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A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

Faunes F, Hayward P, Descalzo SM, Chatterjee SS, Balayo T, Trott J, Christoforou A, Ferrer-Vaquer A, Hadjantonakis AK, Dasgupta R, Arias AM - Development (2013)

Bottom Line: Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency.A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain.Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

ABSTRACT
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

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Related in: MedlinePlus

Effects of Wnt/β-catenin signaling on Nanog and Oct4. (A) Representative confocal images of E14Tg2A cells treated for 4 hours with Wnt3A-CM or CM (in N2B27) and stained for total β-catenin (green), Nanog (red) and Oct4 (blue) used for quantitative immunofluorescence (QIF). DAPI was used to identify the nuclei (white). Scale bar: 50 μm. (B-D) Line plot distributions of Nanog (B), Oct4 (C) and total β-catenin (D) obtained by QIF analysis from single sections confocal images as described in A. Fluorescence levels (grayscale) were quantified for each individual cell from four colonies (see Material and methods), binned in 20 classes spaced logarithmically (x-axis); the frequency of each bin is shown on the y-axis. Wnt3A treatment of the cells for 4 hours increases the mean levels of Nanog (mCM=38.68; mWnt=51.35), Oct4 (mCM=81.4; mWnt=135.4) and total β-catenin (mCM=8.41; mWnt=19.34). There is also a decrease in the variability of the levels upon Wnt3A treatment, as reflected by the coefficient of variation of Nanog (CVCM=0.7; CVWnt=0.54), Oct4 (CVCM=0.52; CVWnt=0.43) and total β-catenin (CVCM=0.73; CVWnt=0.57). (E) Scatterplot of the expression of Nanog and Oct4 after 24-hour exposure to Wnt3A-CM relative to serum+LIF. The black line is the linear fitting of the data for the cells in serum+LIF medium (R2: 0.1822); the red line is the linear fitting of the data for Wnt-treated cells (R2: 0.2931), indicating that Wnt signaling increases the correlation between the expression of the proteins across the population (P value=0.017, Fisher r-to-z transformation). (F) Line-plot distribution of Tcf3 QIF analysis in E14Tg2A cells treated with Wnt-CM (red) or CM (pink) for 4 hours. Quantifications were as described in Fig. 4C-E. (G) Scatterplots showing the analysis of Nanog, Oct4 or total β-catenin (x-axis) versus GFP in TLG2 (y-axis) cells grown in serum+LIF. The black lines are the linear fitting of the data (TLG2 versus Nanog R: R2=0.1530; TLG2 versus Oct4: R2=0.1428).
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Figure 5: Effects of Wnt/β-catenin signaling on Nanog and Oct4. (A) Representative confocal images of E14Tg2A cells treated for 4 hours with Wnt3A-CM or CM (in N2B27) and stained for total β-catenin (green), Nanog (red) and Oct4 (blue) used for quantitative immunofluorescence (QIF). DAPI was used to identify the nuclei (white). Scale bar: 50 μm. (B-D) Line plot distributions of Nanog (B), Oct4 (C) and total β-catenin (D) obtained by QIF analysis from single sections confocal images as described in A. Fluorescence levels (grayscale) were quantified for each individual cell from four colonies (see Material and methods), binned in 20 classes spaced logarithmically (x-axis); the frequency of each bin is shown on the y-axis. Wnt3A treatment of the cells for 4 hours increases the mean levels of Nanog (mCM=38.68; mWnt=51.35), Oct4 (mCM=81.4; mWnt=135.4) and total β-catenin (mCM=8.41; mWnt=19.34). There is also a decrease in the variability of the levels upon Wnt3A treatment, as reflected by the coefficient of variation of Nanog (CVCM=0.7; CVWnt=0.54), Oct4 (CVCM=0.52; CVWnt=0.43) and total β-catenin (CVCM=0.73; CVWnt=0.57). (E) Scatterplot of the expression of Nanog and Oct4 after 24-hour exposure to Wnt3A-CM relative to serum+LIF. The black line is the linear fitting of the data for the cells in serum+LIF medium (R2: 0.1822); the red line is the linear fitting of the data for Wnt-treated cells (R2: 0.2931), indicating that Wnt signaling increases the correlation between the expression of the proteins across the population (P value=0.017, Fisher r-to-z transformation). (F) Line-plot distribution of Tcf3 QIF analysis in E14Tg2A cells treated with Wnt-CM (red) or CM (pink) for 4 hours. Quantifications were as described in Fig. 4C-E. (G) Scatterplots showing the analysis of Nanog, Oct4 or total β-catenin (x-axis) versus GFP in TLG2 (y-axis) cells grown in serum+LIF. The black lines are the linear fitting of the data (TLG2 versus Nanog R: R2=0.1530; TLG2 versus Oct4: R2=0.1428).

Mentions: To explore this, we compared the levels of Oct4 and Nanog protein 4 hours after stimulation of Wnt signaling by Wnt3A under self-renewing conditions. Measuring the protein levels using quantitative immunofluorescence (QIF), we observed changes in the mean levels and profiles of Oct4 and Nanog (Fig. 5A-E). This supports our contention that the immediate effects of β-catenin on pluripotency are not likely to be effected through transcriptional modulation of the pluripotency network (Fig. 1). Use of QIF reveals correlations between the expression of different proteins in single cells and avoids the population averaging associated with western blotting. This analysis reveals a correlation between Nanog and Oct4 at the level of single cells that is increased by Wnt/β-catenin signaling (Fig. 5E; supplementary material Fig. S7).


