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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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Notch signaling regulates Slug expression through a CSL-dependent pathway. (A) qRT-PCR analysis demonstrating efficient knockdown of CSL in HMEC with two different shRNAs targeting CSL (shCSL) compared with a random control sequence (shRan). (B) qRT-PCR of Slug and HeyL in vector- or Dll4-activated HMEC transduced with shCSL constructs (n = 3). *, P < 0.05 vector shRandom versus HA-D114 shRandom; **, P < 0.05 HA-D114 shRandom versus HA-D114 shCSL-A or shCSL-B. (C) Immunoblotting for Slug, VE-cadherin, and CD31 in vector- or Dll4-activated HMEC transduced with shCSL or shSlug constructs. (D) qRT-PCR of vector- or CSL-VP16–expressing HMEC for Slug and HeyL (n = 3). *, P < 0.05. (E) PCR after ChIP with anti–FLAG-M2 antibody on HMEC-expressing vector (vec) or FLAG-CSL (CSL) to demonstrate CSL binding to the human Slug promoter. The negative (-ve) control represents PCR of the ZNF3 promoter after ChIP using FLAG-M2. (F) EMSA using nuclear lysates collected from vector- or FLAG-CSL–expressing HMEC and 32P-labeled double-stranded oligonucleotides spanning each of the two CSL binding sites in the human Slug promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies, and competition assays with 50× wild-type (wt) or mutant probes are also shown. Error bars show SEM.
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fig5: Notch signaling regulates Slug expression through a CSL-dependent pathway. (A) qRT-PCR analysis demonstrating efficient knockdown of CSL in HMEC with two different shRNAs targeting CSL (shCSL) compared with a random control sequence (shRan). (B) qRT-PCR of Slug and HeyL in vector- or Dll4-activated HMEC transduced with shCSL constructs (n = 3). *, P < 0.05 vector shRandom versus HA-D114 shRandom; **, P < 0.05 HA-D114 shRandom versus HA-D114 shCSL-A or shCSL-B. (C) Immunoblotting for Slug, VE-cadherin, and CD31 in vector- or Dll4-activated HMEC transduced with shCSL or shSlug constructs. (D) qRT-PCR of vector- or CSL-VP16–expressing HMEC for Slug and HeyL (n = 3). *, P < 0.05. (E) PCR after ChIP with anti–FLAG-M2 antibody on HMEC-expressing vector (vec) or FLAG-CSL (CSL) to demonstrate CSL binding to the human Slug promoter. The negative (-ve) control represents PCR of the ZNF3 promoter after ChIP using FLAG-M2. (F) EMSA using nuclear lysates collected from vector- or FLAG-CSL–expressing HMEC and 32P-labeled double-stranded oligonucleotides spanning each of the two CSL binding sites in the human Slug promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies, and competition assays with 50× wild-type (wt) or mutant probes are also shown. Error bars show SEM.

Mentions: We next determined whether Notch induces Slug through the canonical CSL-dependent pathway or the less well-defined CSL-independent route (Ramain et al., 2001). Dll4-mediated induction of Slug mRNA and protein was dramatically reduced when CSL was knocked down using either of two lentiviral-delivered short hairpin RNA (shRNA) constructs, which target distinct regions of CSL (Fig. 5, A–C). As expected, induction of the Notch target HeyL was also abolished by CSL knockdown (Fig. 5 B). In addition, the ability of Notch activation to down-regulate the endothelial markers VE-cadherin and CD31 (Fig. 5 C) was abrogated when CSL was knocked down. We also targeted Slug using two distinct lentiviral-delivered shRNAs and found that the ability of Dll4-activated Notch to down-regulate VE-cadherin and CD31 was also reversed by Slug knockdown (Fig. 5 C and Fig. S3), thus demonstrating the requirement of CSL-mediated induction of Slug for Notch-mediated EMT. Furthermore, activation of CSL using a constitutively active CSL mutant (CSL-VP16; MacKenzie et al., 2004) demonstrated that CSL activation alone was sufficient to up-regulate Slug expression as well as the Notch target HeyL (Fig. 5 D). However, enforced expression of the Notch targets Hey1 or Hey2, which have been implicated in cardiac EMT, did not up-regulate Slug or repress VE-cadherin (Fig. S3). Together, these findings indicate that Notch, via CSL, directly up-regulates Slug expression and that Slug is the Notch target responsible for repressing VE-cadherin expression.


