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First published online 11 March 2009
doi: 10.1242/dev.033951

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Activin/Nodal signalling maintains pluripotency by controlling Nanog expression

Ludovic Vallier^1,*, Sasha Mendjan¹, Stephanie Brown¹, Zhenzhi Chng¹, Adrian Teo¹, Lucy E. Smithers^2,, Matthew W. B. Trotter^1,2, Candy H.-H. Cho¹, Amelie Martinez³, Peter Rugg-Gunn^1,, Gabrielle Brons¹ and Roger A. Pedersen¹

¹ Department of Surgery and Laboratory For Regenerative Medicine, West Forvie Building, Robinson Way, University of Cambridge, Cambridge CB2 0SZ, UK.
² CR-UK Viral Oncology Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK.
³ Laboratoire de transfert de gènes dans le foie: applications thérapeutiques. Inserm U804, Université Paris XI, 94276 Le Kremlin Bicêtre, France.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Expression of NANOG in hESCs depends on Activin/Nodal signalling. (A) Microarray gene expression heat map comparing hESCs grown in CDM supplemented with Activin and FGF (hESC) with hESCs grown for 48 hours in CDM supplemented with FGF and SB431542 (hESCs+SB). For each gene (row), the heat map colours depict gene expression in units of standard deviation from the mean across all samples (columns). Upregulation is coloured in shades of red and downregulation in shades of blue. Gene names marked with an asterisk denote genes that did not pass a significant differential regulation threshold with a false discovery rate (FDR) of 1% when global sample group expression profiles were compared. (B) Promoter regions of the NANOG gene responsive to Activin/Nodal signalling. Luciferase reporter genes containing different sized fragments of the NANOG promoter were co-transfected into H9 cells along with renilla expression vector in the presence of Activin and FGF (A + F), or in the presence of Activin and FGF along with an expression vector for SMAD3 (A + F + S3), or in the presence of SB431542 (negative control, SB). Firefly luciferase activity (normalised to renilla luciferase activity) is expressed as mean±s.d. from three independent experiments. (C) Mutation of putative Smad2/3-binding sites in the NANOG promoter inhibits the transcriptional activation induced by Activin/Nodal signalling. Luciferase reporter genes containing the promoter of the human NANOG gene (-379 to +18) with or without mutated Smad2/3-binding sites were co-transfected into H9 cells along with the renilla expression vector in the presence of Activin and FGF (A + F), or in the presence of SB431542 (negative control, SB). Firefly luciferase activity (normalised to renilla luciferase activity) is expressed as mean±s.d. from three independent experiments. (D) Genomic regions of the NANOG gene bound by NANOG and SMAD2/3 proteins. ChIP assays were performed using antibodies directed against SMAD2/3 or NANOG. The immunoprecipitated DNA was then amplified using quantitative PCR and specific primers to detect enrichment in the denoted genomic regions. Results were normalised against control region H (-6237 to +6414) and are expressed as mean±s.d. from three experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Constitutive expression of Nanog is necessary and sufficient to prevent neuroectoderm differentiation of hESCs induced by inhibition of Activin/Nodal signalling. (A) Constitutive expression of NANOG blocks neuroectoderm differentiation. H9 cells (hESCs) and NANOG-hESC subline 2 (Nanog) were grown for 7 days or for 28 days in CDM supplemented with SB431542 and FGF2 and then real-time PCR was performed to detect the expression of the genes denoted. H9 cells grown in Activin and FGF2 were used as normalisation controls. (B) Immunofluorescence analysis for the co-expression of the pluripotency markers OCT4, NANOG and SOX2 in hESCs (left panels) and in NANOG-hESCs (right panels) grown for 7 days in CDM supplemented with SB431542 and FGF2. Scale bar: 50 µm. (C) FACS analysis showing the percentage of cells expressing the pluripotency marker Tra-1-60 (upper panel) or the neuroectoderm marker NCAM (lower panel). The first column shows wild-type hESCs cultured in conditions maintaining pluripotency (CDM supplemented with Activin and FGF2), and the second column in neuroectoderm-inducing conditions (CDM supplemented with SB431542 and FGF2). The subsequent six columns show NANOG-hESCs (sublines 1, 2 and 3) grown either in pluripotency-maintaining or neuroectoderm-inducing (SB) conditions. (D) Neuroectoderm differentiation depends on FGF signalling, and the Nanog inhibitory effect is independent of BMP4 signalling. H9 cells (hESCs) and NANOG-hESC subline 2 (Nanog-hESCs) were grown for 7 days in CDM supplemented with SB431542 and FGF2; 10 µM SB431542 and 10 µM SU5402; 10 µM SB431542, 12 ng/ml FGF2 and 10 ng/ml BMP4; or 10 µM SB431542, 12 ng/ml FGF2 and 200 ng/ml Noggin. Real-time PCR was then performed to detect the expression of the genes denoted. H9 cells grown in CDM supplemented with Activin and FGF2 were used as normalisation controls.