Functional contribution of Metabolism in embryonic development

新陈代谢在胚胎发育中的功能贡献

• 批准号: 10701781
• 负责人: Mamiko Yajima
• 金额: $ 23.93万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-12 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10701781
• 关键词: Address; Antisense Oligonucleotides; Area; Biological; Biological Models; Biology; Biosensor; Body Size; Calcium; Cell Differentiation process; Cell Lineage; Cell division; Cell physiology; Cells; Cellular biology; Chemicals; Confocal Microscopy; DNA biosynthesis; Data; Development; Developmental Biology; Embryo; Embryonic Development; Endomesoderm; Event; Fatty Acids; Fertilization; Genes; Glucose; Glycolysis; Hour; Image; Mediating; Metabolic; Metabolic Pathway; Metabolism; Modeling; Molecular; Morphology; Organism; Outcome; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen; Pathway interactions; Pattern; Phenotype; Physiologic pulse; Physiological; Play; Protein Biosynthesis; Proteins; Pyruvate; Regulation; Reporting; Research; Role; Sea Urchins; Signal Transduction; Specific qualifier value; Testing; Time; Visualization; Warburg Effect; Work; aerobic glycolysis; beta catenin; biosynthetic product; blastocyst; blastomere structure; cancer cell; cell fate specification; cell type; cellular development; embryo cell; experimental study; gastrulation; gene regulatory network; in vivo; inhibitor; membrane synthesis; metabolomics; protein biomarkers; protein expression; sensor; temporal measurement; tool

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.

项目摘要 有氧糖酵解首先在癌细胞中发现（瓦尔堡效应）。这种不寻常的代谢过程 被认为是提供维持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仍然很大程度上 未知最近的报告表明，有氧糖酵解可能直接影响细胞功能， 发展超出了细胞的生物合成需求。事实上，在我们初步的代谢组学中， 结果利用海胆胚胎作为模型系统，动态代谢调节似乎是 存在于整个胚胎发生过程中，并且对于在胚胎发育过程中发生的特定细胞信号传导事件进一步至关重要。 16-胚胎的细胞阶段（受精后5小时; 5 hpf）。这个信号事件被称为“微节 信号传导”，并且已知在胚胎发育后两天驱动整个胚胎的内中胚层特化。 受精（2dpf）。基于这些初步发现，我们假设动态代谢 调节作为细胞特化和信号传导的另一层机制， 胚胎发生为了证明这一假设，在拟议的研究中，我们将首先可视化代谢 动态在真实的时间和体内整个胚胎发生，使用GFP标记的代谢传感器。 将通过4D共聚焦显微镜进行成像，以最大限度地提高空间和时间分辨率。 代谢动力学的分辨率，这将进一步对每个细胞进行定量分析 和每个发展阶段。第二，我们将测试每个功能的重要性， 代谢途径（糖酵解，脂肪酸合成，氧化磷酸化），特别是在 在16-细胞阶段的微粒体信号事件。每种代谢途径的多种抑制剂将 在整个胚胎或特别是在16细胞阶段的微粒体上施用约0.5小时。 后者将通过构建嵌合胚胎来实现，其中微节是 用经过处理的微粒代替。然后将通过分析表型进行评分， 驱动微粒的极性因子和命运决定因素的时空表达， 特异性基因调控网络，是内中胚层特化的关键，以及 所得胚胎的总体形态（例如成功的原肠胚形成）。这些实验将 确定每个代谢途径对整个胚胎模式的重要性。总体看 拟议中的研究将揭示代谢、基因和蛋白质调控的功能相互作用， 胚胎发育，这在细胞和发育生物学领域仍然研究不足。