A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

Faunes F, Hayward P, Descalzo SM, Chatterjee SS, Balayo T, Trott J, Christoforou A, Ferrer-Vaquer A, Hadjantonakis AK, Dasgupta R, Arias AM - Development (2013)

Effects of Wnt/β-catenin signaling on Nanog and Oct4. (A) Representative confocal images of E14Tg2A cells treated for 4 hours with Wnt3A-CM or CM (in N2B27) and stained for total β-catenin (green), Nanog (red) and Oct4 (blue) used for quantitative immunofluorescence (QIF). DAPI was used to identify the nuclei (white). Scale bar: 50 μm. (B-D) Line plot distributions of Nanog (B), Oct4 (C) and total β-catenin (D) obtained by QIF analysis from single sections confocal images as described in A. Fluorescence levels (grayscale) were quantified for each individual cell from four colonies (see Material and methods), binned in 20 classes spaced logarithmically (x-axis); the frequency of each bin is shown on the y-axis. Wnt3A treatment of the cells for 4 hours increases the mean levels of Nanog (mCM=38.68; mWnt=51.35), Oct4 (mCM=81.4; mWnt=135.4) and total β-catenin (mCM=8.41; mWnt=19.34). There is also a decrease in the variability of the levels upon Wnt3A treatment, as reflected by the coefficient of variation of Nanog (CVCM=0.7; CVWnt=0.54), Oct4 (CVCM=0.52; CVWnt=0.43) and total β-catenin (CVCM=0.73; CVWnt=0.57). (E) Scatterplot of the expression of Nanog and Oct4 after 24-hour exposure to Wnt3A-CM relative to serum+LIF. The black line is the linear fitting of the data for the cells in serum+LIF medium (R2: 0.1822); the red line is the linear fitting of the data for Wnt-treated cells (R2: 0.2931), indicating that Wnt signaling increases the correlation between the expression of the proteins across the population (P value=0.017, Fisher r-to-z transformation). (F) Line-plot distribution of Tcf3 QIF analysis in E14Tg2A cells treated with Wnt-CM (red) or CM (pink) for 4 hours. Quantifications were as described in Fig. 4C-E. (G) Scatterplots showing the analysis of Nanog, Oct4 or total β-catenin (x-axis) versus GFP in TLG2 (y-axis) cells grown in serum+LIF. The black lines are the linear fitting of the data (TLG2 versus Nanog R: R2=0.1530; TLG2 versus Oct4: R2=0.1428).
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Figure 5: Effects of Wnt/β-catenin signaling on Nanog and Oct4. (A) Representative confocal images of E14Tg2A cells treated for 4 hours with Wnt3A-CM or CM (in N2B27) and stained for total β-catenin (green), Nanog (red) and Oct4 (blue) used for quantitative immunofluorescence (QIF). DAPI was used to identify the nuclei (white). Scale bar: 50 μm. (B-D) Line plot distributions of Nanog (B), Oct4 (C) and total β-catenin (D) obtained by QIF analysis from single sections confocal images as described in A. Fluorescence levels (grayscale) were quantified for each individual cell from four colonies (see Material and methods), binned in 20 classes spaced logarithmically (x-axis); the frequency of each bin is shown on the y-axis. Wnt3A treatment of the cells for 4 hours increases the mean levels of Nanog (mCM=38.68; mWnt=51.35), Oct4 (mCM=81.4; mWnt=135.4) and total β-catenin (mCM=8.41; mWnt=19.34). There is also a decrease in the variability of the levels upon Wnt3A treatment, as reflected by the coefficient of variation of Nanog (CVCM=0.7; CVWnt=0.54), Oct4 (CVCM=0.52; CVWnt=0.43) and total β-catenin (CVCM=0.73; CVWnt=0.57). (E) Scatterplot of the expression of Nanog and Oct4 after 24-hour exposure to Wnt3A-CM relative to serum+LIF. The black line is the linear fitting of the data for the cells in serum+LIF medium (R2: 0.1822); the red line is the linear fitting of the data for Wnt-treated cells (R2: 0.2931), indicating that Wnt signaling increases the correlation between the expression of the proteins across the population (P value=0.017, Fisher r-to-z transformation). (F) Line-plot distribution of Tcf3 QIF analysis in E14Tg2A cells treated with Wnt-CM (red) or CM (pink) for 4 hours. Quantifications were as described in Fig. 4C-E. (G) Scatterplots showing the analysis of Nanog, Oct4 or total β-catenin (x-axis) versus GFP in TLG2 (y-axis) cells grown in serum+LIF. The black lines are the linear fitting of the data (TLG2 versus Nanog R: R2=0.1530; TLG2 versus Oct4: R2=0.1428).
Mentions: To explore this, we compared the levels of Oct4 and Nanog protein 4 hours after stimulation of Wnt signaling by Wnt3A under self-renewing conditions. Measuring the protein levels using quantitative immunofluorescence (QIF), we observed changes in the mean levels and profiles of Oct4 and Nanog (Fig. 5A-E). This supports our contention that the immediate effects of β-catenin on pluripotency are not likely to be effected through transcriptional modulation of the pluripotency network (Fig. 1). Use of QIF reveals correlations between the expression of different proteins in single cells and avoids the population averaging associated with western blotting. This analysis reveals a correlation between Nanog and Oct4 at the level of single cells that is increased by Wnt/β-catenin signaling (Fig. 5E; supplementary material Fig. S7).

Bottom Line: Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency.A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain.Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

ABSTRACT
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

Show MeSH
Related in: MedlinePlus