Harnessing intrinsic cell clocks to control growth & regeneration

项目摘要 再生医学技术已经成为许多患者的重要选择 肌肉骨骼损伤愈合不良。从干细胞疗法中观察到的益处在一定程度上归因于 它们的旁分泌活动在受损组织中启动了适当的细胞间通讯。尽管很棒 在理解组织构图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差距 存在关于计时在协调的组织反应中的作用的问题。时间守恒基因参与了 分子钟主要是用来同步细胞的，特别是在昼夜节律中。然而， 计时基因的振荡性质也可能使它们对多能细胞的特性做出贡献 一旦受伤就会被激活。尽管有证据表明时钟基因在细胞同步化中发挥作用 肠道再生过程中的状态，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，时钟系统在多种细胞类型中的作用尚不清楚。这项建议 旨在揭示和利用时钟基因和发育过程中的再生之间的关系， 控制非洲爪哇组织再生的速度和能力的目标。非洲爪哇幼虫是 能够再生尾巴，并表现出再生和再生能力不强的发育阶段， 使它们成为询问组织修复过程的有用模型。非洲爪哇也发展得很快， 活体，允许容易地监测形态和遗传操作的结果。尽管哺乳动物 具有更有限的自我再生能力，了解非洲爪哇计时基因的作用可以 为我们如何控制人类的组织再生提供重要的见解。在目标1中，生物时间- 保存机械将在再生和非再生的非洲爪哇尾巴细胞中使用 体内DNA记者和单细胞转录组。单细胞转录学将被用来定义哪个细胞 类型(S)作为计时过程的驱动力，维持超过40种细胞类型的集体行动 在尾巴再生期间。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加速再生。再生性 当时钟基因网络使用小分子处理被放大或抑制时，容量将被评估。 CRISPR/Cas9时钟基因的过表达和下调将被用来确定五个核心时间的作用- 保留已知的影响其他组织再生的基因。小分子与时钟基因的作用 将使用单细胞测序来监测细胞同步环境中的基因敲除。这个项目将 是第一个密切描述整个肢体再生时钟系统的人，可能会带来更大的洞察力 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。

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.