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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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Induction of Snail by TGF-β2 is synergistically enhanced in Dll4-activated endothelial cells. (A) qRT-PCR for Snail mRNA in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2 for the indicated times (n = 3). *, P < 0.05. (B) Immunoblots for Snail and Slug in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2. (C) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 10 μM DMSO or DAPT for 16 h followed by treatment with 2.5 ng/ml TGF-β2 for 3 h (n = 3). *, P < 0.05. (D) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 20 or 50 ng/ml BMP2 (n = 3). Error bars show SEM.
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fig6: Induction of Snail by TGF-β2 is synergistically enhanced in Dll4-activated endothelial cells. (A) qRT-PCR for Snail mRNA in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2 for the indicated times (n = 3). *, P < 0.05. (B) Immunoblots for Snail and Slug in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2. (C) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 10 μM DMSO or DAPT for 16 h followed by treatment with 2.5 ng/ml TGF-β2 for 3 h (n = 3). *, P < 0.05. (D) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 20 or 50 ng/ml BMP2 (n = 3). Error bars show SEM.

Mentions: It has previously been suggested that EMT initiated by Notch may proceed through the induction of Snail; however, the degenerate primers used in that study amplify both Snail and Slug (Timmerman et al., 2004). To clarify which Snail family members are regulated by Notch signaling, we activated Notch in endothelial cells by ectopically expressing the Notch ligands Jagged1 or Dll4 or the constitutively active form of Notch1 or Notch4 (Notch1ICD and Notch4ICD, respectively), all of which are expressed in the cardiac cushion (Loomes et al., 1999; Noseda et al., 2004). Activated Notch up-regulated Slug, but not Snail, in all endothelial cells tested, as demonstrated by RT-PCR (Fig. 1 A), quantitative RT-PCR (qRT-PCR; Fig. 1 B), and immunoblotting (Fig. 1, C and D; and see Fig. 6 B). As a positive control, we confirmed that the known Notch targets Hey1, Hey2, and HeyL were induced by NotchICD (Fig. 1, A and B). Additional experiments with NotchICD deletion constructs revealed that the Ankyrin repeats of Notch are required for induction of Slug (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1). These findings indicate that Notch activation induces Slug but not Snail expression in endothelial cells.


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)

Induction of Snail by TGF-β2 is synergistically enhanced in Dll4-activated endothelial cells. (A) qRT-PCR for Snail mRNA in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2 for the indicated times (n = 3). *, P < 0.05. (B) Immunoblots for Snail and Slug in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2. (C) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 10 μM DMSO or DAPT for 16 h followed by treatment with 2.5 ng/ml TGF-β2 for 3 h (n = 3). *, P < 0.05. (D) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 20 or 50 ng/ml BMP2 (n = 3). Error bars show SEM.
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fig6: Induction of Snail by TGF-β2 is synergistically enhanced in Dll4-activated endothelial cells. (A) qRT-PCR for Snail mRNA in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2 for the indicated times (n = 3). *, P < 0.05. (B) Immunoblots for Snail and Slug in vector- or Dll4-activated HMEC treated with 2.5 ng/ml TGF-β2. (C) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 10 μM DMSO or DAPT for 16 h followed by treatment with 2.5 ng/ml TGF-β2 for 3 h (n = 3). *, P < 0.05. (D) qRT-PCR for Snail, Slug, Hey1, and Smad7 mRNA in vector- or Dll4-activated HMEC treated with 20 or 50 ng/ml BMP2 (n = 3). Error bars show SEM.
Mentions: It has previously been suggested that EMT initiated by Notch may proceed through the induction of Snail; however, the degenerate primers used in that study amplify both Snail and Slug (Timmerman et al., 2004). To clarify which Snail family members are regulated by Notch signaling, we activated Notch in endothelial cells by ectopically expressing the Notch ligands Jagged1 or Dll4 or the constitutively active form of Notch1 or Notch4 (Notch1ICD and Notch4ICD, respectively), all of which are expressed in the cardiac cushion (Loomes et al., 1999; Noseda et al., 2004). Activated Notch up-regulated Slug, but not Snail, in all endothelial cells tested, as demonstrated by RT-PCR (Fig. 1 A), quantitative RT-PCR (qRT-PCR; Fig. 1 B), and immunoblotting (Fig. 1, C and D; and see Fig. 6 B). As a positive control, we confirmed that the known Notch targets Hey1, Hey2, and HeyL were induced by NotchICD (Fig. 1, A and B). Additional experiments with NotchICD deletion constructs revealed that the Ankyrin repeats of Notch are required for induction of Slug (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1). These findings indicate that Notch activation induces Slug but not Snail expression in endothelial cells.

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