Intracellular pH controls WNT downstream of glycolysis in amniote embryos

Abstract

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic β-catenin acetylation downstream of WNT signalling. As acetylated β-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: A glycolysis-dependent pHi gradient in the tail bud.
Fig. 2: pHi and glycolysis decrease during in vitro differentiation of human iPS-derived PSM cells.
Fig. 3: Regulation of β-catenin acetylation by glycolysis and pHi.

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information). Source data are provided with the paper.

Code availability

The custom MATLAB code used to process the 3D segmentations of pH measurements is available at https://github.com/amichaut/pHanalysis.

References

  1. 1.

    Bénazéraf, B. et al. A random cell motility gradient downstream of FGF controls elongation of an amniote embryo. Nature 466, 248–252 (2010).

    ADS  Article  Google Scholar 

  2. 2.

    Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    ADS  Article  Google Scholar 

  3. 3.

    Oginuma, M. et al. A gradient of glycolytic activity coordinates FGF and Wnt signaling during elongation of the body axis in amniote embryos. Dev. Cell 40, 342–353.e10 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Henrique, D., Abranches, E., Verrier, L. & Storey, K. G. Neuromesodermal progenitors and the making of the spinal cord. Development 142, 2864–2875 (2015).

    CAS  Article  Google Scholar 

  5. 5.

    Kimelman, D. Tales of tails (and trunks): forming the posterior body in vertebrate embryos. Curr. Top. Dev. Biol. 116, 517–536 (2016).

    CAS  Article  Google Scholar 

  6. 6.

    Parks, S. K., Chiche, J. & Pouysségur, J. Disrupting proton dynamics and energy metabolism for cancer therapy. Nat. Rev. Cancer 13, 611–623 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Hoffmeyer, K., Junghans, D., Kanzler, B. & Kemler, R. Trimethylation and acetylation of β-catenin at lysine 49 represent key elements in ESC pluripotency. Cell Rep. 18, 2815–2824 (2017).

    CAS  Article  Google Scholar 

  8. 8.

    Webb, B. A., Chimenti, M., Jacobson, M. P. & Barber, D. L. Dysregulated pH: a perfect storm for cancer progression. Nat. Rev. Cancer 11, 671–677 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    Miesenböck, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    ADS  Article  Google Scholar 

  10. 10.

    Grillo-Hill, B. K., Webb, B. A. & Barber, D. L. Ratiometric imaging of pH probes. Methods Cell Biol. 123, 429–448 (2014).

    Article  Google Scholar 

  11. 11.

    Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195, 231–272 (1992).

    CAS  Article  Google Scholar 

  12. 12.

    Balgi, A. D. et al. Regulation of mTORC1 signaling by pH. PLoS ONE 6, e21549 (2011).

    ADS  CAS  Article  Google Scholar 

  13. 13.

    Chiche, J. et al. In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH. Int. J. Cancer 130, 1511–1520 (2012).

    CAS  Article  Google Scholar 

  14. 14.

    Fellenz, M. P. & Gerweck, L. E. Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity. Radiat. Res. 116, 305–312 (1988).

    ADS  CAS  Article  Google Scholar 

  15. 15.

    Chambard, J. C. & Pouyssegur, J. Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0→G1 transition of fibroblasts. Exp. Cell Res. 164, 282–294 (1986).

    CAS  Article  Google Scholar 

  16. 16.

    Yoon, J. K. & Wold, B. The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm. Genes Dev. 14, 3204–3214 (2000).

    CAS  Article  Google Scholar 

  17. 17.

    Chal, J. et al. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat. Biotechnol. 33, 962–969 (2015).

    CAS  Article  Google Scholar 

  18. 18.

    Chal, J. et al. Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nat. Protoc. 11, 1833–1850 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Diaz-Cuadros, M. et al. In vitro characterization of the human segmentation clock. Nature 580, 113–118 (2020).

    ADS  CAS  Article  Google Scholar 

  20. 20.

    Nusse, R. & Clevers, H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 169, 985–999 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Chalamalasetty, R. B. et al. Mesogenin 1 is a master regulator of paraxial presomitic mesoderm differentiation. Development 141, 4285–4297 (2014).

    CAS  Article  Google Scholar 

  22. 22.

    Aulehla, A. et al. Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395–406 (2003).

    CAS  Article  Google Scholar 

  23. 23.

    Nowotschin, S., Ferrer-Vaquer, A., Concepcion, D., Papaioannou, V. E. & Hadjantonakis, A. K. Interaction of Wnt3a, Msgn1 and Tbx6 in neural versus paraxial mesoderm lineage commitment and paraxial mesoderm differentiation in the mouse embryo. Dev. Biol. 367, 1–14 (2012).

