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Article |
Slug is a direct Notch target required for initiation of cardiac cushion cellularization
Correspondence to Aly Karsan: akarsan{at}bccrc.ca
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 β (TGF-β) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-β 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.
© 2008 Niessen et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Recent studies have demonstrated a critical role of the Notch signaling pathway during cardiac EMT, and disruption of this pathway has been implicated in the pathogenesis of various cardiovascular diseases (Iso et al., 2003; Niessen and Karsan, 2007). In the mouse, targeted deletion of Notch1 or its key nuclear partner CSL (CBF1/Suppressor of Hairless/Lag-1) results in cardiac cushion EMT defects (Oka et al., 1995; Timmerman et al., 2004). Further, targeted deletion of the downstream Notch/CSL effector Hey2 or double-deficiency of Hey1 and Hey2 or Hey1 and HeyL results in various congenital heart anomalies including cardiac cushion defects (Donovan et al., 2002; Fischer et al., 2004, 2007). In humans, mutations at the Notch1 locus are associated with bicuspid aortic valve disease as well as mitral valve anomalies and tetralogy of Fallot (Garg et al., 2005). Further, patients with mutations of the Notch ligand Jagged1 develop Alagille syndrome, a polymalformative disorder which includes cardiac cushion defects (Li et al., 1997; Oda et al., 1997; Eldadah et al., 2001).
TGF-β–related pathways have also been shown to be essential for proper heart development through their role in regulating EMT (Azhar et al., 2003). Of particular interest, BMP2 and TGF-β2 are expressed by the AV canal cushion myocardium (Dickson et al., 1993; Zhang and Bradley, 1996). BMP2-deficient mice die before cardiac cushion development (Zhang and Bradley, 1996); however, deficiency of the BMP2 receptor Alk2 results in AV canal EMT defects (Wang et al., 2005). TGF-β2–deficient mouse embryos do not display obvious cardiac cushion EMT defects, although later remodeling of the AV canal cushion is impaired (Dickson et al., 1993; Sanford et al., 1997; Bartram et al., 2001; Molin et al., 2002, 2003). However, using an ex vivo AV canal explant assay, TGF-β2–blocking antibodies or blocking antibodies for its coreceptor TβRIII inhibit AV canal EMT, suggesting redundancy of this pathway in vivo (Brown et al., 1999; Camenisch et al., 2002a). These data and others have established a clear role of TGF-β–related pathways during mammalian cardiac cushion development.
The Snail family members Snail (also known as Snai1) and Slug (also known as Snai2) encode zinc finger–containing transcriptional repressors that trigger EMT during embryonic development and tumor progression, in part by regulating expression of junctional proteins, most notably E-cadherin (Nieto, 2002). In the mouse, Snail has been shown to be expressed in the cardiac cushions from E9.5 onwards (Timmerman et al., 2004). Mice deficient for Snail die at E7.5, before cardiac development, and display defects in mesoderm formation (Carver et al., 2001). Conditional deletion of Snail after E8 results in lethality by E9.5, partially because of severe cardiovascular defects, but before the initiation of cardiac cushion EMT (Murray and Gridley, 2006). In the mouse, Slug is expressed in the cardiac cushions at E13.5, and mice deficient for Slug are viable but are growth retarded and display defects in pigmentation and hematopoiesis (Jiang et al., 1998; Inoue et al., 2002). To date, there is no direct evidence demonstrating the requirement for any Snail family member during mammalian heart development.
In this paper, we demonstrate that Slug is first expressed by a subset of endothelial cells as well as mesenchymal cells of the AV canal at E9.5, at the initiation of EMT. In keeping with a requirement for Slug during the initiation of cardiac EMT, the AV canal cushions show markedly reduced cellularization at E9.5, which normalizes by E10.5. Concordant with the in vivo findings, AV canal explant assays demonstrate that EMT in Slug-deficient embryos is impaired at E9.5 but not E10.5, as EMT in Slug-deficient embryos is rescued by an increase in Snail expression by E10.5. Accordingly, abolishing both Slug and Snail expression results in EMT defects at E10.5. In contrast to a previous study, we show that Notch signaling, through CSL, directly regulates the Slug promoter, resulting in the up-regulation of Slug, but not Snail, in endothelial cells (Timmerman et al., 2004). We further show that Slug directly binds and represses the vascular endothelial cadherin (VE-cadherin) promoter. Slug also promotes increased migration toward PDGF. In contrast, TGF-β2 and BMP2 induce Snail expression but minimal Slug expression. However, Notch synergistically induces Snail in concert with TGF-β2. Our data demonstrate that Notch-induced expression of Slug plays an important role in the initiation of EMT in the heart but that increased Snail compensates for the lack of Slug in Slug-targeted embryos as cardiac cushion morphogenesis progresses.
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Slug is necessary for EMT in the cardiac cushions
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).
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To determine whether Slug is sufficient to promote a motile phenotype in endothelial cells, endothelial cells were transduced with vector or Slug, and an in vitro wound healing (scratch) assay was performed. The scratch assay revealed increased migration of Slug-expressing endothelial cells as early as 4 h and up to 24 h after wounding of the endothelial monolayer, resulting in Slug-expressing cells migrating almost twice as far after 24 h (Fig. 3 G). PDGF has been shown to be expressed in the cardiac cushions during EMT (Van Den Akker et al., 2005). Using a modified Boyden chamber assay with 20 ng/ml PDGF-BB present in the lower chamber, we found that Slug-expressing endothelial cells showed significantly increased directed migration toward PDGF-BB (Fig. 3 H). Thus, Slug expression in the endothelium appears to be sufficient for endothelial motility and directed migration.
Slug represses endothelial phenotype
Given our findings demonstrating the requirement of Slug in cardiac EMT, we sought to examine the role that Slug plays in modulating the endothelial phenotype. Enforced expression of Slug repressed expression of key endothelial genes such as VE-cadherin, CD31, and Tie2 as determined by qRT-PCR, immunoblotting, and immunofluorescence (Fig. 4, A and B; and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1).
However, in contrast to activated Notch, Slug did not induce the mesenchymal markers smooth muscle 
-actin and h1-calponin (Fig. 4 B). These findings suggest that Slug expression promotes the initial phases of EMT associated with the loss of endothelial phenotype but is not sufficient to complete the transition into a mesenchymal cell that is mediated by Notch activation.
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Notch acts through CSL to induce Slug and repress the endothelial phenotype
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.
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Notch and TGF-β act synergistically to induce Snail
Components of the TGF-β pathway have been shown to be required for EMT and for the regulation of Snail family genes during heart development (Romano and Runyan, 2000; Camenisch et al., 2002a; Wang et al., 2005). Additionally, the Notch and TGF-β pathways have been shown to coregulate target gene expression in various cell types (Blokzijl et al., 2003; Zavadil et al., 2004). To investigate the relationship between the Notch and TGF-β pathways and Snail family member expression, endothelial cells cocultured with vector- or Dll4-transduced cells were treated with 2.5 ng/ml TGF-β2 or 20 or 50 ng/ml BMP2. TGF-β2 stimulation induced maximal induction of Snail mRNA and protein expression after 2 h of treatment in vector-transduced cells, followed by rapid down-regulation (Fig. 6, A and B).
Although Dll4 stimulation alone did not induce Snail, combined activation of the Notch and TGF-β pathways resulted in a synergistic increase of Snail mRNA levels and maintenance of expression for at least 8 h after stimulation with TGF-β2 (Fig. 6 A). Protein expression of Snail peaked slightly later (4 h) and was sustained at a much higher level in the context of Dll4 and TGF-β2 costimulation compared with TGF-β2 stimulation alone (Fig. 6 B). In contrast, there was minimal induction of Slug by TGF-β2, whereas Notch activation alone dramatically up-regulated Slug (Fig. 6, B and C). Costimulation by Dll4 and TGF-β2 did not increase the level of Slug induction over that seen with Dll4 alone (Fig. 6, B and C).
