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Metabolic regulation of species-specific developmental rates

Nature volume 613, pages 550–557 (2023)Cite this article

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An Author Correction to this article was published on 22 March 2023

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Abstract

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1,2,3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

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Fig. 1: Cell-autonomous differences in developmental rate between differentiating mouse and human PSM cells.
Fig. 2: Mass-specific metabolic rates are higher in mouse PSM cells compared with human PSM cells.
Fig. 3: Regulation of the segmentation clock by the NAD+/NADH ratio.
Fig. 4: The global rate of protein synthesis acts downstream of the ETC to regulate developmental speed.

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Data availability

All data are available within the article, supplementary files and source data files. All materials used in this study, including stem cell lines carrying knock-in reporters, are available by request from the corresponding author. Source data are provided with this paper.

Code availability

The MATLAB code for Hilbert analysis of oscillation properties can be found at GitHub: https://github.com/md2981/Hilbert-Segmentation-Clock. The R custom package, mzrtools, for correction of natural isotope abundance in raw mass isotopomer distributions can also be found at GitHub: https://github.com/wmoldham/mzrtools.

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Acknowledgements

We thank members of the Pourquié laboratory and J. Locasale, G. Ruvkun, C. Tabin, N. Perrimon, S. Rath and V. Mootha for critical reading of the manuscript and discussions; the NeuroTechnology Studio at Brigham and Women’s Hospital for providing access to the Agilent Seahorse XF96 Extracellular Flux Analyzer and Zeiss LSM880 confocal microscope, and for consultation on data acquisition and data analysis; the Center for Neurologic Diseases (ARCND) Flow Cytometry Core Facility at Brigham and Women’s Hospital for access to the BD Fortessa flow cytometer; the Seahorse core facility at Brigham and Women’s hospital for training and access to the Agilent Seahorse XFe24 Extracellular Flux Analyzer; S. Oh and the Kirschner laboratory at Harvard Medical School for providing access to the Moxi Go II for cell volume measurements; O. Goldberger and V. Mootha for providing the LbNOX plasmids; and M. Bharadwaj for guidance in the implementation of Seahorse measurements on isolated mitochondria. The Pourquié laboratory and M.D.-C. were funded by grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) under award numbers 2R01HD085121-06A and F31HD100033, respectively. M.D.-C. is a recipient of the NASEM Ford Foundation Dissertation fellowship. This work was supported by the Cancer Systems Biology Consortium funding (U54-CA217377) from the National Cancer Institute (S.R.M.) and the Virginia and D. K. Ludwig Fund for Cancer Research (S.R.M.). O.S.S. was supported by the F32GM133047 from the NIH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Author notes

  1. Margarete Diaz-Cuadros

    Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

  2. Carlos Manlio Díaz-García

    Present address: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Margarete Diaz-Cuadros, Dylan Sheedy, Svetlana Gapon, Alexis Hubaud & Olivier Pourquié

  2. Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA

    Margarete Diaz-Cuadros, Dylan Sheedy, Svetlana Gapon, Alexis Hubaud & Olivier Pourquié

  3. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Teemu P. Miettinen & Scott R. Manalis

  4. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Owen S. Skinner

  5. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Owen S. Skinner

  6. Broad Institute, Cambridge, MA, USA

    Owen S. Skinner

  7. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Carlos Manlio Díaz-García & Gary Yellen

  8. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Scott R. Manalis

  9. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Scott R. Manalis

  10. Department of Medicine, Harvard Medical School, Boston, MA, USA

    William M. Oldham

  11. Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    William M. Oldham

  12. Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA

    Olivier Pourquié

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Contributions

M.D.-C. and O.P. conceptualized the study. M.D.-C. performed the experiments and analysed the data. T.P.M. and S.R.M. carried out SMR experiments and analysed the resulting data. O.S.S. performed the lactate and pyruvate measurements in spent media. D.S. generated the AAVS1-CAG-Peredox-mCherry-NLS human PSC line. C.M.D.-G. and G.Y. contributed to the functional validation of the AAVS1-CAG-Peredox-mCherry-NLS human PSC line. S.G. helped maintain mouse and human PSC cultures and seeded cells for differentiation. A.H. performed experiments involving primary mouse PSM tissue. W.M.O. performed LC–MS/MS for stable isotope metabolic tracing, helped with Seahorse experiments, and provided guidance on the project. M.D.-C. and O.P. wrote the manuscript. O.P. supervised the project. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Margarete Diaz-Cuadros or Olivier Pourquié.

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Competing interests

O.P. is scientific founder of Anagenesis Biotechnologies. S.R.M. is a co-founder of Travera and Affinity Biosensors, which develop technologies relevant to the research presented in this work. All other authors declare no competing interests.

