Abstract
In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.
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Data availability
Deep sequencing and ChIP–seq data that support the findings of this study have been deposited in the Gene Expression Omnnibus under accession code GSE102831. Previously published sequencing data that were re-analysed here are available under accession code E-MTAB-2958, E-MTAB-2959 (ref. 31), GSE81285 (ref. 30) and GSE31381 (ref. 52). All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
We are grateful to the PBES Lyon for technical assistance. We thank V. Azuara and D. Stupack for critical reading of the manuscript and L. Favre-Louis for technical assistance. This work was supported by institutional grants from INSERM/CNRS, Atip-avenir, Plan cancer, La ligue contre le cancer nationale et régionale (F.L.), INCa (F.L.), Fondation ARC (F.L., G.F. and D.O.), Centre Léon Bérard (F.L. and A.H.), Fondation pour la recherche médicale (F.L.), National Institutes of Health (R01-HD081534 (B.J.M.)), ANR (P.M.), ERC (P.M.) Max Planck Society (A.M.) and the DFG Forschergruppe 2722 (M.K.).
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Contributions
A.H. and G.F. performed most of the experiments in Figs. 1–7. D.O. performed experiments in Figs. 1, 5 and 7. X.G. and N.C. performed experiments in Figs. 1, 3 and 5. A.M., C.G. and C.R. performed and analysed ChIP–seq experiments. J.C. and N.R. carried out the bioinformatic analyses. M.K. and T.I. produced recombinant Netrin-1 used in Figs. 4 and 5. P.W. performed teratoma experiments. F.L., A.H., G.F. and D.O. designed experiments. F.L. initiated, designed and supervised the study. F.L. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Netrin-1 is expressed in naive pluripotent cells in vitro.
(a) Data present FPKM values for Ntn1, Ntn4, Ntn5, NtnG1 and NtnG2 in serum/Lif mESCs treated or not with Mek1/2-inh (PD), Gsk3α/β-inh (CHIR) or both (2i). 2 independent experiments. (b) Western blot depicting netrin-1 levels in human iPS cells treated similarly as (a) (3 independent experiments). (c) Netrin-1 transcripts level in indicated mESCs. Q-RTPCR data are expressed relative to mESCs as the mean ± s.d. (n = 3 independent experiments). Student’s t-test was used and two-tailed p-values are indicated. (d) Netrin-1 western blot in indicated mESCs (3 independent experiments). (e) Netrin-1 expression in single mESCs in Serum/Lif and 2i. Single-cell transcriptomic data are extracted from Kumar et al., 2014, ref. 28. n = number of cells analysed in each condition. (f) Netrin-1 mean expression in single mESCs. Data are extracted from Kumar et al., 2014, ref. 28. n = number of cells analysed in each condition. The bar represents the mean ± s.d. of netrin-1 expression in the 2 conditions. Student t-test was used and two-sided p-value is indicated.
Extended Data Fig. 2 Netrin-1 triggers pluripotency features partially overlapping with the ground state.
(a) Netrin-1 receptors transcript levels in mouse ES and iPS cells. RNA-seq data are presented as FPKM values and expressed as the mean ± s.d. (n = 3 independent experiments). (b) Netrin-1 receptors expression during early mouse development. Data, extracted from Boroviak. et al., 2015, ref. 31, present transcripts level in FPKM. Data are presented as the mean ± s.d. (n = 3 independent experiments). (c) Western blot performed in the indicated cell lines (3 independent experiments). (d) Western blot performed in the indicated mESCs grown in serum/Lif (3 independent experiments). (e) FACS analysis (FSC/SSC) of the different populations grown in serum/Lif. (f) netrin-1 expression in ES cells subpopulations. Data are extracted from Guo et al. 2016, ref. 29. Esrrb expression is used to distinguish quartiles of Esrrbhigh (>Q1) and Esrrblow (<Q1) cells. netrin-1 expression is analysed in the corresponding quartile. n = 48 total cells were analysed, 12 cells for <Q1 and 36 cells for >Q1. Student’s t-test was used and two-tailed p-value is indicated. (g) Scheme depicting exit from pluripotency assays. (h) Pictures of a single experiment representative of three independent ones. (i) Colony counting. Data are the mean ± s.d. (n = 3 independent experiments). Student’s t-test was used and two-sided p-values are indicated. (j) Q-RTPCR depicts transcript level in mESCs and EBs generated with indicated cell lines. Data are the mean ± s.d. n = 3 independent experiments with exclusion of outliers. (k) Statistical overrepresentation analysis. Panther DB was used to detect overrepresented GO within differentially expressed genes. A Fisher’s exact two-sided test was used to calculate p-values. n = 3 independent samples. (l) Proliferation curves. Data are the mean ± s.d. of 2 independent experiments. (m) Cell cycle features. Data are the mean ± s.d. (n = 2 independent experiments). (n) Violin plots displaying methylation levels for n = 1.3 M matched CpGs. Bold line indicates 25-75th percentile, white dot indicates median. (o) Dnmt3A and Dnmt3B expression levels. Data are the mean ± s.d. (n = 3 independent experiments) of normalized counts. Student’s T test was used and two-sided p-values are indicated.