    CAS  Article  Google Scholar 

  24. 24.

    Aulehla, A. et al. A β-catenin gradient links the clock and wavefront systems in mouse embryo segmentation. Nat. Cell Biol. 10, 186–193 (2008).

    CAS  Article  Google Scholar 

  25. 25.

    Yamaguchi, T. P., Takada, S., Yoshikawa, Y., Wu, N. & McMahon, A. P. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev. 13, 3185–3190 (1999).

    CAS  Article  Google Scholar 

  26. 26.

    Wagman, A. S., Johnson, K. W. & Bussiere, D. E. Discovery and development of GSK3 inhibitors for the treatment of type 2 diabetes. Curr. Pharm. Des. 10, 1105–1137 (2004).

    CAS  Article  Google Scholar 

  27. 27.

    White, K. A. et al. β-Catenin is a pH sensor with decreased stability at higher intracellular pH. J. Cell Biol. 217, 3965–3976 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    Wagner, G. R. & Hirschey, M. D. Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases. Mol. Cell 54, 5–16 (2014).

    CAS  Article  Google Scholar 

  29. 29.

    Paik, W. K., Pearson, D., Lee, H. W. & Kim, S. Nonenzymatic acetylation of histones with acetyl-CoA. Biochim. Biophys. Acta 213, 513–522 (1970).

    CAS  Article  Google Scholar 

  30. 30.

    Melnik, S. et al. Cancer cell specific inhibition of Wnt/β-catenin signaling by forced intracellular acidification. Cell Discov. 4, 37 (2018).

    Article  Google Scholar 

  31. 31.

    Chapman, S. C., Collignon, J., Schoenwolf, G. C. & Lumsden, A. Improved method for chick whole-embryo culture using a filter paper carrier. Dev. Dyn. 220, 284–289 (2001).

    CAS  Article  Google Scholar 

  32. 32.

    Denans, N., Iimura, T. & Pourquié, O. Hox genes control vertebrate body elongation by collinear Wnt repression. eLife 4, e04379 (2015).

    Article  Google Scholar 

  33. 33.

    Henrique, D. et al. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790 (1995).

    ADS  CAS  Article  Google Scholar 

  34. 34.

    Krol, A. J. et al. Evolutionary plasticity of segmentation clock networks. Development 138, 2783–2792 (2011).

    CAS  Article  Google Scholar 

  35. 35.

    Buchberger, A., Bonneick, S. & Arnold, H. Expression of the novel basic-helix-loop-helix transcription factor cMespo in presomitic mesoderm of chicken embryos. Mech. Dev. 97, 223–226 (2000).

    CAS  Article  Google Scholar 

  36. 36.

    Diez del Corral, R., Breitkreuz, D. N. & Storey, K. G. Onset of neuronal differentiation is regulated by paraxial mesoderm and requires attenuation of FGF signalling. Development 129, 1681–1691 (2002).

    PubMed  Google Scholar 

  37. 37.

    Iimura, T. & Pourquié, O. Manipulation and electroporation of the avian segmental plate and somites in vitro. Methods Cell Biol. 87, 257–270 (2008).

    CAS  Article  Google Scholar 

  38. 38.

    Xiong, F. et al. Specified neural progenitors sort to form sharp domains after noisy Shh signaling. Cell 153, 550–561 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Chal, J. et al. Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro. Development 145, dev157339 (2018).

    Article  Google Scholar 

Download references

Acknowledgements

We thank N. Perrimon and members of the Pourquié laboratory for critical reading of the manuscript and discussions. Y.H. acknowledges Grant-in-Aid for JSPS Fellows (29-456). Research in the Pourquié laboratory was funded by a grant from the US National Institutes of Health (5R01HD085121). F.X. acknowledges the National Institutes of Health K99 award HD092582. This work was supported by AMED (JP19gm5010001 to T.I.), a Grant-in-Aid for Scientific Research on Innovative Areas (25117720 to T.I. and 19H04768 to M.O.), Scientific Research (B) (16H05141 and 19H03412 to T.I.) and Scientific Research (C) (19K06673 to M.O.).

Author information

Affiliations

Authors

Contributions

M.O. designed, performed and analysed the chicken embryo experiments and the in vitro acetylation study of purified β-catenin with help from O.A.T. Y.H. designed, performed and analysed the human iPS cell experiments with help from M.D.-C. and O.A.T. A.M. performed the in vivo BCECF pH measurements. F.X. performed the in vivo quantitative analysis of pHi. O.P. wrote the manuscript and supervised the project. All authors discussed and agreed on the results and commented on the manuscript.

Corresponding author

Correspondence to Olivier Pourquié.