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Nature thanks Joseph Baur, Katharina Sonnen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 The segmentation clock-period is cell-autonomous even in chimeric conditions.

a. Representative micrographs of MSGN1-Venus fluorescence in mouse (left) and human (right) PSC-derived PSM cells on day 2 of differentiation. Note the reporter is cytoplasmic in mouse cells but nuclear in human cells. Similar results were obtained n = 15 times. Scale bar = 400 μm. b. Period of segmentation clock oscillations in primary PSM tissue dissected from E9.5 mouse embryos carrying the LuVeLu reporter (n = 18) and PSC-derived PSM expressing the Hes7-Achilles reporter (n = 24). Mean ±SD. Unpaired two-sided t-test. c. Cell cycle duration in primary PSM tissue dissected from E9.5 mouse embryos (n = 27) and PSC-derived PSM (n = 33). Mean ±SD. Unpaired two-sided t-test. d. Instantaneous HES7-Achilles oscillatory period over the course of 16 h as calculated by Hilbert transformation for mouse PSM cells co-cultured with mouse (n = 10) or human (n = 7) non-reporter PSM cells. Mean ±SEM. e. Instantaneous HES7-Achilles oscillatory period over the course of 16 h as calculated by Hilbert transformation for human PSM cells co-cultured with human (n = 11) or mouse (n = 8) non-reporter PSM cells. Mean ±SEM.f. Mean segmentation clock period as calculated by Hilbert transformation for mouse (left) or human (right) PSM cells co-cultured with either mouse or human non-reporter PSM cells. Mean ±SD. n = 10 (mouse-mouse), n = 9 (mouse-human), n = 8 (human-mouse), n = 10 (human-human). One-way ANOVA with Šidák correction. g. Mean amplitude of HES7-Achilles oscillations in mouse (left) or human (right) PSM cells co-cultured with either mouse or human non-reporter PSM cells. Mean ±SD. n = 10 (mouse-mouse), n = 9 (mouse-human), n = 8 (human-mouse), n = 10 (human-human). Kruskal-Wallis test with Dunn’s correction. h. Representative single-cell tracks of HES7-Achilles fluorescence for mouse PSM cells cultured with non-reporter mouse PSM cells. i. Representative single-cell tracks of HES7-Achilles fluorescence for mouse PSM cells cultured with non-reporter human PSM cells. j. Representative single-cell tracks of HES7-Achilles fluorescence for human PSM cells cultured with non-reporter human PSM cells. k. Representative single-cell tracks of HES7-Achilles fluorescence for human PSM cells cultured with non-reporter mouse PSM cells.

Source data

Extended Data Fig. 2 Comparison of metabolic and physical parameters in mouse vs. human PSM and neural progenitor cells.

a. HES7-Achilles oscillatory period in human PSM cells under control (DMSO; n = 22) or 5 μM aphidicolin (n = 20) conditions. Cultures were pre-treated with DMSO or aphidicolin for 24 h to induce cell cycle arrest. Mean ±SD. Unpaired two-sided t-test. b. Quantification of immunofluorescence staining for histone H3 phosphorylated at Ser10 in human PSM cells treated with vehicle control (DMSO) or 5 μM aphidicolin for 24 or 48 h. Mean ±SD. n = 5 biological replicates. c. Glycolytic proton efflux rate per cell for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Same data as Fig. 2d but normalized by cell. Mean ±SD. n = 15. Unpaired two-sided t-test. d. Oxygen consumption rate per cell for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Same data as Fig. 2d but normalized by cell. Mean ±SD. n = 12. Unpaired two-sided t-test. e. Total cell volume as measured in a suspended microchannel resonator for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Each datapoint represents the mean of >200 individual cells. Mean ±SD. n = 3 independent experiments. Unpaired two-sided t-test: p = 5.9 × 10−5. f. Total cell density of MSGN1-Venus+ PSC-derived mouse and human PSM cells as measured on a suspended microchannel resonator. Each datapoint represents the mean of >200 individual cells. Mean ±SD. n = 3 independent experiments. Unpaired two-sided t-test. g. Dry mass as measured in a suspended microchannel resonator for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Each datapoint represents the mean of >200 individual cells. Mean ±SD. n = 3 independent experiments. Unpaired two-sided t-test. h. Dry volume as measured in a suspended microchannel resonator for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Each datapoint represents the mean of >200 individual cells. Mean ±SD. n = 3 independent experiments. Unpaired two-sided t-test. i. Dry density as measured in a suspended microchannel resonator for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Each datapoint represents the mean of >200 individual cells. Mean ±SD. n = 3 independent experiments. Unpaired two-sided t-test. j. Mass-specific extracellular acidification rate (ECAR) in MSGN1-Venus+ PSC-derived mouse and human PSM cells. Mean ±SD. n = 15. Unpaired two-sided t-test: p = 8.7 × 10−23. k. Relative mass-specific glutamine consumption after 12 h of culture for MSGN1-Venus+ PSC-derived mouse and human PSM cells. Mean ±SD. n = 4 biological replicates. Unpaired two-sided t-test. l. Percent PAX6+ cells in mouse (day 5) and human (day 7) neural progenitor cultures as measured by intracellular staining and flow cytometry. Mean ±SD. n = 3 independent experiments. m. Representative micrographs of immunofluorescence staining for PAX6 in PSC-derived mouse (left) and human (right) neural progenitor cells on days 5 and 7 of differentiation, respectively. Similar results were obtained n = 3 times. Scale bar = 200 μm. n. Volume of mouse (day 5) and human (day 7) neural progenitor cells as measured by a coulter counter. Mean ±SD. n = 3 independent experiments. Unpaired two-sided t-test. o. Volume-specific oxygen consumption rate for PSC-derived mouse and human neural progenitor cells. Mean ±SD. n = 30. Unpaired two-sided t-test: p = 1.69 × 10−51. p. Volume-specific extracellular acidification rate for PSC-derived mouse and human neural progenitor cells. Mean ±SD. n = 36. Unpaired two-sided t-test: p = 3.75 × 10−18.

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Extended Data Fig. 3 Stable isotope tracing of glucose and glutamine utilization patterns in mouse and human PSM cells.

a-f. Stable isotope labeling with 25 mM U13C6-Glucose over the course of 48 h for PSC-derived mouse and human PSM cells. Total fraction labeled by any isotopomer is expressed as 1-M0 for pyruvate (a), lactate (b), citrate (c), succinate (d), malate (e), and glutamate (f). Mean ±SD. n = 3 independent experiments. g-l. Mass isotopomer distribution, adjusted for natural abundance, for pyruvate (e), lactate (f), citrate (g), succinate (h), malate (i), and glutamate (j) after 24 h of labeling with 25 mM U13C6-Glucose in mouse and human PSM cells. Labels in the x-axis correspond to distinct mass isotopomers with increasing number of heavy carbons. Mean ±SD. n = 3 independent experiments. m-r. Mass isotopomer distribution, adjusted for natural abundance, for pyruvate (k), lactate (l), citrate (m), succinate (n), malate (o), and glutamate (p) after 24 h of labeling with 4 mM U13C5-Glutamine in mouse and human PSM cells. Labels in the x-axis correspond to distinct mass isotopomers with increasing number of heavy carbons. Mean ±SD. n = 3 independent experiments.

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Extended Data Fig. 4 Mitochondrial properties of mouse and human PSM cells.

a. Mass-specific oxygen consumption rate measured over the course of the mitochondrial stress test for MSGN1-Venus+ PSC-derived mouse (n = 9) and human (n = 7) PSM cells. 1 μM oligomycin, 1 μM FCCP, and 0.5 μM Rotenone + 0.5 μM Antimycin A were added at the timepoints marked by dotted lines. The first three timepoints denote basal respiration;, respiration after oligomycin addition corresponds to proton leak; FCCP induces maximal respiration; and rotenone/antimycin reveal non-mitochondrial respiration. Spare capacity refers to the difference between maximal and basal respiration rates. Mean ±SD. b. Spare respiratory capacity in MSGN1-Venus+ PSC-derived mouse and human PSM cells. Mean ±SD. n = 7 biological replicates. Unpaired two-sided t-test: p = 2.7 × 10−9. c. Oxygen consumption rate profiles for mitochondria isolated from mouse and human PSC-derived PSM cells. Rates correspond to 10 ug mitochondria seeded per assay well. ETC complex I substrates pyruvate and malate were provided to fuel respiration. 2 mM ADP, 5 μM Oligomycin, 6 μM FCCP, and 1 μM Rotenone with 1 μM Antimycin A were injected at the timepoints marked by dotted lines. First two timepoints correspond to basal respiration (state 2), followed by ADP-stimulated respiration (state 3), then leak respiration (state 4o), followed by maximal FCCP-stimulated respiration (state 3u), and finally non-mitochondrial respiration. Mean ±SD. n = 5 biological replicates. d. Whole-cell NAD+/NADH ratio in MSGN1-Venus+ PSC-derived mouse and human PSM cells. Each datapoint represents the average of 3 technical replicates. Mean ±SD. n = 6 biological replicates. Unpaired two-sided t-test: p = 1.2 × 10−7. e. Whole-cell ADP/ATP ratio in MSGN1-Venus+ PSC-derived mouse and human PSM cells. Mean ±SD. n = 9. Each datapoint represents the average of 3 technical replicates. Unpaired two-sided t-test: p = 2.42 × 10−7. f. Percent viable cells as measured by trypan blue staining in human PSM cells treated with the indicated inhibitors for 24 h. n = 3 independent experiments.

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Extended Data Fig. 5 Effect of electron transport chain inhibitors on the segmentation clock.

