Functional contribution of Metabolism in embryonic development

项目总结 首先在癌细胞中发现了有氧糖酵解(Warburg效应)。这种不寻常的新陈代谢过程 被认为是维持DNA、蛋白质和膜合成所必需的生物合成产物 在高度增殖的细胞中。然而，糖酵解的实际生理意义在很大程度上仍然存在。 未知。最近的报道表明，有氧糖酵解可能直接影响细胞功能和 发展超越了细胞的生物合成需求。事实上，在我们的初步代谢组学中 结果以海胆胚胎为模型系统，建立了动态代谢调节模型 在整个胚胎发育过程中都存在，并且对发生在 胚胎的16细胞期(受精后5小时；5hpf)。这一信号事件被称为“微米” 信号“，并且已知在两天后驱动整个胚胎的内胚层规格 受精(2dpf)。基于这些初步发现，我们假设动态新陈代谢 调控是细胞规范和信号传递的另一层机制 胚胎发生。为了证明这一假设，在拟议的研究中，我们将首先可视化新陈代谢 使用绿色荧光蛋白标记的代谢传感器，在整个胚胎发育过程中的实时和活体动态。 成像将由4D-共焦显微镜进行，以最大化空间和时间 分解代谢动力学，这将进一步对每个细胞进行定量分析 血统和每个发育阶段。其次，我们将测试每个组件的功能意义 代谢途径(糖酵解、脂肪酸合成、氧化磷酸化)，特别是在 16-细胞期的微丝信号事件。每条代谢途径的多个抑制剂将 应用于整个胚胎或16-细胞期的微粒，持续约0.5小时。 后者将通过构建嵌合胚胎来实现，在嵌合胚胎中微粒是 取而代之的是经抑制剂处理的微球。然后通过分析对表型进行评分 驱动微米的极性因素和命运决定因素的时间和空间表达- 特定的基因调控网络，对于内胚层的规范以及 结果胚胎的整体形态(例如，成功的原肠形成)。这些实验将 确定每条代谢途径对整个胚胎构型的重要性。总体而言， 拟议的研究将揭示代谢、基因和蛋白质调节的功能相互作用 对于胚胎发育，这在细胞和发育生物学领域仍未得到充分的研究。

PROJECT SUMMARY Aerobic glycolysis was first identified in cancer cells (Warburg effect). This unusual metabolic process is considered to supply necessary biosynthetic products to sustain DNA, protein, and membrane synthesis in highly proliferative cells. However, the actual physiological significance of glycolysis remains largely unknown. Recent reports suggest that aerobic glycolysis may directly influence cellular function and development beyond matching a cell’s biosynthetic demands. Indeed, in our preliminary metabolomics results using the sea urchin embryo as a model system, dynamic metabolic regulation appears to be present throughout embryogenesis and further critical for a specific cell signaling event that occurs at the 16-cell stage of the embryo (5 hours post fertilization; 5hpf). This signaling event is called “micromere signaling” and known to drive endomesodermal specification in the entire embryo at two days post fertilization (2dpf). Based on these preliminary findings, we hypothesize that dynamic metabolic regulation serves as another layer of mechanism for cell specification and signaling during embryogenesis. To prove this hypothesis, in the proposed research, we will first visualize metabolic dynamics in real time and in vivo throughout embryogenesis, using GFP-tagged metabolic sensors. Imaging will be performed by 4D-confocal microscopy to maximize the spatial and temporal resolution of metabolic dynamics, which will be further subject to quantitative analysis for each cell lineage and for each developmental stage. Second, we will test the functional significance of each metabolic pathway (Glycolysis, Fatty Acid Synthesis, Oxidative Phosphorylation), especially in the event of micromere signaling at the 16-cell stage. Multiple inhibitors for each metabolic pathway will be applied for ~0.5 hour to the entire embryo or specifically to the micromeres at the 16-cell stage. The latter will be accomplished by constructing chimeric embryos in which the micromeres are replaced with the inhibitor-treated micromeres. The phenotypes will be then scored by analyzing temporal and spatial expression of polarity factors and fate determinants that drive the micromere- specific Gene Regulatory Network and is critical for endomesodermal specification, as well as overall morphology (e.g. successful gastrulation) in the resultant embryos. These experiments will identify the essentiality of each metabolic pathway to entire embryonic patterning. Overall, the proposed research will reveal the functional interplay of metabolic, gene and protein regulations essential for embryonic development, which is still understudied in the field of cell and developmental biology.