Elucidating the Role of Metabolism in Regulating the Vertebrate Segmentation Clock During vertebrate embryogenesis, segmentation is established by the sequential and rhythmic formation of somites from paraxial mesoderm. The periodicity of somite formation is controlled by traveling waves of gene expression collectively known as the segmentation clock. Despite considerable progress in identifying the signaling dynamics that sustain and synchronize oscillations in the presomitic mesoderm, much less is known about the pacemaker mechanisms that initiate and control the period of the segmentation clock. Current models focus on splicing and nuclear export delay kinetics of individual Hes/Her cyclic genes. However, not all oscillating genes depend on Hes/Her activity and partial segmentation is still observed in Hes/Her mutant embryos. Thus, the identity of the clock pacemaker remains largely elusive. We hypothesize that the segmentation clock might instead be regulated by a metabolic pacemaker. In recent years, it has become clear that oscillatory cells in the presomitic mesoderm exhibit specialized metabolic properties, including high levels of oxidative glycolysis reminiscent of the Warburg effect in cancer cells. A posterior to anterior gradient of glycolytic activity is present in the presomitic mesoderm and might control the dynamics of segmentation clock waves. Furthermore, treatment of embryos with glycolysis and oxidative respiration inhibitors specifically blocks oscillations of the segmentation clock in posterior presomitic mesoderm cells. We propose to test the role of metabolism in regulating the period of the segmentation clock by making use an in vitro pluripotent stem cell-derived model of the human segmentation clock that we recently developed. We will use fluorescent metabolic sensors to determine whether metabolic oscillations take place in human presomitic mesoderm cells and test the effect of metabolic perturbations on the oscillatory period of these cells in vitro. We will furthermore develop a parallel mouse system based on embryonic stem cells and use it to perform cross-species comparisons. Given that the mouse segmentation clock operates more than two times faster than its human counterpart, comparing these two species is likely to provide insights into period control. Using the Seahorse technology and metabolic flux analysis, we will compare the metabolic properties of mouse and human presomitic mesoderm cells to find parameters that scale with clock period. The proposed experiments should inform the regulation of segmentation clock period, with important implications for the understanding of evolutionary processes and human disease.