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Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells.

Kim H, Kim S, Song Y, Kim W, Ying QL, Jho EH - Stem Cells Int (2015)

Bottom Line: Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells.Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation.Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul 130-743, Republic of Korea ; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

ABSTRACT
Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus

Increase in neural differentiation of precursor cells by GSK3β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μM) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μM) from days 0 to 3. (g) FACS analysis of Sox1-GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1-GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μM) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μm.
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fig3: Increase in neural differentiation of precursor cells by GSK3β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μM) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μM) from days 0 to 3. (g) FACS analysis of Sox1-GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1-GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μM) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μm.

Mentions: Based on data described above (Figures 1 and 2), we asked whether time window of Wnt/β-catenin signaling affects neural differentiation. For this, we sequentially treated with BIO (GSK3β inhibitor) for indicated duration as described in Figure 3 [24]. Addition of BIO in 46C ES cells from day 0 to day 6 completely diminished EGFP expression compared to MeBIO treatment, a control analog of BIO which displays minimal activity against GSK3β (Figures 3(a) and 3(b)). These results were corroborated by FACS analysis (Figures 3(g) and 3(h)). Similar to report that activation of canonical Wnt signaling by GSK3β inhibition maintains pluripotency of ES cell, we found that BIO treatment could enhance the expression of stemness marker gene such as Nanog under differentiation conditions (Figure 3(i)) [18–20, 28]. On the other hand, treatment with BIO from day 4 to day 6 surprisingly increased EGFP intensity and EGFP-positive cells (Figures 3(f)–3(h)). To test whether or not the increase in EGFP expression by BIO can be attributed to actual neural differentiation, we compared the mRNA expression levels of marker genes in cells treated or untreated with BIO. We observed that the expression Sox1 mRNA was increased in ES cells after BIO treatment from day 4 compared to untreated cells (Figure 3(i)). These results imply that activation of Wnt/β-catenin signaling for different time periods has differential effects on neural differentiation of mES cells.


Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells.

Kim H, Kim S, Song Y, Kim W, Ying QL, Jho EH - Stem Cells Int (2015)

Increase in neural differentiation of precursor cells by GSK3β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μM) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μM) from days 0 to 3. (g) FACS analysis of Sox1-GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1-GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μM) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4402205&req=5

fig3: Increase in neural differentiation of precursor cells by GSK3β inhibitor (BIO) during days 4 to 6 in N2B27 medium. (a)–(f) 46C ES cells were cultured in N2B27 medium for 6 days. GFP expression was elevated by BIO treatment (0.75 μM) from days 4 to 6, whereas GFP expression was reduced by BIO treatment (0.75 μM) from days 0 to 3. (g) FACS analysis of Sox1-GFP expression during monolayer differentiation in N2B27 medium. (h) Proportions of Sox1-GFP expressing cells as determined by FACS. (i) RT-PCR analysis showed that Nanog and Sox1 expression were elevated by BIO treatment (0.75 μM) under nondifferentiation and neural differentiation conditions, respectively. Scale bars, 100 μm.
Mentions: Based on data described above (Figures 1 and 2), we asked whether time window of Wnt/β-catenin signaling affects neural differentiation. For this, we sequentially treated with BIO (GSK3β inhibitor) for indicated duration as described in Figure 3 [24]. Addition of BIO in 46C ES cells from day 0 to day 6 completely diminished EGFP expression compared to MeBIO treatment, a control analog of BIO which displays minimal activity against GSK3β (Figures 3(a) and 3(b)). These results were corroborated by FACS analysis (Figures 3(g) and 3(h)). Similar to report that activation of canonical Wnt signaling by GSK3β inhibition maintains pluripotency of ES cell, we found that BIO treatment could enhance the expression of stemness marker gene such as Nanog under differentiation conditions (Figure 3(i)) [18–20, 28]. On the other hand, treatment with BIO from day 4 to day 6 surprisingly increased EGFP intensity and EGFP-positive cells (Figures 3(f)–3(h)). To test whether or not the increase in EGFP expression by BIO can be attributed to actual neural differentiation, we compared the mRNA expression levels of marker genes in cells treated or untreated with BIO. We observed that the expression Sox1 mRNA was increased in ES cells after BIO treatment from day 4 compared to untreated cells (Figure 3(i)). These results imply that activation of Wnt/β-catenin signaling for different time periods has differential effects on neural differentiation of mES cells.

Bottom Line: Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells.Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation.Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul 130-743, Republic of Korea ; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

ABSTRACT
Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus