Metabolic activities are selective modulators for individual segmentation clock processes

Abstract

A sequence of cellular and molecular processes unfolding during embryonic development prompts fundamental questions of how the tempo of multiple processes is coordinated and whether a common global modulator exists. The oscillation of the segmentation clock is a well-studied model of developmental tempo. While the clock period is known to scale with the kinetics of gene expression and degradation processes of the core clock gene Hes7 across mammalian species, how these key molecular processes are coordinated remains unclear. In this study, we investigated if metabolic activities act as a global modulator for the segmentation clock, finding that they are rather selective modulators. While several metabolic inhibitions extended the clock period, their effects on the key processes varied. Inhibition of glycolysis decelerated the protein degradation of Hes7 and extended the production delay but did not influence the intron delay. Electron transport chain inhibition extended Hes7 intron delay without influencing the other two processes. Combinations of distinct metabolic inhibitions exhibited synergistic effects. By contrast, temperature changes affected the clock period and all three key processes simultaneously. These results highlight the selective effects of metabolic activities on segmentation clock processes, hinting that their scaled kinetics across species may be achieved through combinations of multiple modulators.

Systematic analysis of protein stability associated with species-specific developmental tempo

“…As described previously 34 , mouse EpiSCs stably expressing the mHes7 promoter-Fluc-NLS-PEST-UTR reporter were used. After the PSM induction, the medium was changed into the SCDF medium containing a lower dosage of CHIR99021 (1 µM) and D-luciferin to monitor the oscillations of the Hes7 promoter-luciferase reporter signal.…”

“…The measurements were performed with Seahorse XF analyzer (Agilent) as described previously 34 . iPSM cells were re-seeded on a fibronectin-coated Seahorse plate (Agilent) at a density of 9.1 × 10 5 cells/cm 2 in 100 μl of Seahorse XF DMEM (Agilent) supplemented with glucose (20 mM), pyruvate (1 mM), and glutamine (2 mM).…”

“…As described previously 34 , the mouse EpiSCs and human iPSCs stably expressing the rTetOne promoter-mHes7 (w/o intron)-Nluc or individual localization reporters were used. PSM cells were induced in the presence of Doxycycline (Dox; 100 ng/ml).…”

“…The cell culture methods were described previously 34 . Human iPSCs (201B7 line, from RIKEN BRC #HPS0063) 39 were cultured on Matrigel-coated dishes in the StemFit medium (Ajinomoto) without feeder cells.…”

“…We next investigated the effect of metabolism on the protein stability profile. While both glycolytic activity and electron transport chain activity are involved in the segmentation clock and somitogenesis 12,18,[20][21][22][23][24] , we have recently found that a glycolysis inhibitor, 2-Deoxy-D-glucose (2DG), and an electron transport chain inhibitor, sodium azide, decelerate the segmentation clock through different mechanisms: 2DG decelerates HES7 protein degradation whereas sodium azide elongates HES7 intron processing delay 34 .…”