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)

Notch signaling regulates Slug expression through a CSL-dependent pathway. (A) qRT-PCR analysis demonstrating efficient knockdown of CSL in HMEC with two different shRNAs targeting CSL (shCSL) compared with a random control sequence (shRan). (B) qRT-PCR of Slug and HeyL in vector- or Dll4-activated HMEC transduced with shCSL constructs (n = 3). *, P < 0.05 vector shRandom versus HA-D114 shRandom; **, P < 0.05 HA-D114 shRandom versus HA-D114 shCSL-A or shCSL-B. (C) Immunoblotting for Slug, VE-cadherin, and CD31 in vector- or Dll4-activated HMEC transduced with shCSL or shSlug constructs. (D) qRT-PCR of vector- or CSL-VP16–expressing HMEC for Slug and HeyL (n = 3). *, P < 0.05. (E) PCR after ChIP with anti–FLAG-M2 antibody on HMEC-expressing vector (vec) or FLAG-CSL (CSL) to demonstrate CSL binding to the human Slug promoter. The negative (-ve) control represents PCR of the ZNF3 promoter after ChIP using FLAG-M2. (F) EMSA using nuclear lysates collected from vector- or FLAG-CSL–expressing HMEC and 32P-labeled double-stranded oligonucleotides spanning each of the two CSL binding sites in the human Slug promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies, and competition assays with 50× wild-type (wt) or mutant probes are also shown. Error bars show SEM.
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Related In: Results  -  Collection

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fig5: Notch signaling regulates Slug expression through a CSL-dependent pathway. (A) qRT-PCR analysis demonstrating efficient knockdown of CSL in HMEC with two different shRNAs targeting CSL (shCSL) compared with a random control sequence (shRan). (B) qRT-PCR of Slug and HeyL in vector- or Dll4-activated HMEC transduced with shCSL constructs (n = 3). *, P < 0.05 vector shRandom versus HA-D114 shRandom; **, P < 0.05 HA-D114 shRandom versus HA-D114 shCSL-A or shCSL-B. (C) Immunoblotting for Slug, VE-cadherin, and CD31 in vector- or Dll4-activated HMEC transduced with shCSL or shSlug constructs. (D) qRT-PCR of vector- or CSL-VP16–expressing HMEC for Slug and HeyL (n = 3). *, P < 0.05. (E) PCR after ChIP with anti–FLAG-M2 antibody on HMEC-expressing vector (vec) or FLAG-CSL (CSL) to demonstrate CSL binding to the human Slug promoter. The negative (-ve) control represents PCR of the ZNF3 promoter after ChIP using FLAG-M2. (F) EMSA using nuclear lysates collected from vector- or FLAG-CSL–expressing HMEC and 32P-labeled double-stranded oligonucleotides spanning each of the two CSL binding sites in the human Slug promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies, and competition assays with 50× wild-type (wt) or mutant probes are also shown. Error bars show SEM.
Mentions: We next determined whether Notch induces Slug through the canonical CSL-dependent pathway or the less well-defined CSL-independent route (Ramain et al., 2001). Dll4-mediated induction of Slug mRNA and protein was dramatically reduced when CSL was knocked down using either of two lentiviral-delivered short hairpin RNA (shRNA) constructs, which target distinct regions of CSL (Fig. 5, A–C). As expected, induction of the Notch target HeyL was also abolished by CSL knockdown (Fig. 5 B). In addition, the ability of Notch activation to down-regulate the endothelial markers VE-cadherin and CD31 (Fig. 5 C) was abrogated when CSL was knocked down. We also targeted Slug using two distinct lentiviral-delivered shRNAs and found that the ability of Dll4-activated Notch to down-regulate VE-cadherin and CD31 was also reversed by Slug knockdown (Fig. 5 C and Fig. S3), thus demonstrating the requirement of CSL-mediated induction of Slug for Notch-mediated EMT. Furthermore, activation of CSL using a constitutively active CSL mutant (CSL-VP16; MacKenzie et al., 2004) demonstrated that CSL activation alone was sufficient to up-regulate Slug expression as well as the Notch target HeyL (Fig. 5 D). However, enforced expression of the Notch targets Hey1 or Hey2, which have been implicated in cardiac EMT, did not up-regulate Slug or repress VE-cadherin (Fig. S3). Together, these findings indicate that Notch, via CSL, directly up-regulates Slug expression and that Slug is the Notch target responsible for repressing VE-cadherin expression.

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