a. Basal oxygen consumption rate in human PSM cells treated with vehicle control (DMSO), 20nM rotenone, 50 nM atpenin A5, 100 nM antimycin A, 1 mM sodium azide, 1 μM oligomycin or 1 μM FCCP. Mean ±SD. n = 10 biological replicates. One-way ANOVA with Šidák correction: control vs. rotenone p = 5.4 × 10−12, control vs. atpenin p = 4.3 × 10−11, control vs. antimycin p = 3.8 × 10−18, control vs. azide p = 1.04 × 10−20, control vs. oligomycin p = 5.9 × 10−8, control vs. FCCP p = 0.999. b. Number of HES7-Achilles oscillations observed in 25 h for human PSM cells treated with vehicle control (DMSO, n = 29), 20 nM rotenone (n = 10), 50 nM atpenin A5 (n = 17), 100 nM antimycin A (n = 11), 1 mM sodium azide (n = 15), 1 μM oligomycin (n = 11), or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. rotenone p = 1.1 × 10−10, control vs. atpenin p = 2.3 × 10−12, control vs. antimycin p = 6.1 × 10−15, control vs. azide p = 2.0 × 10−15, control vs. oligomycin p = 4.7 × 10−10. c. Mean amplitude expressed as a percent of control for HES7-Achilles oscillations in human PSM cells treated with vehicle control (DMSO, n = 53), 20 nM rotenone (n = 10), 50 nM atpenin A5 (n = 18), 100 nM antimycin A (n = 11), 1 mM sodium azide (n = 14), 1 μM oligomycin (n = 11), or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction: control vs. oligomycin p = 3.4 × 10−8. d. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with vehicle control (DMSO, n = 53), 20 nM rotenone (n = 10), 50 nM atpenin A5 (n = 18), 100 nM antimycin A (n = 11), 1 mM sodium azide (n = 14), 1 μM oligomycin (n = 11), or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. rotenone p = 8.9 × 10−21, control vs. atpenin p = 1.8 × 10−18, control vs. antimycin p = 2 × 10−15, control vs. azide p = 1.4 × 10−33. e. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 10) or 20 nM rotenone (n = 11). n denotes independent experiments. Mean ±SEM. f. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 12) or 50 nM atpenin A5 (n = 18). n denotes independent experiments. Mean ±SEM. g. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 11) or 100 nM antimycin A (n = 13). n denotes independent experiments. Mean ±SEM. h. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 9) or 1 mM sodium azide (n = 15). n denotes independent experiments. Mean ±SEM. i. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 12) or 1 μM oligomycin (n = 17). n denotes independent experiments. Mean ±SEM. j. HES7-Achilles oscillatory profile in human PSM cultures treated with DMSO control (n = 3) or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SEM. k. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 10) or 20 nM rotenone (n = 11). n denotes independent experiments. Mean ±SEM. l. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 50 nM atpenin A5 (n = 18). n denotes independent experiments. Mean ±SEM. m. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 11) or 100 nM antimycin A (n = 13). n denotes independent experiments. Mean ±SEM. n. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 9) or 1 mM sodium azide (n = 15). n denotes independent experiments. Mean ±SEM. o. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 1 μM oligomycin (n = 17). n denotes independent experiments. Mean ±SEM. p. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 3) or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SEM. q. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 10) or 20 nM rotenone (n = 11). n denotes independent experiments. Mean ±SEM. r. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 50 nM atpenin A5 (n = 18). n denotes independent experiments. Mean ±SEM. s. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 11) or 100 nM antimycin A (n = 13). n denotes independent experiments. Mean ±SEM. t. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 9) or 1 mM sodium azide (n = 15). n denotes independent experiments. Mean ±SEM. u. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 12) or 1 μM oligomycin (n = 17). n denotes independent experiments. Mean ±SEM. v. Instantaneous HES7-Achilles oscillatory amplitude over the time as calculated by Hilbert transformation in human PSM cultures treated with DMSO control (n = 3) or 1 μM FCCP (n = 7). n denotes independent experiments. Mean ±SEM. w. Inner mitochondrial membrane potential (ΔΨm) in human PSM cells under control conditions or treated acutely with 1 μM FCCP. TMRM fluorescence was normalized by mitochondrial content (MitoTracker Green) following flow cytometry. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test: p = 6.4 × 10−7.

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Extended Data Fig. 6 Increased ATP concentrations do not accelerate the segmentation clock.

