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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 canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β-catenin) and β-actin antibodies. Active β-catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4-Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μM). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μm.
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fig2: Increase in canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β-catenin) and β-actin antibodies. Active β-catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4-Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μM). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μm.

Mentions: Next, we asked whether Wnt signaling activity can be regulated under self-renewal and differentiation conditions. To this end, we measured endogenous Wnt/β-catenin signaling activity under self-renewal and differentiation conditions using TOP and Wnt target genes Axin2 promoters (hereafter called Ax2P)-driven reporter construct that contain responsive TCF binding elements [26, 27]. Notably, reporter activities were low in undifferentiated mouse ES cells, whereas it was increased under absence of LIF conditions (Figure 2(a)). To monitor the change status of Wnt signaling during neural differentiation, we established reporter ES cell lines (Oct4-Gip/TOP or Ax2P-mCherry). As expected, we observed only GFP expression but not mCherry expression in self-renewal condition due to low Wnt activity (Figure 2(d)). As shown in Figure 2(e), addition of BIO, GSK3β inhibitor, enhanced expression of mCherry, indicating that this cell line reflects Wnt/β-catenin signaling. After stable cells were cultured in N2B27 medium for neural differentiation, expression of mCherry was significantly increased in neural precursor cells, whereas it was decreased in fully differentiated neurons (Figures 2(f)-2(g)). Consistently, the active β-catenin was more abundant during differentiation induced by the removal of LIF or N2B27 medium, which induces embryonic stem cells differentiation toward neuronal lineage, reaching a maximum on day 4 (Figure 2(b)). LRP6 phosphorylation on S1490, used an indicator for initial activation of Wnt/β-catenin signaling, was increased during neural differentiation (Figure 2(c)). Taken together, these data suggest that Wnt/β-catenin signaling is regulated during ES cell differentiation and neural precursor differentiation.


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 canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β-catenin) and β-actin antibodies. Active β-catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4-Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μM). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4402205&req=5

fig2: Increase in canonical Wnt signaling during neural differentiation. (a) Both TOP and Axin2 promoter luciferase activities in E14 ES cells were induced at 48 h after LIF removal. (b) Western blot analysis using ABC (active β-catenin) and β-actin antibodies. Active β-catenin level was the highest on day 4 of neural differentiation. (c) p-LRP level was the highest on day 4 of neural differentiation. (d)-(e) In Oct4-Gip/Ax2P-mCherry cells, only GFP expression was detected. mCherry expression was increased after 24 h addition of BIO (0.75 μM). (f)-(g) mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) and Ax2p-mCherry ((f) and (g)) stable cell lines during neuronal differentiation. Stable cell lines were cultured in N2B27 medium for 14 days. Scale bars, 100 μm.
Mentions: Next, we asked whether Wnt signaling activity can be regulated under self-renewal and differentiation conditions. To this end, we measured endogenous Wnt/β-catenin signaling activity under self-renewal and differentiation conditions using TOP and Wnt target genes Axin2 promoters (hereafter called Ax2P)-driven reporter construct that contain responsive TCF binding elements [26, 27]. Notably, reporter activities were low in undifferentiated mouse ES cells, whereas it was increased under absence of LIF conditions (Figure 2(a)). To monitor the change status of Wnt signaling during neural differentiation, we established reporter ES cell lines (Oct4-Gip/TOP or Ax2P-mCherry). As expected, we observed only GFP expression but not mCherry expression in self-renewal condition due to low Wnt activity (Figure 2(d)). As shown in Figure 2(e), addition of BIO, GSK3β inhibitor, enhanced expression of mCherry, indicating that this cell line reflects Wnt/β-catenin signaling. After stable cells were cultured in N2B27 medium for neural differentiation, expression of mCherry was significantly increased in neural precursor cells, whereas it was decreased in fully differentiated neurons (Figures 2(f)-2(g)). Consistently, the active β-catenin was more abundant during differentiation induced by the removal of LIF or N2B27 medium, which induces embryonic stem cells differentiation toward neuronal lineage, reaching a maximum on day 4 (Figure 2(b)). LRP6 phosphorylation on S1490, used an indicator for initial activation of Wnt/β-catenin signaling, was increased during neural differentiation (Figure 2(c)). Taken together, these data suggest that Wnt/β-catenin signaling is regulated during ES cell differentiation and neural precursor differentiation.

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