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Knockdown of NANOG expression in hESCs induces the expression of neuroectoderm markers. (A) Expression of NANOG in shRNA-NANOG-hESC sublines. shRNA-NANOG-hESCs (SiNanog) were grown for two passages in CDM supplemented with Activin and FGF and then real-time PCR analyses were performed to determine the level of Nanog transcripts. hESC lines expressing a scrambled shRNA were used as negative controls (SiControl). (B) Absence of NANOG protein in hESCs stably expressing shRNA directed against NANOG mRNA sequence (shRNA-treated, top; DAPI-stained nuclei, bottom). Scale bar: 100 µm. (C) Knockdown of NANOG mRNA increased the expression of neuroectoderm markers. The expression of the genes denoted was analysed in shRNA-NANOG-hESCs using real-time PCR. (D) Expression of neuroectoderm markers in shRNA-NANOG-hESCs. shRNA-NANOG-hESCs (SiNanog) were grown for 10 passages in CDM supplemented with Activin and FGF and then immunostaining analyses were performed to detect the expression of OCT4, PAX6 and SOX1 proteins. Scale bar: 100 µm. (E) The expression of neuroectoderm markers provoked by NANOG knockdown depends on FGF signalling. Control H9 shRNA-hESCs (Scramble) and shRNA-NANOG-hESC subline 1 (SiNanog) were grown for 7 days in CDM supplemented with Activin and FGF2, or with Activin and 10 µM SU5402, and then real-time PCR was performed to detect the expression of the genes denoted.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Nanog is not sufficient to prevent BMP4-induced extraembryonic differentiation. (A) Immunofluorescence analysis for the co-expression of Nanog (top row) and OCT4 or extraembryonic markers (middle row) in NANOG-hESCs grown in the presence of BMP4. Bottom row shows DAPI-stained nuclei. Scale bar: 50 µm. (B) Expression of extraembryonic markers in wild-type and NANOG-hESCs grown in the presence of BMP4. H9 cells (hESCs) and NANOG-hESCs (subline 11) were grown for 4 days in culture conditions maintaining pluripotency or for 8 days in the presence of BMP4 to induce extraembryonic differentiation. Then real-time PCR was performed to detect the expression of the genes denoted.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Nanog inhibits endoderm differentiation by limiting the transcriptional activity of the Activin/Nodal signalling pathway. (A) Nanog expression is compatible with mesendoderm specification. Immunofluorescence analysis for the co-expression of NANOG and the mesendoderm marker brachyury in hESCs and in NANOG-hESCs grown in culture conditions inducing mesendoderm differentiation. Scale bar: 50 µm. (B) Expression of mesendoderm markers in wild-type and NANOG-hESCs differentiated into mesendoderm-like cells. H9 cells (hESCs) and NANOG-hESCs (subline 11) were grown for 4 days in culture conditions maintaining pluripotency or for 8 days in culture conditions inducing mesendoderm differentiation. Then, real-time PCR was performed to detect the expression of the genes denoted. H9 cells grown in CDM supplemented with Activin and FGF2 were used as normalisation controls. (C) FACS analysis showing the percentage of hESCs expressing the definitive endoderm marker CXCR4 and the mesendoderm/mesoderm marker PDGFR. H9 cells and NANOG-hESCs (sublines 1, 2) were grown for 7 days in culture conditions driving the differentiation of hESCs into mesendoderm progenitors and then the expression of CXCR4 and PDGFR was analysed using FACS. hESCs and NANOG-hESCs grown in CDM supplemented with Activin and FGF2 were used as negative controls. (D) A high dose of Activin, BMP or FGF is not sufficient to bypass the inhibitory effect of Nanog on endoderm differentiation. H9 cells (hESCs) and NANOG-hESCs (subline 11) were grown for 4 days in culture conditions maintaining pluripotency or for 8 days in culture conditions inducing mesendoderm differentiation with increasing doses of Activin (100 ng/ml, 250 ng/ml, 500 ng/ml), BMP (10 ng/ml, 50 ng/ml) and FGF2 (20 ng/ml, 100 ng/ml). Then, real-time PCR was performed to detect the expression of the genes denoted. H9 cells grown in CDM supplemented with Activin and FGF2 were used as normalisation controls. (E) Effect of Nanog on Smad transcriptional activity. A reporter gene for the transcriptional activity of Activin/Nodal signalling (containing four Smad-binding elements, SBE4) was co-transfected into H9 cells, in CDM supplemented with Activin or in CDM supplemented with SB431542, along with the renilla expression vector and with the expression vectors listed below the chart. Firefly luciferase activity (normalised to renilla luciferase activity) is expressed as mean±s.d. from three independent experiments. (F) Nanog protein interacts with Smad2/3. Co-immunoprecipitation of endogenous NANOG (hNanog) with SMAD2/3 (left panel) and of transfected HA-Flag-hNanog with Smad2/3 (right panels). Immunoprecipitations (IPs) were performed on nuclear extracts of hESCs grown in CDM supplemented with Activin and FGF2, or in the presence of SB431542. Input nuclear extracts (Input NE) and IP lanes were probed with the indicated antibodies. Loading percentage of the total material is indicated.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Model explaining the regulation of Nanog in hESCs/mEpiSCs and its function in both cell types. Nanog expression in hESCs, mEpiSCs and post-implantation embryos is controlled by Activin/Nodal signalling and, in turn, Nanog prevents neuroectoderm differentiation induced by FGF signalling. However, Nanog also decreases the transcriptional activity of Smad2/3 proteins to limit the positive effect of the Activin/Nodal signalling on the progression of mesendoderm differentiation towards definitive endoderm. This feedback loop generates stasis of both neuroectoderm and mesendoderm differentiation, resulting in pluripotency of hESCs and mEpiSCs.

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