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Slug is a direct Notch target required for initiation of cardiac cushion cellularization.

Niessen K, Fu Y, Chang L, Hoodless PA, McFadden D, Karsan A - J. Cell Biol. (2008)

Bottom Line: Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT.We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells.Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, British Columbia Cancer Agency, Vancouver V5Z 1L3, Canada.

ABSTRACT
Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

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Related in: MedlinePlus

Slug−/− embryos display defects in AV canal EMT at E9.5. (A) Phase contrast (left) and DAPI (right) images of AV canal explants from wild-type and Slug−/− embryos. Bars, 250 μm. (B) Quantitative analysis of EMT in AV canal explants from E9.5 wild-type (wt), Slug+/−, and Slug−/− embryos after 48 h in culture. Results represent the distance of a positive pixel (DAPI-stained nucleus) to the closest point of the AV canal normalized to the area of the AV canal tissue. *, P < 0.05. (C) Slug expression in the AV canal explant assay as visualized by β-galactosidase staining of wild-type and Slug-lacZ+/− AV explants. The rounded morphology of most of the LacZ+ cells is shown on the right. The black square indicates the region of higher magnification shown to the right. Bars, 50 μm. (D) Representative sections of wild-type and Slug−/− hearts counterstained with Nuclear Fast Red used for analysis in E. Dotted blue lines highlight the superior and inferior AV cushions. Bars, 50 μm. (E) Quantitation of the cellularity of the superior and inferior cushions in E9.5 wild-type (wt; n = 3) and Slug−/− (n = 3) embryos. Error bars show SEM. (F) BrdU analysis on the percentage of proliferating cells in wild-type (n = 4) and Slug−/− (n = 6) AV canal cardiac cushions (total), the AV canal endocardium (Endo), and AV canal mesenchymal cells (Mesen; 10–15 sections per embryo). Error bars show SEM. (G) Vector- or Slug-transduced HMEC were subjected to an endothelial wounding assay. Bars represent the distance migrated after 24 h (n = 4). *, P < 0.05. Error bars show SD. (H) Vector- or Slug-transduced HMEC were evaluated in a modified Boyden chamber assay with 20 ng/ml PDGF-BB present in the lower chamber. Bars represent the total number of cells migrated after 4 h (n = 6). *, P < 0.05. Error bars show SD.
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fig3: Slug−/− embryos display defects in AV canal EMT at E9.5. (A) Phase contrast (left) and DAPI (right) images of AV canal explants from wild-type and Slug−/− embryos. Bars, 250 μm. (B) Quantitative analysis of EMT in AV canal explants from E9.5 wild-type (wt), Slug+/−, and Slug−/− embryos after 48 h in culture. Results represent the distance of a positive pixel (DAPI-stained nucleus) to the closest point of the AV canal normalized to the area of the AV canal tissue. *, P < 0.05. (C) Slug expression in the AV canal explant assay as visualized by β-galactosidase staining of wild-type and Slug-lacZ+/− AV explants. The rounded morphology of most of the LacZ+ cells is shown on the right. The black square indicates the region of higher magnification shown to the right. Bars, 50 μm. (D) Representative sections of wild-type and Slug−/− hearts counterstained with Nuclear Fast Red used for analysis in E. Dotted blue lines highlight the superior and inferior AV cushions. Bars, 50 μm. (E) Quantitation of the cellularity of the superior and inferior cushions in E9.5 wild-type (wt; n = 3) and Slug−/− (n = 3) embryos. Error bars show SEM. (F) BrdU analysis on the percentage of proliferating cells in wild-type (n = 4) and Slug−/− (n = 6) AV canal cardiac cushions (total), the AV canal endocardium (Endo), and AV canal mesenchymal cells (Mesen; 10–15 sections per embryo). Error bars show SEM. (G) Vector- or Slug-transduced HMEC were subjected to an endothelial wounding assay. Bars represent the distance migrated after 24 h (n = 4). *, P < 0.05. Error bars show SD. (H) Vector- or Slug-transduced HMEC were evaluated in a modified Boyden chamber assay with 20 ng/ml PDGF-BB present in the lower chamber. Bars represent the total number of cells migrated after 4 h (n = 6). *, P < 0.05. Error bars show SD.

Mentions: To determine whether targeted disruption of the Slug gene has a functional effect on cardiac cushion development, the AV canal of E9.5 embryos were placed on collagen gels to monitor EMT ex vivo, as previously described (Camenisch et al., 2002a; Chang et al., 2004). Occasional endothelial cell outgrowths occur proximal to 100 μm of the AV canal explant. Therefore, only the morphologically distinct mesenchymal cells distal to 100 μm from the AV canal were quantitated to determine the degree of EMT. Homozygous Slug-LacZ mutants behave as Slug−/− (Slug-deficient) animals (Jiang et al., 1998; Inoue et al., 2002), and Slug−/− AV canal explants had significantly reduced migration and invasion compared with Slug+/− or wild-type controls at E9.5 (Fig. 3, A and B). Of the few Slug−/− cells that did migrate, many had a rounded morphology, and were not able to differentiate into spindled mesenchymal cells. Analysis of β-galactosidase activity in Slug+/− AV explants revealed Slug expression in the migrating cells as well as the proximal cardiac endothelial cells (Fig. 3 C). Interestingly, the majority of β-galactosidase staining was seen in rounded cells, which is consistent with morphology of cells that are intermediate between endothelial and mesenchymal phenotype, as previously described (Camenisch et al., 2002b).


