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Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

Mora-Castilla S, Tejedo JR, Tapia-Limonchi R, Díaz I, Hitos AB, Cahuana GM, Hmadcha A, Martín F, Soria B, Bedoya FJ - Stem Cells Int (2014)

Bottom Line: Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment.Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression.Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), University Pablo de Olavide, Biomedical Research Network (CIBER) of Diabetes and Related Metabolic Diseases, Red-Tercel, Avenida Américo Vespucio S/N, 41092 Seville, Spain ; Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.

ABSTRACT
The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

No MeSH data available.


Related in: MedlinePlus

Nanog is expressed in differentiated cells. (a) Western blot analysis shows endoderm markers and Nanog reexpression after NO treatment. Blots are representative of three independent experiments. C: control cells cultured with LIF for 4 days; T: cells cultured with LIF for 3 days and then exposed to 1 mM DETA/NO for 19 h. Days 5 and 6 refer to DETA/NO treated cells cultured in the absence of LIF for additional periods of 1-2 days. (b) Endoderm markers are expressed in both Oct4 positive and negative cell populations after in vitro differentiation. Real-time PCR analysis for expression of pluripotent (Oct4 and Nanog) and endoderm (Gata4, FoxA2, Cxcr4, Pdx1, Sox17) markers in control (undifferentiated cells grown for 4 days in the presence of LIF), treated and DE differentiated GFP positive and GFP negative cells. D3-Oct4 cells were grown, differentiated, and sorted for GFP expression according to the protocol described in Section 2. Control cells indicate cells grown for 4 days in the presence of LIF; NO-treated cells indicate cells maintained for 3 days in the absence of LIF and treated for 19 h with 1 mM DETA/NO. DE-differentiated cells indicate cells 2 days after treatment in the absence of LIF.  *Significant difference from GFP+;  **significantly different from control condition. (c) Quantitative real-time PCR for mesodermal lineage markers expressed before and after DETA/NO treatment.
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fig3: Nanog is expressed in differentiated cells. (a) Western blot analysis shows endoderm markers and Nanog reexpression after NO treatment. Blots are representative of three independent experiments. C: control cells cultured with LIF for 4 days; T: cells cultured with LIF for 3 days and then exposed to 1 mM DETA/NO for 19 h. Days 5 and 6 refer to DETA/NO treated cells cultured in the absence of LIF for additional periods of 1-2 days. (b) Endoderm markers are expressed in both Oct4 positive and negative cell populations after in vitro differentiation. Real-time PCR analysis for expression of pluripotent (Oct4 and Nanog) and endoderm (Gata4, FoxA2, Cxcr4, Pdx1, Sox17) markers in control (undifferentiated cells grown for 4 days in the presence of LIF), treated and DE differentiated GFP positive and GFP negative cells. D3-Oct4 cells were grown, differentiated, and sorted for GFP expression according to the protocol described in Section 2. Control cells indicate cells grown for 4 days in the presence of LIF; NO-treated cells indicate cells maintained for 3 days in the absence of LIF and treated for 19 h with 1 mM DETA/NO. DE-differentiated cells indicate cells 2 days after treatment in the absence of LIF.  *Significant difference from GFP+;  **significantly different from control condition. (c) Quantitative real-time PCR for mesodermal lineage markers expressed before and after DETA/NO treatment.

Mentions: In order to assess the impact of Nanog and Oct4 reexpression on the differentiation process, the expression of definitive endoderm (DE) markers was analyzed in a defined protocol for generation of DE cells. Figure 3(a) shows a remarkable downregulation of Nanog and an increase in Pdx1 expression upon DETA/NO treatment in mESC. In addition, differentiated cell populations reexpress Nanog and increase expression of DE factors such as Gata4 and Sox17 (Figure 3(a)) 1 and 2 days after NO treatment. Pdx1 expression levels, however, decrease after NO treatment (Figure 3(a)). To assess the relevance of Oct4 reexpression on upregulation of DE markers, a mESC line expressing GFP under the control of Oct4 promoter was used. Following differentiation, sorted GFP positive and GFP negative cells were analyzed for expression of Nanog, Oct4, and different endoderm markers (Figure 3(b)). NO treatment induces downregulation of Oct4 and Nanog and upregulation of endoderm markers Gata4, FoxA2, Cxcr4, and Pdx1 in both GFP negative cells and in residual GFP positive cells (Figure 3(b), upper and lower left graphs). Only two days after exposure to differentiating stimulus, both GFP positive and negative cells upregulate Gata4, FoxA2, Hnf1β, and Sox17, with significantly higher levels in GFP negative cell population (Figure 3(b), upper and lower right graphs). In addition, in order to help understand the differentiation phase, mesodermal markers Flk1 and Mef2c were analyzed by quantitative real-time PCR. In the presence of LIF the expression of both markers decreases following exposure to DETA/NO, and upregulates after 2 days. On the other hand, in absence of LIF the treatment induces Flk1 expression and maintains the increase for 2 days more; nevertheless, Mef2c also increases its expression after NO but the increase is not sustained at day 6 (Figure 3(c)).


Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

Mora-Castilla S, Tejedo JR, Tapia-Limonchi R, Díaz I, Hitos AB, Cahuana GM, Hmadcha A, Martín F, Soria B, Bedoya FJ - Stem Cells Int (2014)

Nanog is expressed in differentiated cells. (a) Western blot analysis shows endoderm markers and Nanog reexpression after NO treatment. Blots are representative of three independent experiments. C: control cells cultured with LIF for 4 days; T: cells cultured with LIF for 3 days and then exposed to 1 mM DETA/NO for 19 h. Days 5 and 6 refer to DETA/NO treated cells cultured in the absence of LIF for additional periods of 1-2 days. (b) Endoderm markers are expressed in both Oct4 positive and negative cell populations after in vitro differentiation. Real-time PCR analysis for expression of pluripotent (Oct4 and Nanog) and endoderm (Gata4, FoxA2, Cxcr4, Pdx1, Sox17) markers in control (undifferentiated cells grown for 4 days in the presence of LIF), treated and DE differentiated GFP positive and GFP negative cells. D3-Oct4 cells were grown, differentiated, and sorted for GFP expression according to the protocol described in Section 2. Control cells indicate cells grown for 4 days in the presence of LIF; NO-treated cells indicate cells maintained for 3 days in the absence of LIF and treated for 19 h with 1 mM DETA/NO. DE-differentiated cells indicate cells 2 days after treatment in the absence of LIF.  *Significant difference from GFP+;  **significantly different from control condition. (c) Quantitative real-time PCR for mesodermal lineage markers expressed before and after DETA/NO treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Nanog is expressed in differentiated cells. (a) Western blot analysis shows endoderm markers and Nanog reexpression after NO treatment. Blots are representative of three independent experiments. C: control cells cultured with LIF for 4 days; T: cells cultured with LIF for 3 days and then exposed to 1 mM DETA/NO for 19 h. Days 5 and 6 refer to DETA/NO treated cells cultured in the absence of LIF for additional periods of 1-2 days. (b) Endoderm markers are expressed in both Oct4 positive and negative cell populations after in vitro differentiation. Real-time PCR analysis for expression of pluripotent (Oct4 and Nanog) and endoderm (Gata4, FoxA2, Cxcr4, Pdx1, Sox17) markers in control (undifferentiated cells grown for 4 days in the presence of LIF), treated and DE differentiated GFP positive and GFP negative cells. D3-Oct4 cells were grown, differentiated, and sorted for GFP expression according to the protocol described in Section 2. Control cells indicate cells grown for 4 days in the presence of LIF; NO-treated cells indicate cells maintained for 3 days in the absence of LIF and treated for 19 h with 1 mM DETA/NO. DE-differentiated cells indicate cells 2 days after treatment in the absence of LIF.  *Significant difference from GFP+;  **significantly different from control condition. (c) Quantitative real-time PCR for mesodermal lineage markers expressed before and after DETA/NO treatment.
Mentions: In order to assess the impact of Nanog and Oct4 reexpression on the differentiation process, the expression of definitive endoderm (DE) markers was analyzed in a defined protocol for generation of DE cells. Figure 3(a) shows a remarkable downregulation of Nanog and an increase in Pdx1 expression upon DETA/NO treatment in mESC. In addition, differentiated cell populations reexpress Nanog and increase expression of DE factors such as Gata4 and Sox17 (Figure 3(a)) 1 and 2 days after NO treatment. Pdx1 expression levels, however, decrease after NO treatment (Figure 3(a)). To assess the relevance of Oct4 reexpression on upregulation of DE markers, a mESC line expressing GFP under the control of Oct4 promoter was used. Following differentiation, sorted GFP positive and GFP negative cells were analyzed for expression of Nanog, Oct4, and different endoderm markers (Figure 3(b)). NO treatment induces downregulation of Oct4 and Nanog and upregulation of endoderm markers Gata4, FoxA2, Cxcr4, and Pdx1 in both GFP negative cells and in residual GFP positive cells (Figure 3(b), upper and lower left graphs). Only two days after exposure to differentiating stimulus, both GFP positive and negative cells upregulate Gata4, FoxA2, Hnf1β, and Sox17, with significantly higher levels in GFP negative cell population (Figure 3(b), upper and lower right graphs). In addition, in order to help understand the differentiation phase, mesodermal markers Flk1 and Mef2c were analyzed by quantitative real-time PCR. In the presence of LIF the expression of both markers decreases following exposure to DETA/NO, and upregulates after 2 days. On the other hand, in absence of LIF the treatment induces Flk1 expression and maintains the increase for 2 days more; nevertheless, Mef2c also increases its expression after NO but the increase is not sustained at day 6 (Figure 3(c)).

Bottom Line: Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment.Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression.Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), University Pablo de Olavide, Biomedical Research Network (CIBER) of Diabetes and Related Metabolic Diseases, Red-Tercel, Avenida Américo Vespucio S/N, 41092 Seville, Spain ; Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.

ABSTRACT
The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

No MeSH data available.


Related in: MedlinePlus