Ethics declarations

Competing interests

O.P. is a scientific founder of Anagenesis Biotechnologies. The other authors declare no competing interests.

Additional information

Peer review information Nature thanks Matthew Hirschey, Jacques Pouyssegur and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Analysis of the pHi in the chicken embryo in vivo.

a, Ratiometric live expression of pHluorin (488/405 nm) detected in the posterior domain of electroporated embryos exposed to different pH buffers and nigericin and valinomycin (n = 7). Fluorescence intensity is shown by a pseudocolour image (Fire colour) using ImageJ. The yellow signal indicates lower pH. The ventral view shows the anterior region to the left. Scale bars, 100 μm. b, Each dot represents the average 488/405-nm signal ratio of about 300 single cells segmented in one embryo (n = 2 for pH 5.5, n = 3 for pH 6.5 and n = 2 for pH 7.5). Embryos were treated for 20 min in different pH buffers with the protonophores nigericin and valinomycin, before live imaging. The red horizontal bar is the mean and error bar is the s.d. c, Micro-dissected posterior PSM incubated with 20 μM BCECF (n = 7). Fluorescence intensities for excitation at 405 nm (blue) and 488 nm (green) are shown (left). In addition, the 488/405-nm ratio is shown (right). Scale bars, 100 μm. A, anterior; P, posterior. d, Fluorescence 488/405-nm ratios along the PSM. Each coloured line corresponds to an explant (n = 7 from two independent experiments). med, medial PSM; pos, posterior region. Two-sided, paired t-test. **P = 0.009. e, f, Whole-mount in situ hybridization of 2-day-old chicken embryos cultured at different pH and hybridized with MSGN1 (pH 5.3: n = 4, pH 7.2: n = 3 and pH 7.6: n = 3) (e) and SAX1 (pH 5.3: n = 6, pH 7.2: n = 4 and pH 7.6: n = 5) (f). The ventral view shows the anterior region to the top. Scale bars, 100 μm. Source Data

Extended Data Fig. 2 Lowering the pHi can reversibly slow down embryo elongation.

a, b, Snapshots of 2-day-old chicken embryos cultured in minimal medium at pH 7.2 with 8.3 mM (n = 6) glucose (a) or 0.83 mM glucose (n = 3) (b). c, d, Snapshots of 2-day-old chicken embryos cultured in minimal medium with 8.3 mM glucose in acidic conditions (pH 6.0: n = 6 (c) and pH 5.3: n = 6 (d)). e, Snapshots of a 2-day-old chicken embryo first cultured in minimal medium with 8.3 mM glucose at pH 5.3 showing the arrest of elongation after 9 h, and returned to control medium after 10.5 h showing the rescue of elongation (n = 6). All panels show bright-field micrographs of the posterior region of chicken embryos taken at 1.5-h intervals. Somites formed at the last time point are indicated by asterisks on the right. The yellow asterisks mark the last somite at the beginning of the culture and the white asterisks are the somites produced during the culture period. The ventral views show the anterior region to the top. Scale bars, 100 μm.

Extended Data Fig. 3 Inhibiting lactate transporters downregulates WNT signalling.

a, Whole-mount in situ hybridization of a 2-day-old chicken embryo hybridized with MCT1 (n = 4). Scale bar, 100 μm. b, Comparison of lactate amounts in cellular extracts of the posterior region of 2-day-old chicken embryos cultured for 10 h in chemically defined medium with or without 5 mM CNCn (n = 3). Mean ± s.d. is shown. Two-sided, unpaired t-test, P = 0.0292. *P < 0.05. c, Whole-mount in situ hybridization of 2-day-old chicken embryos cultured with 0 mM (control (CTL)) or 5 mM CNCn and hybridized with AXIN2 (control: n = 8, 5 mM CNCn: n = 7). Scale bars, 100 μm. d, qPCR analysis of MSGN1, SAX1, SOX2, T and AXIN2 expression in the posterior region of 2-day-old chicken embryos cultured with or without 5 mM CNCn (n = 3 for each gene). Data were normalized by control samples. Mean ± s.d. is shown. Two-sided, unpaired t-test. AXIN2: P = 0.0197, T: P = 0.0270, SOX2: P = 0.0458. *P < 0.05. e, Comparison of AXIN2 and SOX1 mRNA expression in day 2 human iPS cells differentiated in vitro and cultured for 24 h in CL medium containing 5 mM CNCn or vehicle control (DMSO). n = 3 biological replicates. Mean ± s.d. is shown. Two-way ANOVA followed by Tukey’s multiple comparisons test: ***P = 0.0004 and **P = 0.0067. n = 3. f, Western blot analysis using anti-acetylated K49 β-catenin, anti-active β-catenin, anti-actin and anti-β-catenin of whole-cell extracts of 2-day-old chicken embryos cultured in chemically defined medium with 0 or 5 mM CNCn for 10 h (n = 3). For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 4 Kinetics of WNT–β-catenin signalling during human iPS cell differentiation to the PSM.