a. Illustration depicting the alternate fates of pyruvate and their regulation by metabolic enzymes. Lactate dehydrogenase (LDH) converts pyruvate to lactate and regenerates NAD+ from NADH. Pyruvate dehydrogenase (PDH) oxidizes pyruvate to acetyl coenzyme-A (acetyl-CoA) in the mitochondria and consumes NAD+. Acetyl -CoA then enters the tricarboxylic acid (TCA) cycle, which also consumes NAD+. Pyruvate dehydrogenase kinase (PDK) inhibits PDH by phosphorylating it. DCA is a PDK inhibitor that promotes the conversion of pyruvate to acetyl-CoA by relieving PDH inhibition. Created with BioRender.com. b. ATP content per well for human PSM cells in control (water; n = 6), 25 mM succinate supplementation (n = 6), 10 mM galactose in the absence of glucose (n = 6), and 6.25 mM DCA (n = 8) conditions after 24 h of culture. In each case, 30,000 cells were seeded per assay well. Each datapoint represents the average of 3 technical replicates. Mean ±SD. One-way ANOVA with Šidák correction: control vs. succinate p = 2 × 10−5, control vs. galactose p = 1.6 × 10−8, control vs. DCA p = 7.4 × 10−6. c. Ratio of Oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) in human PSM cells treated with water control (n-18), 25 mM succinate (n = 15), 6.25 mM DCA (n = 18), or cultured with 10 mM galactose instead of glucose (n = 18) for 24 h. Mean ±SD. One-way Brown-Forsythe and Welch ANOVA with Dunnett T3 correction: control vs. galactose p = 2.5 × 10−12. d. Oxygen consumption rate (OCR) in human PSM cells treated with water control (n = 18), 25 mM succinate (n = 15), 6.25 mM DCA (n = 18), or cultured with 10mM galactose instead of glucose (n = 18) for 24 h. Mean ±SD. One-way ANOVA with Dunnett correction: control vs. DCA p = 3.8 × 10−18. e. Extracellular acidification rate (ECAR) in human PSM cells treated with water control (n = 18), 25 mM succinate (n = 15), 6.25 mM DCA (n = 18), or cultured with 10 mM galactose instead of glucose (n = 18) for 24 h. Mean ±SD. One-way ANOVA with Šidák correction: control vs. galactose p = 3.05 × 10−51, control vs. DCA p = 1 × 10−28. f. Percent of total ATP production corresponding to glycolysis (glycoATP) or mitochondrial respiration (mitoATP) in human PSM cells treated with water control (n = 6), 25 mM succinate (n = 5), 6.25 mM DCA (n = 6), or cultured with 10 mM galactose instead of glucose (n = 6) for 24 h. Mean ±SD. g. HES7-Achilles oscillatory period in human PSM cells treated with vehicle control (water; n = 36), 25 mM succinate (n = 23), 10 mM galactose in the absence of glucose (n = 43), and 6.25 mM DCA (n = 34). Mean ±SD. One-way ANOVA with Šidák correction: control vs. galactose p = 5.9 × 10−7, control vs. DCA p = 3.1 × 10−5. h. HES7-Achilles oscillatory profile in human PSM cells cultured under control conditions or supplemented with 25 mM succinate. Mean ±SEM. n = 8. i. HES7-Achilles oscillatory profile in human PSM cells cultured with either 10 mM glucose or 10 mM galactose. Mean ±SEM. n = 6. j. HES7-Achilles oscillatory profile in human PSM cultures under control conditions (n = 9) or 6.25 mM DCA (n = 8). Mean ±SEM. k. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cells cultured under control conditions or supplemented with 25 mM succinate. Mean ±SEM. n = 8. l. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cells cultured with either 10 mM glucose or 10 mM galactose. Mean ±SEM. n = 6. m. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cells cultured under control conditions (n = 9) or supplemented with 6.25 mM DCA (n = 7). Mean ±SEM. n. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cells cultured under control conditions or supplemented with 25 mM succinate. Mean ±SEM. n = 8. o. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cells cultured with either 10 mM glucose or 10 mM galactose. Mean ±SEM. n = 6. p. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cells cultured under control conditions (n = 9) or supplemented with 6.25 mM DCA (n = 8). Mean ±SEM. q. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with vehicle control (water; n = 21), 25 mM succinate (n = 8), 10 mM galactose in the absence of glucose (n = 6), and 6.25 mM DCA (n = 8). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: contro vs. galactose p = 3.3 × 10−12, control vs. DCA p = 1.4 × 10−10. r. Mean amplitude expressed as a percent of control for HES7-Achilles oscillations in human PSM cells treated with vehicle control (water; n = 22), 25 mM succinate (n = 8), 10 mM galactose in the absence of glucose (n = 6), and 6.25 mM DCA (n = 7). n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction. s. Number of HES7-Achilles oscillations observed in 25 h for human PSM cells treated with vehicle control (water; n = 23), 25 mM succinate (n = 8), 10 mM galactose in the absence of glucose (n = 6), and 6.25 mM DCA (n = 8). n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction. t. Duration of the cell cycle in hours for human PSM cells treated with vehicle control (DMSO; n = 42), 25 mM succinate (n = 44), 10 mM galactose in the absence of glucose (n = 18), and 6.25 mM DCA (n = 30). Mean ±SD. One-way ANOVA with Dunnett correction: control vs. galactose p = 1.2 × 10−12.

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Extended Data Fig. 7 Rescue of the segmentation clock period by restoration of the NAD+/NADH ratio.

