; Xenopus; Xenopus laevis; blastema; cartilage degradation; cartilage regeneration; cell type; gene network; gene therapy; genetic manipulation; healing; improved; in vivo; injured; insight; intestinal epithelium; knock-down; knockout gene; limb regeneration; loss of function; molecular clock; multipotent cell; musculoskeletal injury; nobiletin; novel; overexpression; paracrine; regenerative; regenerative cell; response; response to injury; single cell analysis; single cell sequencing; single-cell RNA sequencing; small molecule; stem cell division; stem cell therapy; stem cells; tissue regeneration; tissue repair; transcriptome sequencing; transcriptomics

Project Summary Regenerative medicine techniques have become an important option for the many patients suffering from poorly healing musculoskeletal injuries. Benefits observed from stem cells therapies have, in part, been attributed to their paracrine actions that initiate appropriate cell-cell communication in the injured tissue. Despite great strides in understanding molecular controls of spatial cues during tissue patterning, a major knowledge gap exists regarding the role of timekeeping in coordinated tissue responses. Time-keeping genes involved in the molecular clock are principally organized to synchronize cells, especially in the day-night rhythms. However, the oscillatory nature of timekeeping genes may also allow them to contribute to the properties of multipotent cells that activate upon injury. Despite evidence that clock genes have been shown to play a role in synchronizing cell states during intestinal regeneration and contribute to the regenerative capacity of basal epithelium and cartilage, the role of the clock system across multiple cell types during whole limb regeneration is unknown. This proposal aims to uncover and exploit the relationship between clock genes and regeneration during development, with the goal of controlling the speed and capacity for tissue regeneration in the Xenopus laevis. Xenopus larvae are capable of tail regeneration and exhibit regenerative and regeneration-incompetent developmental stages, making them a useful model for interrogating the process of tissue repair. Xenopus also develop quickly and ex vivo, permitting easy monitoring of morphology and the outcomes of genetic manipulation. Although mammals have a more limited capacity for self-regeneration, understanding the role of timekeeping genes in Xenopus can provide important insights into how we may control tissue regeneration in humans. In Aim 1, biological time- keeping machinery will be characterized in cells of the regenerative and non-regenerative Xenopus tail using in vivo DNA reporters and single-cell transcriptomics. Single-cell transcriptomics will be used to define which cell type(s) act as drivers of time keeping processes to maintain the collective actions of greater than 40 cell types during tail regeneration. Aim 2 will determine how the clock gene system affects regenerative capacity and the speed of regrowth by assessing timekeeping control at both network and single-gene levels. Regenerative capacity will be evaluated when the clock gene network is amplified or damped using small molecule treatments. CRISPR/Cas9 clock gene overexpression and knock downs will be used to determine the role of five core time- keeping genes known to affect regeneration in other tissues. The effects of small molecules and clock gene knockouts on the cell synchronization landscape will be monitored using single-cell sequencing. This project will be the first to closely characterize the clock system in whole-limb regeneration and may lead to greater insights for improving tissue repair and reprogramming cells during integration, like that required in clinical stem cell and living tissue transplantations.

项目概要 再生医学技术已成为众多患有此病的患者的重要选择 肌肉骨骼损伤愈合不良。从干细胞疗法中观察到的益处部分归因于 它们的旁分泌作用可以在受损组织中启动适当的细胞间通讯。尽管很棒 在理解组织图案化过程中空间线索的分子控制方面取得了长足进步，这是一个主要的知识差距 存在关于协调组织反应中计时的作用。计时基因参与 分子钟主要是为了使细胞同步，特别是在昼夜节律方面。然而， 计时基因的振荡性质也可能使它们有助于多能细胞的特性 受伤时激活。尽管有证据表明时钟基因在同步细胞中发挥作用 肠道再生过程中的状态并有助于基底上皮和软骨的再生能力， 在整个肢体再生过程中，多种细胞类型的时钟系统的作用尚不清楚。这个提议 旨在揭示和利用发育过程中的时钟基因与再生之间的关系， 控制非洲爪蟾组织再生的速度和能力的目标。爪蟾幼虫是 能够尾部再生并表现出再生和无再生能力的发育阶段， 使它们成为研究组织修复过程的有用模型。非洲爪蟾也发育得很快 体内，可以轻松监测形态和基因操作的结果。虽然哺乳动物 自我再生能力较为有限，了解非洲爪蟾计时基因的作用可以 为我们如何控制人类组织再生提供重要见解。在目标 1 中，生物时间- 保持机制将在再生和非再生爪蟾尾部细胞中进行表征，用于 体内 DNA 报告基因和单细胞转录组学。单细胞转录组学将用于定义哪些细胞 类型充当计时过程的驱动程序，以维持超过 40 种细胞类型的集体行动 尾巴再生期间。目标 2 将确定时钟基因系统如何影响再生能力和 通过评估网络和单基因水平的计时控制来评估再生速度。再生性 当使用小分子治疗放大或抑制时钟基因网络时，将评估其能力。 CRISPR/Cas9时钟基因过表达和敲低将用于确定五个核心时间的作用- 保留影响其他组织再生的基因。小分子和时钟基因的影响 将使用单细胞测序来监测细胞同步景观的敲除。该项目将 成为第一个详细描述全肢再生时钟系统特征的人，并可能带来更深入的见解 用于在整合过程中改善组织修复和重新编程细胞，就像临床干细胞和 活体组织移植。