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.
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Hypoxia is fine-tuned by Hif-1α and regulates mesendoderm differentiation through the Wnt/β-Catenin pathway
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The custom MATLAB code used to process the 3D segmentations of pH measurements is available at https://github.com/amichaut/pHanalysis.
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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.).
O.P. is a scientific founder of Anagenesis Biotechnologies. The other authors declare no competing interests.
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
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.
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.
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.
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. c–e, 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.
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.
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.
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.
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Oginuma, M., Harima, Y., Tarazona, O.A. et al. Intracellular pH controls WNT downstream of glycolysis in amniote embryos. Nature 584, 98–101 (2020). https://doi.org/10.1038/s41586-020-2428-0
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