a. Whole-cell NAD+/NADH ratio in vehicle-treated human PSM cells and cells treated with either 6.25 mM DCA alone or DCA in combination with 1 mM sodium pyruvate or 10 nM FCCP for 24 h. Each datapoint represents the average of 3 technical replicates. Mean ±SD. n = 4. One-way ANOVA with Dunnett correction. b. Inner mitochondrial membrane potential (ΔΨm) in human PSM cells under control conditions or treated with 6.25 mM DCA for 24 h. TMRM fluorescence was normalized by mitochondrial content (MitoTracker Green) following flow cytometry. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test. c. Peredox-mCherryNLS fluorescence lifetime in human PSM cells cultured acutely in a balanced salt solution and supplemented with the indicated concentrations of glucose, lactate or pyruvate. Mean ±SD. n = 4 biological replicates. d. Ratiometric Peredox-to-mCherry fluorescence signal in human PSM cells cultured acutely in a balanced salt solution and supplemented with the indicated concentrations of glucose, lactate or pyruvate. Mean ±SD. n = 6 biological replicates. e. Ratiometric Peredox/mCherry signal in vehicle-treated human PSM cells and cells treated with either 6.25 mM DCA alone or DCA in combination with 1 mM sodium pyruvate or 10 nM FCCP for 24 h. Each datapoint represents the average of >200 individual cells analyzed within a biological replicate. Mean ±SD. n = 6 biological replicates. One-way ANOVA with Dunnett correction. f. HES7-Achilles oscillatory period in human PSM cells treated with vehicle control (water; n = 78), 6.25 mM DCA alone (n = 68), DCA with 1 mM sodium pyruvate (n = 73), and DCA with 10 nM FCCP (n = 85). Mean ±SD. One-way ANOVA with Dunnett correction. g. Inner mitochondrial membrane potential (ΔΨm) in human PSM cells under control conditions or treated with 25 mM succinate for 24 h. TMRM fluorescence was normalized by mitochondrial content (MitoTracker Green) following flow cytometry. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test. h. Whole-cell NAD+/NADH ratio in vehicle-treated human PSM cells and cells treated with either 25 mM succinate alone or succinate in combination with 1 mM sodium pyruvate for 24 h. Each datapoint represents the average of 3 technical replicates. Mean ±SD. n = 3 biological replicates. One-way ANOVA with Dunnett correction. i. HES7-Achilles oscillatory period in human PSM cells treated with vehicle control (water; n = 62), 25 mM succinate alone (n = 46), or succinate with 1 mM sodium pyruvate (n = 46). Mean ±SD. One-way ANOVA with Dunnett correction. j. Ratiometric Peredox/mCherry signal in DMSO-treated human PSM cells and cells treated with 20 nM rotenone, 100 nM antimycin A, 1 mM sodium azide alone, azide with 1 mM sodium pyruvate, and azide with 5 μM duroquinone (DQ) for 24 h. Each datapoint represents the average of >200 individual cells analyzed in a biological replicate. Mean ±SD. n = 6. One-way ANOVA with Šidák correction: control vs. rotenone p = 2.9 × 10-15, control vs. antimycin p = 7.1 × 10−17, control vs. azide p = 4.4 × 10−22, control vs. azide+pyr p = 3.3 × 10−16, control vs. azide+DQ p = 2.2 × 10−19, azide vs. azide+pyr p = 9.7 × 10−10, azide vs. azide+DQ p = 5.3 × 10−6. k. HES7-Achilles oscillatory profile in human PSM cells cultures treated with DMSO control (n = 10), 1 mM sodium azide alone (n = 13), and azide with 1mM sodium pyruvate (n = 13). Mean ±SEM. l. Whole-cell NAD+/NADH ratio in human PSM cells treated with vehicle-control (n = 5), 1 mM sodium azide alone (n = 5), or azide with 5 μM duroquinone (DQ) (n = 6) for 24 h. Each datapoint represents the average of 3 technical replicates. Mean ±SD. One-way ANOVA with Tukey correction: control vs. azide p = 3.5 × 10−8, control vs. azide+DQ p = 1.8 × 10−8. m. HES7-Achilles oscillatory profile in human PSM cells cultures treated with DMSO control (n = 11), 1 mM sodium azide alone (n = 19), and azide with 5 μM duroquinone (n = 21). Mean ±SEM.

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Extended Data Fig. 8 Modulation of the segmentation clock period by direct manipulation of the NAD+/NADH ratio.

a. Whole-cell NAD+/NADH ratio human PSM cells under control conditions or treated acutely with 10mM oxamate. Each datapoint represents the average of 3 technical replicates. Mean ±SD. n = 6. Unpaired two-sided t-test. b. Ratiometric Peredox/mCherry signal in human PSM cells cultured under control conditions or treated acutely with 10 mM oxamate. Each datapoint represents the average of >200 individual cells analyzed within a biological replicate. Mean ±SD. n = 3. Unpaired two-sided t-test, p = 8.3 × 10−5. c. Period of HES7-Achilles oscillations in human PSM cells cultures treated with water control (n = 73) or 10 mM sodium oxamate (n = 17). Mean ±SEM. Unpaired two-sided t-test. d. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells cultures treated with water control (n = 10) or 10 mM sodium oxamate (n = 6). n denotes independent experiments. Mean ±SD. Unpaired two-sided t-test. e. HES7-Achilles oscillatory profile in human PSM cells cultures treated with water control (n = 10) or 10 mM sodium oxamate (n = 3). n denotes independent experiments. Mean ±SEM. f. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cells cultures treated with water control (n = 10) or 10 mM sodium oxamate (n = 3). n denotes independent experiments. Mean ±SEM. g. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cells cultures treated with water control (n = 10) or 10 mM sodium oxamate (n = 3). n denotes independent experiments. Mean ±SEM. h. Number of HES7-Achilles oscillations observed in 25 h in human PSM cells cultures treated with water control (n = 10) or 10 mM sodium oxamate (n = 6). n denotes independent experiments. Mean ±SD. Unpaired two-sided t-test. i. Representative micrographs of DAPI nuclear stain, mCherry endogenous fluorescence, and anti-FLAG immunofluorescence (LbNOX is flag-tagged in the C terminus28) in human PSM cells subjected to mock transduction (top) or transduced with LbNOX-mCherry (bottom). Similar results were obtained n = 15 times. Scale bar = 100 μm. j. Non-mitochondrial oxygen consumption in human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 12) or LbNOX in combination with mCherry (n = 14). OCR after addition of 0.5 μM rotenone and 0.5 μM antimycin A is expressed as fraction of basal OCR. Mean ±SD. Unpaired two-sided t-test: p = 1.04 × 10−8. k. HES7-Achilles oscillatory profile over the course of 40 h for human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 16) or LbNOX in combination with mCherry (n = 14). Mean ±SEM. l. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 13) or LbNOX in combination with mCherry (n = 12). Mean ±SD. Unpaired two-sided t-test: p = 4.87 × 10−8. m. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation in human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 13) or LbNOX in combination with mCherry (n = 12). Mean ±SD. n. Mean HES7-Achilles oscillation amplitude in human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 16) or LbNOX in combination with mCherry (n = 14). Mean ±SD. Unpaired two-sided t-test. o. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation in human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 16) or LbNOX in combination with mCherry (n = 14). Mean ±SD. p. Number of oscillations (peaks) observed in 25 h for human PSM cells transduced with a lentivirus expressing either mCherry alone (n = 16) or LbNOX in combination with mCherry (n = 14). Mean ±SD. Unpaired two-sided t-test.

