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

项目摘要/摘要 拟议的项目集中在新陈代谢和细胞命运之间的串扰在发展过程中 近轴中胚层，形成骨骼肌和脊椎的组织。这其中有一个显著的特点 组织被分割成被称为体节的重复结构，这是一个由分子振荡器驱动的过程 称为分段时钟[1]。轴旁中胚层发育缺陷可导致严重畸形 如先天性脊柱侧弯、脊柱裂或尾部发育不全。近轴中胚层起源于一个种群 位于原始条纹和尾芽中的祖先。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外pH梯度，这是Warburg效应的特征 癌细胞[2，3]。在尾芽中，我们证明了糖酵解增加细胞内的pH以促进 -连环蛋白的乙酰化和Wnt的激活，最终导致近轴中胚层的诱导[3]。就像这些 过程很难在体内研究，我们已经开发出体外系统，在其中胚胎干细胞(ES) 或诱导多能干细胞(IPS)可以有效地分化为近轴向中胚层的命运。 概述其新陈代谢的正常特征，信号，甚至分段时钟的振荡 [4-7]。我们现在将利用这些体外系统，以及小鼠和鸡的胚胎，来 详细描述有氧糖酵解在近轴中胚层发育中的作用，以了解它与 华宝效应。在这一应用中，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 描述代谢转变对基因表达、蛋白质功能和细胞调节的影响 命运。由于沃堡效应的生理意义还没有被很好地理解，所以仔细剖析了它的作用 可能有助于阐明它在癌症中的作用。最后，我们建议在体内、体外和体外使用 系统重述分段时钟的振荡以研究新陈代谢在控制中的作用 振荡周期的变化。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。 在两个物种之间有显著差异，并可作为发育时间的替代，试图了解 为什么人类的发展比老鼠的发展要慢。我们预计这些实验将 揭示了发育时间的分子基础，这与哺乳动物的寿命密切相关。

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.