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.