Harnessing intrinsic cell clocks to control growth & regeneration

项目摘要 再生医学技术已成为许多患有癌症的患者的重要选择。 愈合不良的肌肉骨骼损伤从干细胞疗法中观察到的益处部分归因于 它们的旁分泌作用在受损组织中启动适当的细胞间通讯。虽然做了大量 在理解组织形成过程中空间线索的分子控制方面取得了进展，这是一个主要的知识空白 存在关于在协调的组织响应中计时的作用的问题。与时间相关的基因 分子钟主要是为了使细胞同步，特别是在昼夜节律方面。但 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 一旦受伤就会激活尽管有证据表明，时钟基因已被证明在同步细胞中发挥作用， 在肠再生过程中的状态，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，跨多种细胞类型的时钟系统的作用是未知的。这项建议 旨在揭示和利用生物钟基因与发育过程中再生之间的关系， 目的是控制非洲爪蟾组织再生的速度和能力。非洲爪蟾幼虫 能够尾部再生并表现出再生和不能再生的发育阶段， 使它们成为研究组织修复过程的有用模型。非洲爪蟾也发展迅速， 体内，允许容易地监测形态学和遗传操作的结果。但哺乳动物 自我再生的能力更为有限，了解非洲爪蟾中计时基因的作用， 为我们如何控制人体组织再生提供了重要的见解。在目标1中，生物时间- 保持机械将在再生和非再生爪蟾尾巴的细胞中使用， 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确定哪种细胞 类型充当计时过程的驱动器，以维持超过40种细胞类型的集体行动 在尾部再生过程中。目标2将确定时钟基因系统如何影响再生能力， 通过评估网络和单基因水平上的计时控制来评估再生速度。再生 当使用小分子处理扩增或衰减时钟基因网络时，将评估能力。 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- 保持已知影响其他组织再生的基因。小分子和时钟基因的作用 将使用单细胞测序监测细胞同步化景观上的敲除。该项目将 成为第一个密切表征全肢再生中的时钟系统的人，并可能导致更大的见解 在整合过程中改善组织修复和重编程细胞，如临床干细胞所需， 活体组织移植

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.