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Extended Data Fig. 9 The segmentation clock is sensitive to the rate of translation.

a. Mass-specific OPP-Puromycin incorporation as a measure of global translation rate in MSGN1-Venus+ PSC-derived mouse and human PSM cells. OPP-Puromycilated peptides were detected by click chemistry with AlexaFluor647-Picoyl Azide. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test. b. Translation rate as measured by puromycin incorporation expressed as percent of control for human PSM cells treated with DMSO, 40 nM, 80 nM or 160 nM cycloheximide (CHX) for 24 h. Mean ±SD. n = 3 biological replicates. One-way ANOVA with Dunnett correction. c. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with DMSO (n = 6), 40 nM (n = 4), 80 nM (n = 5) or 160 nM (n = 4) cycloheximide (CHX) for 24 h. n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. 40 nM p = 7.6 × 10−5, control vs. 80 nM p = 4.5 × 10−6, control vs. 160 nM p = 4.2 × 10−5. d. Number of HES7-Achilles oscillations observed in 25 h in human PSM cells treated with DMSO (n = 6), 40 nM (n = 4), 80 nM (n = 5) or 160 nM (n = 5) cycloheximide (CHX) for 24 h. n denotes independent experiments. Mean ±SD. e. Mean amplitude expressed as a percent of control in human PSM cells treated with DMSO (n = 6), 40 nM (n = 4), 80 nM (n = 5) or 160 nM (n = 4) cycloheximide (CHX) for 24 h. n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction. f. HES7-Achilles oscillatory profile for human PSM cells treated with DMSO-control (n = 3) or 80 nM cycloheximide (CHX; n = 5). n denotes independent experiments. Mean ±SEM. g. Instantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 3) or 80 nM cycloheximide (CHX, n = 5). n denotes independent experiments. Mean ±SEM. h. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 3) or 80 nM cycloheximide (CHX, n = 5). n denotes independent experiments. Mean ±SEM. i. Translation rate as measured by incorporation of the methionine analog AHA in human PSM cells treated with either DMSO control or 1mM sodium azide for one hour. Mean ±SD. n = 3 biological replicates. Unpaired two-sided t-test. j. Oxygen consumption rate measured over the course of the mitochondrial stress test for human PSM cells treated with DMSO control or 100 nM cycloheximide for 24 h. 1 μM Oligomycin, 1 μM FCCP, and 0.5 μM Rotenone + 0.5 μM Antimycin A were added at the timepoints marked by dotted lines. Mean ±SD. n = 9 biological replicates. k. Spare respiratory capacity in human PSM cells treated with vehicle control (DMSO) or 100 nM cycloheximide (CHX) for 24 h. Mean ±SD. n = 8 biological replicates. Unpaired two-sided t-test. l. Coupling efficiency shown as the percent of basal oxygen consumption that is linked to ATP production in human PSM cells treated with vehicle control (DMSO) or 100 nM cycloheximide (CHX) for 24 h. Mean ±SD. n = 8 biological replicates. Unpaired two-sided t-test.

Source data

Extended Data Fig. 10 Protein stability differences between mouse and human PSM cells.