Extended Data Fig. 3 Molecular cascade downstream of Netrin-1 in mESCs.
(a) Knockdown efficiency of Fak in mESCs. Q-RTPCR depicts Fak transcript level following transfection of netrin-1 mESCs with independent siRNA. Data, normalised to si#control mESCs, are the mean +/- sd of n = 3 independent experiments. Student T-test was used and two-sided p-values are indicated. (b) Effect of netrin-1 signalling on Lif sensitivity. Control and netrin-1 (WT) mESCs were serum-starved ON and stimulated with Lif for 10 mins prior to samples collection (3 independent experiments). (c) Knockdown efficiency of Ppp2ca and Ppp2r2c in mESCs. Similar settings as (a). Data, normalised to si#control mESCs, are the mean +/− sd of n = 3 independent experiments. Student T-test was used and two-sided p-values are indicated.
Extended Data Fig. 4 Endogenous Netrin-1 controls pluripotency features.
(a) Western blot in Ntn1fl/fl mESCs treated or not with 4′OH-tamoxifen (TAM) for 3 days before collection (3 independent experiments). (b) Q-RTPCR depicts Fgf5, Otx2, Gata4 and Gata6 transcript level following netrin-1 depletion in mESCs. Data are the mean +/− sd of n = 3 independent experiments. Student T-test was used and two-sided p-values are indicated. (c) Proliferation curves. The Ntn1fl/fl mESCs, treated or not with TAM, were counted at each passage in serum/Lif. Data are the mean +/- sd of n = 3 independent experiments. (d) Cell death analysis. The Ntn1fl/fl mESCs, treated or not with TAM, were grown for 2 days in N2B27+Lif before PI-AnnexinV staining was performed. The left panel presents a representative FACS profile and the right panel a graph of mean data ± s.d. (n = 3 independent experiments). Value 100% is given to the percentage of live cells in untreated Ntn1fl/fl mESCs. Student T-test was used and two-sided p-values are indicated. (e) Brightfield pictures of Ntn1fl/fl mESCs treated or not with 4’OH-tamoxifen and subsequently maintained in culture for 22 passages. Bars: 50 µm. 3 independent experiments. (f) Scheme of the crispr/cas9 guides. The grey boxes correspond to exons, and pink arrows indicate the 2 independent guides for each locus. (g) Self-renewal assay. Control and netrin-1 KO mESCs are plated at clonal density in serum+Lif (left panel) or serum+Lif+2i (right panel) for 7 days before AP positive colonies was scored. Data are mean ± s.d. (n = 3 independent experiments). Student’s t-test was used and two-sided p-values are indicated. (h) Gene expression in single blastomeres. Data, extracted from Nakamura et al., 2016 (ref. 46), correspond to FPKM values. n = 9 nanog positive cells n = 12 gata6 positive blastomeres. Each dot corresponds to a cell, the bar is the mean ± s.d. Student T-test was used and two-sided p-values are indicated.
Extended Data Fig. 5 Netrin-1 controls coordinated differentiation.
(a) Neo1 and Unc5B expression in epiblast-like cells (EpiLC). Q-RTPCR data are expressed relative to mESCs as the mean ± s.d. (n = 3 independent experiments). (b) Pictures of teratoma obtained following injection of Ntn1fl/fl mESCs treated (right panel) or not (left panel) with TAM 24 hours prior to injection. 4 independent teratoma per condition were analysed. (c) Q-RTPCR depicts Nestin and βIII-tubulin levels at day 8 of differentiation in N2B27-Lif. Data are normalized to housekeeping genes and value 1 is given to day8 Ctrl mESCs. Data are the mean ± s.d. (n = 3 independent experiments). Student’s t-test was used and two-sided p-values are indicated. (d) Cell death analysis. The Ntn1fl/fl mESCs, treated or not with TAM, were grown for 2 days in N2B27-Lif before PI-AnnexinV staining was performed. The left panel present a representative FACS profile and the right panel a graph of mean data ± s.d. (n = 3 independent experiments). Value 100% is given to the percentage of live cells in untreated Ntn1fl/fl mESCs. Student T-test was used and two-sided p-values are indicated.
Supplementary information
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Huyghe, A., Furlan, G., Ozmadenci, D. et al. Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling. Nat Cell Biol 22, 389–400 (2020). https://doi.org/10.1038/s41556-020-0483-2
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DOI: https://doi.org/10.1038/s41556-020-0483-2
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