a, Immunohistochemistry showing the dynamic expression of β-catenin and Venus (YFP) proteins in human MSGN1-Venus iPS reporter cells differentiated to the PSM fate in vitro (n = 3). Hoechst labelling of the nuclei is shown in blue. Scale bars, 30 μm. b, Quantification of the intensity of nuclear localization of β-catenin shown in a using Fiji. Mean ± s.d. is shown (D0, D1 and D4: n = 3; D2 and D3: n = 4). One-way ANOVA followed by Tukey’s multiple comparisons test: D1 versus D2: P = 0.0411, D2 versus D3: P = 0.0038, D2 versus D4: P = 0.0009. *P < 0.05, **P < 0.01 and ***P < 0.001. ce, qPCR analysis comparing the expression level of MSGN1 (c), TBX6 (d) and AXIN2 (e) of human iPS cells differentiating to the PSM fate in vitro. Values were normalized by the results of differentiation at D0. Mean ± s.d. is shown (n = 3). One-way ANOVA followed by Tukey’s multiple comparisons test: TBX6 D0 versus D2: P < 0.0001, D0 versus D3: P < 0.0001, D3 versus D4: P < 0.0001, D4 versus D10: P = 0.0466; AXIN2 D0 versus D1: P = 0.0006, D0 versus D3: P < 0.0001, D3 versus D4: P = 0.0003, D4 versus D10: P = 0.0318 *P < 0.05, ***P < 0.001 and ****P < 0.0001. Source Data

Extended Data Fig. 5 Sodium acetate treatment increases WNT–β-catenin signalling in vivo and in vitro.

a, Western blot analysis using anti-acetylated K49 β-catenin, anti-active β-catenin, anti-actin and anti-β-catenin. Whole-cell extracts of day 2 human iPS cells differentiated to PSM in vitro in CL medium and treated with sodium acetate (SA) for 24 h (n = 3). b, qPCR analysis of SOX1 and MSGN1 mRNA expression in day 2 human iPS cells differentiated to PSM in vitro and treated with SA in CL medium for 24 h. Mean ± s.d. is shown (n = 3). Two-way ANOVA followed by Tukey’s multiple comparisons test. MSGN1 control versus 10 mM SA: P = 0.003. **P < 0.01. c, Western blot analysis using anti-acetylated K49 β-catenin, anti-active β-catenin, anti-actin and anti-β-catenin of whole-cell extracts of 2-day-old chicken embryos cultured in chemically defined medium with 0 or 10 mM SA for 10 h (n = 3). d, Whole-mount in situ hybridization of 2-day-old chicken embryos cultured with 0 or 10 mM SA and hybridized with AXIN2 (control: n = 5 and 10 mM SA: n = 7). Scale bars, 100 μm. e, qPCR analysis of AXIN2, MSGN1, SOX2 and SAX1 expression in the posterior region of 2-day-old chicken embryos cultured with 0 or 10 mM SA. Data were normalized by control samples. Mean ± s.d. is shown (n = 4). Two-sided, unpaired t-test. AXIN2: P = 0.0070 and MSGN1: P = 0.0298. *P < 0.01 and **P < 0.001. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 6 β-Catenin acetylation depends on pHi and glycolytic activity.

a, b, Western blot analysis using anti-acetylated K49 β-catenin, anti-active β-catenin, anti-actin and anti-β-catenin. Extracts of 2-day-old chicken embryos cultured in minimal medium with 8.3 mM glucose at various pH (n = 3 per condition) (a) or in minimal medium at pH 7.2 with various glucose concentrations (n = 4 per condition) (b). c, Quantification of acetylated lysine and β-catenin intensity in Fig. 3h using Fiji. The acetylation rate is calculated from the slope of the graph. n = 2 independent experiments. Linear approximation, mean ± s.d. of the slope. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 7 Calibration curve used for quantifying pHi variations as a function of pHe in differentiating human iPS cells.

Calibration curve obtained for the pH measurements in differentiated iPS cells in vitro using BCECF as described in the Methods. n = 6 independent experiments. Source Data

Supplementary information

Supplementary Figure 1

Uncropped data from gels shown in Figure 3a-h and Extended Data Figures 3f, 5a,c, 6a-b.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Oginuma, M., Harima, Y., Tarazona, O.A. et al. Intracellular pH controls WNT downstream of glycolysis in amniote embryos. Nature (2020). https://doi.org/10.1038/s41586-020-2428-0

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.