Role of Energy Metabolism in Patterning the Vertebrate Musculo-Skeletal Axis

项目总结/摘要 拟议的项目是集中在发展过程中的代谢和细胞命运之间的串扰， 近轴中胚层，形成骨骼肌和椎骨的组织。其中一个显著的特点是 组织分裂成称为体节的重复结构，这是由分子振荡器驱动的过程 所谓的分段时钟[1]。近轴中胚层发育缺陷可导致严重畸形 例如先天性脊柱侧凸、脊柱裂或尾部发育不全。近轴中胚层起源于 位于原条和尾芽中的祖细胞。值得注意的是，这些祖细胞表现出有氧 糖酵解和倒置的细胞内与细胞外pH梯度，这是瓦尔堡效应的特征， 癌细胞[2，3]。在尾芽中，我们证明了糖酵解增加了细胞内pH， β-连环蛋白的乙酰化和Wnt活化，这最终导致近轴中胚层诱导[3]。因为这些 过程很难在体内研究，我们已经开发了体外系统，其中胚胎干细胞（ES） 或诱导的多能干（iPS）细胞可以有效地向近轴中胚层分化 概括了其代谢、信号传导甚至节段时钟振荡的正常特征 [4-7]。我们现在将利用这些体外系统，以及小鼠和鸡胚胎， 详细描述有氧糖酵解在近轴中胚层发育中的作用，以了解它如何与 瓦尔堡效应。在此应用中，我们建议进行大规模的多组学实验 （代谢组学、转录组学、蛋白质组学、表观基因组学）和细胞代谢状态的实时成像， 表征代谢转换对基因表达、蛋白质功能和细胞生长调节的影响， 命运由于对瓦尔堡效应的生理学意义还没有很好的理解，仔细剖析其作用 可能有助于阐明它在癌症中的作用。最后，我们建议使用体内，体外和体外 系统重演的振荡的分段时钟研究的作用，代谢的控制 的振荡周期。我们将分析小鼠和人类在代谢调节方面的差异 近轴中胚层细胞，特别关注线粒体呼吸。振荡的周期发散 这两个物种之间的差异很大，可以作为发育时间的代表， 为什么人类的发育比老鼠的慢我们希望这些实验 揭示了与哺乳动物寿命密切相关的发育时间的分子基础。

Project Summary/Abstract The proposed project is focused on the crosstalk between metabolism and cell fate during development of the paraxial mesoderm, the tissue which forms skeletal muscles and vertebrae. One striking characteristic of this tissue is its segmentation into repeated structures termed somites, a process driven by a molecular oscillator called segmentation clock [1]. Defects in paraxial mesoderm development can lead to severe malformations such as congenital scoliosis, spina bifida or caudal agenesis. The paraxial mesoderm arises from a population of progenitors located in the primitive streak and tail bud. Remarkably, these progenitor cells exhibit aerobic glycolysis and an inverted intra- vs. extracellular pH gradient, which are characteristic of the Warburg effect of cancer cells [2, 3]. In the tailbud, we demonstrated that glycolysis increases the intracellular pH to promote acetylation of -catenin and Wnt activation, which ultimately leads to paraxial mesoderm induction [3]. As these processes are difficult to study in vivo, we have developed in vitro systems in which embryonic stem (ES) cells or induced pluripotent stem (iPS) cells can be efficiently differentiated toward the paraxial mesoderm fate recapitulating the normal features of its metabolism, signaling and even oscillations of the segmentation clock [4-7]. We will now take advantage of these in vitro systems, as well as mouse and chicken embryos, to characterize in detail the role of aerobic glycolysis in paraxial mesoderm development to see how it relates to the Warburg effect. In this application, we propose to carry out large scale multi-omics experiments (metabolomics, transcriptomics, proteomics, epigenomics) and live imaging of cellular metabolic state to characterize the impact of metabolic transitions on the regulation of gene expression, protein function and cell fate. As the physiological significance of the Warburg effect is not well understood, carefully dissecting its role in the embryo might help shed light on its role in cancer. Finally, we propose to use in vivo, ex vivo and in vitro systems recapitulating the oscillations of the segmentation clock to study the role of metabolism in the control of the oscillatory period. We will analyze the differences in metabolism regulation between mouse and human paraxial mesoderm cells, with special focus on mitochondrial respiration. The period of the oscillations diverges significantly between the two species and can be used as a proxy for developmental timing to try to understand why human development proceeds more slowly than mouse development. We expect these experiments to shed light on the molecular basis of developmental timing, which is tightly linked to longevity in mammals.