a. Pulse-chase experiment tracking the degradation of puromycilated peptides over the course of 12 h in MSGN1-Venus+ PSC-derived mouse and human PSM cells following a 1-hour pulse with puromycin. Solid line represents best one-phase decay fit with the 95% confidence intervals shown as shaded regions. n = 3 independent experiments. b. Mean amplitude expressed as a percent of control in human PSM treated with DMSO control (n = 9), 2.5 nM (n = 6), 5 nM (n = 13) or 10 nM (n = 12) bortezomib, or 1 μM lactacystin (n = 8). n denotes independent experiments. Mean ±SD. One-way ANOVA with Dunnett correction. c. Number of HES7-Achilles oscillations observed in 25 h in human PSM treated with DMSO control (n = 17), 2.5 nM (n = 6), 5 nM (n = 13) or 10 nM (n = 12) bortezomib, or 1 μM lactacystin (n = 9). n denotes independent experiments. Mean ±SD. One-way ANOVA with Šidák correction: control vs. 5 nM bortezomib p = 2.4 × 10−19, control vs. 10 nM bortezomib p = 1.8 × 10−22. d. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with DMSO control (n = 10) or 2.5 nM bortezomib (BTZ, n = 6). n denotes independent experiments. Mean ±SD. Unpaired two-sided t-test. e. Mean segmentation clock period as calculated by Hilbert transformation for human PSM cells treated with DMSO control (n = 9) or 1 μM lactacystin (n = 8). n denotes independent experiments. Mean ±SD. Unpaired two-sided t-test. f. HES7-Achilles oscillatory profile for human PSM cells treated with DMSO-control (n = 9), 5 nM bortezomib (n = 13), or 1 μM lactacystin (n = 8). Mean ±SEM. g. stantaneous HES7-Achilles oscillatory period over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 9), 2.5 nM bortezomib (n = 6), or 1 μM lactacystin (n = 8). Mean ±SEM. h. Instantaneous HES7-Achilles oscillatory amplitude over time as calculated by Hilbert transformation for human PSM cells treated with DMSO-control (n = 9), 5 nM bortezomib (n = 13), or 1 μM lactacystin (n = 8). Mean ±SEM. i. Proteasome activity cells as measured by cleavage of a luminogenic proteasome substrate in human PSM treated with DMSO control, 2.5 nM, 5 nM or 10 nM bortezomib, or 1 μM lactacystin for 24 h. Mean ±SD. n = 6 biological replicates. One-way ANOVA with Šidák correction: control vs. 2.5 nM bortezomib p = 5.8 × 10−8, control vs. 5 nM bortezomib p = 1.2 × 10−9, control vs. 10 nM bortezomib p = 4.3 × 10−13, control vs. 1 μM lactacystin p = 2.2 × 10−10. j. Proteasome activity cells as measured by cleavage of a luminogenic proteasome substrate in human PSM treated with DMSO control, 100 nM antimycin A, or 1 mM sodium azide for 24 h. Mean ±SD. n = 3 biological replicates. One-way ANOVA with Šidák correction: control vs. antimycin p = 1.3 × 10−5, control vs. azide p = 3.1 × 10−7. k. Pulse-chase experiment tracking the degradation of puromycilated peptides over the course of 12 h in human PSM cells treated with DMSO control or 1 mM sodium azide following a 1-hour pulse with puromycin. Solid line represents best one-phase decay fit with the 95% confidence intervals shown as shaded regions. n = 3 independent experiments. l. Pulse-chase experiment tracking the degradation of AHA-labeled proteins over the course of 30 h in human PSM cells treated with DMSO control of 1 mM sodium azide following a 1-hour pulse with AHA. Solid line represents best one-phase decay fit with the 95% confidence intervals shown as shaded regions. n = 3 independent experiments.

Source data

Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1 and 2 and Supplementary Table 1.

Reporting Summary

Supplementary Table 2

Suspended microchannel resonator data. Individual cell measurements from suspended microchannel resonator experiments. Each row represents one cell. Replicates correspond to independent experiments. Data is summarized in figures as the mean of each independent experiment (rep1, rep2, rep3).

Supplementary Table 3

[U-13C6]Glucose tracing time course. Mass isotopomer distributions for the [U-13C6]glucose stable isotope tracing time course in mouse and human PSM cells.

Supplementary Video 1

Mouse HES7-Achilles oscillations. HES7-Achilles expression over the course of 28 hours in a population of ES cell-derived mouse PSM cells. Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 2

Human HES7-Achilles oscillations. HES7-Achilles expression over the course of 33 hours in a population of iPS cell-derived human PSM cells. Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 3

Mouse cell co-culture assays. ES cell-derived mouse PSM cells expressing the HES7-Achilles (green) and CAG-NLS-BFP (blue) reporters co-cultured with an excess of non-reporter mouse (left) or human (right) PSM cells and imaged for 16 hours. Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 4

Human cell co-culture assays. iPS cell-derived human PSM cells expressing the HES7-Achilles (green) and CAG-H2B-mCherry (red) reporters co-cultured with an excess of non-reporter human (left) or mouse (right) PSM cells and imaged for 18 hours. Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 5

Azide treatment. Time lapse of HES7-Achilles expression over the course of 24 hours in populations of iPS cell-derived human PSM cells treated with either vehicle control (DMSO; left), 1mM sodium azide (center), or 1mM sodium azide supplemented with 1mM sodium pyruvate (right). Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 6

LbNOX overexpression. HES7-Achilles expression over the course of 33 hours in iPS cell-derived human PSM cells transduced with lentiviruses expressing either mCherry alone (left) or LbNOX with mCherry (right). Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 7

Cycloheximide treatment. Time lapse of HES7-Achilles expression over the course of 24 hours in populations of iPS cell-derived human PSM cells treated with either vehicle control (DMSO; left) or 80nM cycloheximide. Time stamp shows hours:minutes. Scale bar = 100 µm.

Supplementary Video 8

Bortezomib treatment. Time lapse of HES7-Achilles expression over the course of 30 hours in populations of iPS cell-derived human PSM cells treated with either vehicle control (DMSO; left) or 2.5nM bortezomib. Time stamp shows hours:minutes. Scale bar = 100 µm.

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Diaz-Cuadros, M., Miettinen, T.P., Skinner, O.S. et al. Metabolic regulation of species-specific developmental rates. Nature 613, 550–557 (2023). https://doi.org/10.1038/s41586-022-05574-4

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