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The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells.

Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y - PLoS ONE (2013)

Bottom Line: The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1.Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression.These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms' tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

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Primary culture of epicardial cells from E12.5 mouse embryos.(A) Representative image of primary epicardial cells generated from E12.5 mouse hearts, as described in the Materials and Methods section. (B) The relative mRNA expression levels of epicardial markers (Tbx18 and Wt1) and a cardiomyocyte marker (Nkx2-5) in primary epicardial cells and cardiomyocytes, as determined by quantitative real-time PCR (n = 3; *P<0.0001 vs. primary epicardial cells). The results are normalized to Gapdh expression, and the relative expression level is given as a ratio to primary epicardial cells. (C) Immunostaining for Wt1 (green) and DAPI nuclear staining (blue) of primary epicardial cells after 4 days in culture. The data are presented as the mean ± SD. Scale bars: 200 µm.
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pone-0057829-g001: Primary culture of epicardial cells from E12.5 mouse embryos.(A) Representative image of primary epicardial cells generated from E12.5 mouse hearts, as described in the Materials and Methods section. (B) The relative mRNA expression levels of epicardial markers (Tbx18 and Wt1) and a cardiomyocyte marker (Nkx2-5) in primary epicardial cells and cardiomyocytes, as determined by quantitative real-time PCR (n = 3; *P<0.0001 vs. primary epicardial cells). The results are normalized to Gapdh expression, and the relative expression level is given as a ratio to primary epicardial cells. (C) Immunostaining for Wt1 (green) and DAPI nuclear staining (blue) of primary epicardial cells after 4 days in culture. The data are presented as the mean ± SD. Scale bars: 200 µm.

Mentions: To investigate the molecular functions of Tbx18 and Wt1 in the epicardial EMT, we developed a primary epicardial cell culture from E12.5 mouse embryos. After 4 days of culture, these primary cells exhibited a cobblestone-like morphology (Figure 1A) and demonstrated high levels of mRNA expression of the epicardial markers Wt1 and Tbx18 but not the cardiomyocyte marker Nkx2-5 (Figure 1B). The epicardial cells were confirmed to be 97% pure by immunofluorescent staining with an anti-Wt1 antibody (Figure 1C). These data indicate that primary embryonic epicardial cells can be successfully cultured with high purity.


The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells.

Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y - PLoS ONE (2013)

Primary culture of epicardial cells from E12.5 mouse embryos.(A) Representative image of primary epicardial cells generated from E12.5 mouse hearts, as described in the Materials and Methods section. (B) The relative mRNA expression levels of epicardial markers (Tbx18 and Wt1) and a cardiomyocyte marker (Nkx2-5) in primary epicardial cells and cardiomyocytes, as determined by quantitative real-time PCR (n = 3; *P<0.0001 vs. primary epicardial cells). The results are normalized to Gapdh expression, and the relative expression level is given as a ratio to primary epicardial cells. (C) Immunostaining for Wt1 (green) and DAPI nuclear staining (blue) of primary epicardial cells after 4 days in culture. The data are presented as the mean ± SD. Scale bars: 200 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585213&req=5

pone-0057829-g001: Primary culture of epicardial cells from E12.5 mouse embryos.(A) Representative image of primary epicardial cells generated from E12.5 mouse hearts, as described in the Materials and Methods section. (B) The relative mRNA expression levels of epicardial markers (Tbx18 and Wt1) and a cardiomyocyte marker (Nkx2-5) in primary epicardial cells and cardiomyocytes, as determined by quantitative real-time PCR (n = 3; *P<0.0001 vs. primary epicardial cells). The results are normalized to Gapdh expression, and the relative expression level is given as a ratio to primary epicardial cells. (C) Immunostaining for Wt1 (green) and DAPI nuclear staining (blue) of primary epicardial cells after 4 days in culture. The data are presented as the mean ± SD. Scale bars: 200 µm.
Mentions: To investigate the molecular functions of Tbx18 and Wt1 in the epicardial EMT, we developed a primary epicardial cell culture from E12.5 mouse embryos. After 4 days of culture, these primary cells exhibited a cobblestone-like morphology (Figure 1A) and demonstrated high levels of mRNA expression of the epicardial markers Wt1 and Tbx18 but not the cardiomyocyte marker Nkx2-5 (Figure 1B). The epicardial cells were confirmed to be 97% pure by immunofluorescent staining with an anti-Wt1 antibody (Figure 1C). These data indicate that primary embryonic epicardial cells can be successfully cultured with high purity.

Bottom Line: The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1.Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression.These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms' tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

Show MeSH
Related in: MedlinePlus