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.

项目摘要 对于许多患有患者的患者，再生医学技术已成为 治愈肌肉骨骼损伤不良。从干细胞疗法中观察到的益处部分归因于 他们在受伤组织中引发适当的细胞通信的旁分泌作用。尽管很棒 在了解组织模式期间空间提示的分子对照方面的步伐，这是一个主要的知识差距 关于计时在协调组织反应中的作用。涉及的时间保存基因 分子时钟主要组织为同步细胞，尤其是在昼夜节奏中。但是， 计时基因的振荡性也可能使它们有助于多能细胞的性质 激活受伤。尽管有证据表明时钟基因已被证明在同步细胞中起作用 肠部再生期间的状态，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，时钟系统跨多种细胞类型的作用尚不清楚。这个建议 旨在发现和利用发育过程中时钟基因与再生之间的关系， 控制爪蟾laevis中组织再生的速度和能力的目的。爪蟾幼虫是 能够尾部再生和表现出再生和再生 - 不足的发育阶段， 使它们成为询问组织修复过程的有用模型。 Xenopus也很快发展 体内，可以轻松监测形态和遗传操作的结果。虽然哺乳动物 具有更有限的自我再生能力，了解计时基因在Xenopus中的作用 提供有关我们如何控制人类组织再生的重要见解。在AIM 1中，生物学时间 - 保持机械的特征是在再生和非再生异武尾尾的细胞中使用 体内DNA记者和单细胞转录组学。单细胞转录组学将用于定义哪个单元 类型是时间的驱动力，以保持流程以维持超过40个单元格的集体行动 在尾部再生期间。 AIM 2将决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级别的计时控制来进行再生速度。再生 使用小分子处理对时钟基因网络进行扩增或阻尼时，将评估容量。 CRISPR/CAS9时钟基因的过表达和敲击将用于确定五个核心时间的作用 保持已知会影响其他组织再生的基因。小分子和时钟基因的影响 将使用单细胞测序监测细胞同步景观上的敲除。这个项目将 成为第一个在全limb再生中紧密表征时钟系统的人，并可能导致更多的见解 为了改善整合过程中的组织修复和重编程细胞，例如在临床干细胞和 活组织移植。