Slug is a direct Notch target required for initiation of cardiac cushion cellularization.

Niessen K, Fu Y, Chang L, Hoodless PA, McFadden D, Karsan A - J. Cell Biol. (2008)

Slug−/− embryos display defects in AV canal EMT at E9.5. (A) Phase contrast (left) and DAPI (right) images of AV canal explants from wild-type and Slug−/− embryos. Bars, 250 μm. (B) Quantitative analysis of EMT in AV canal explants from E9.5 wild-type (wt), Slug+/−, and Slug−/− embryos after 48 h in culture. Results represent the distance of a positive pixel (DAPI-stained nucleus) to the closest point of the AV canal normalized to the area of the AV canal tissue. *, P < 0.05. (C) Slug expression in the AV canal explant assay as visualized by β-galactosidase staining of wild-type and Slug-lacZ+/− AV explants. The rounded morphology of most of the LacZ+ cells is shown on the right. The black square indicates the region of higher magnification shown to the right. Bars, 50 μm. (D) Representative sections of wild-type and Slug−/− hearts counterstained with Nuclear Fast Red used for analysis in E. Dotted blue lines highlight the superior and inferior AV cushions. Bars, 50 μm. (E) Quantitation of the cellularity of the superior and inferior cushions in E9.5 wild-type (wt; n = 3) and Slug−/− (n = 3) embryos. Error bars show SEM. (F) BrdU analysis on the percentage of proliferating cells in wild-type (n = 4) and Slug−/− (n = 6) AV canal cardiac cushions (total), the AV canal endocardium (Endo), and AV canal mesenchymal cells (Mesen; 10–15 sections per embryo). Error bars show SEM. (G) Vector- or Slug-transduced HMEC were subjected to an endothelial wounding assay. Bars represent the distance migrated after 24 h (n = 4). *, P < 0.05. Error bars show SD. (H) Vector- or Slug-transduced HMEC were evaluated in a modified Boyden chamber assay with 20 ng/ml PDGF-BB present in the lower chamber. Bars represent the total number of cells migrated after 4 h (n = 6). *, P < 0.05. Error bars show SD.
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Related In: Results  -  Collection

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fig3: Slug−/− embryos display defects in AV canal EMT at E9.5. (A) Phase contrast (left) and DAPI (right) images of AV canal explants from wild-type and Slug−/− embryos. Bars, 250 μm. (B) Quantitative analysis of EMT in AV canal explants from E9.5 wild-type (wt), Slug+/−, and Slug−/− embryos after 48 h in culture. Results represent the distance of a positive pixel (DAPI-stained nucleus) to the closest point of the AV canal normalized to the area of the AV canal tissue. *, P < 0.05. (C) Slug expression in the AV canal explant assay as visualized by β-galactosidase staining of wild-type and Slug-lacZ+/− AV explants. The rounded morphology of most of the LacZ+ cells is shown on the right. The black square indicates the region of higher magnification shown to the right. Bars, 50 μm. (D) Representative sections of wild-type and Slug−/− hearts counterstained with Nuclear Fast Red used for analysis in E. Dotted blue lines highlight the superior and inferior AV cushions. Bars, 50 μm. (E) Quantitation of the cellularity of the superior and inferior cushions in E9.5 wild-type (wt; n = 3) and Slug−/− (n = 3) embryos. Error bars show SEM. (F) BrdU analysis on the percentage of proliferating cells in wild-type (n = 4) and Slug−/− (n = 6) AV canal cardiac cushions (total), the AV canal endocardium (Endo), and AV canal mesenchymal cells (Mesen; 10–15 sections per embryo). Error bars show SEM. (G) Vector- or Slug-transduced HMEC were subjected to an endothelial wounding assay. Bars represent the distance migrated after 24 h (n = 4). *, P < 0.05. Error bars show SD. (H) Vector- or Slug-transduced HMEC were evaluated in a modified Boyden chamber assay with 20 ng/ml PDGF-BB present in the lower chamber. Bars represent the total number of cells migrated after 4 h (n = 6). *, P < 0.05. Error bars show SD.
Mentions: To determine whether targeted disruption of the Slug gene has a functional effect on cardiac cushion development, the AV canal of E9.5 embryos were placed on collagen gels to monitor EMT ex vivo, as previously described (Camenisch et al., 2002a; Chang et al., 2004). Occasional endothelial cell outgrowths occur proximal to 100 μm of the AV canal explant. Therefore, only the morphologically distinct mesenchymal cells distal to 100 μm from the AV canal were quantitated to determine the degree of EMT. Homozygous Slug-LacZ mutants behave as Slug−/− (Slug-deficient) animals (Jiang et al., 1998; Inoue et al., 2002), and Slug−/− AV canal explants had significantly reduced migration and invasion compared with Slug+/− or wild-type controls at E9.5 (Fig. 3, A and B). Of the few Slug−/− cells that did migrate, many had a rounded morphology, and were not able to differentiate into spindled mesenchymal cells. Analysis of β-galactosidase activity in Slug+/− AV explants revealed Slug expression in the migrating cells as well as the proximal cardiac endothelial cells (Fig. 3 C). Interestingly, the majority of β-galactosidase staining was seen in rounded cells, which is consistent with morphology of cells that are intermediate between endothelial and mesenchymal phenotype, as previously described (Camenisch et al., 2002b).

Bottom Line: Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT.We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells.Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, British Columbia Cancer Agency, Vancouver V5Z 1L3, Canada.

ABSTRACT
Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

Show MeSH
Related in: MedlinePlus