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-secretase inhibitor DAPT to block ligand-activated Notch signaling. TGF-β2 treatment dramatically up-regulated the expression of Snail, but the addition of DAPT did not affect the ability of TGF-β2 to induce Snail, which is consistent with Notch-independent induction (Fig. 6 C). In the context of combined Notch and TGF-β2 activation, the synergistic up-regulation of Snail expression was reduced by DAPT to the level seen by TGF-β2 stimulation alone (Fig. 6 C). TGF-β2 had no effect on Slug levels, and the addition of DAPT abrogated Slug induction by Dll4, suggesting complete dependence on Notch activation for Slug up-regulation (Fig. 6 C). Similar results were observed with TGF-β1 treatment (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1). As expected, stimulation of endothelial cells with TGF-β2 induced expression of Smad7, a TGF-β target gene, to similar levels in control and Notch-activated cells (Fig. 6 C). Addition of DAPT appeared to block the ability of TGF-β2 to induce Smad7, but the results were variable and did not reach statistical significance for TGF-β2 or TGF-β1 (Fig. 6 C and Fig. S4), suggesting a minimal role for Notch activation in TGF-β–induced Smad7 induction. Hey1 expression was induced by Notch signaling and TGF-β2 and was dependent on active Notch signaling (Fig. 6 C). Similar to what has been described for BMP4/6, Hey1 was also synergistically induced to very high levels by TGF-β2 and Dll4 (Fig. 6 C; Dahlqvist et al., 2003; Itoh et al., 2004). Similar to TGF-β2, when endothelial cells were stimulated with BMP2 there was dramatic up-regulation of Snail expression, minimal up-regulation of Slug, and synergistic activation of Hey1 in Notch activated cells (Fig. 6 D). However, unlike TGF-β2, combined activation of the Notch pathway and BMP2 stimulation did not synergistically up-regulate Snail expression (Fig. 6 D). This suggests that a Smad3-dependent process is involved in the synergistic activation of Snail expression by the Notch and TGF-β pathways. These findings clearly confirm that Slug is a direct target of Notch and that Snail is not but that Snail is synergistically induced when Notch activation is superimposed on TGF-β stimulation.
Snail and Slug cooperatively induce cardiac EMT
Given that the cardiac EMT defect seen in Slug–/– mice at E9.5 was reduced by E10.5 (Fig. 3, A and B; and Fig. S2), we sought to determine whether Snail was compensating for the absence of Slug in vivo. qRT-PCR analysis of wild-type and Slug–/– hearts was conducted for Snail and Slug. Slug expression increased from E9.5 to 11.5 in the wild-type heart and its expression was abolished in the Slug–/– hearts. In contrast, Snail expression did not increase from E9.5 to 11.5 in the wild-type hearts. However, in the Slug–/– hearts Snail was up-regulated 3.6-fold by E11.5 (Fig. 7 A).
In situ hybridization at E10.5 and 11.5 revealed Snail expression in the AV canal and OFT in both wild-type and Slug–/– hearts, with increased expression in the Slug–/– embryos (Fig. 7 B), suggesting that the region of Snail expression is not expanded but, rather, that the cells normally expressing Snail do so at a higher level in the Slug–/– hearts.
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Interestingly, endothelial-specific gene targeting of the BMP receptor Alk2 results in cardiac cushion defects that are associated with a decrease in the expression of Snail, but not Hey2 or Slug, in the AV canal (Wang et al., 2005). In contrast, Notch-mediated EMT is cell autonomous and TGF-β independent (Noseda et al., 2004). Collectively, these findings support the data presented herein that Slug is a direct target of the Notch pathway and that Snail is a target of TGF-β–related pathways.
We show for the first time that Slug is expressed in a subset of cardiac endothelial cells and the mesenchyme of the AV canal and OFT from the onset of EMT at E9.5 and is essential for initiating cardiac cushion cellularization. We further demonstrate that Slug binds and represses the VE-cadherin promoter, inducing a motile phenotype. Taken with the defect in AV canal EMT at E9.5, the ability of Slug to bind and repress the VE-cadherin promoter and induce migration suggests that the activation phase of EMT in the endocardium is impaired by loss of Slug.
Using an AV canal explant model, we demonstrate that Slug-deficient hearts have a specific defect in cardiac cushion EMT at E9.5 but not at E10.5. In Slug-deficient hearts at E10.5, cardiac EMT is compensated for by a relative increase in Snail expression. Accordingly, inducing Snail expression by treatment with TGF-β2 at E9.5 rescues the EMT defect in Slug-deficient mice. Conversely, abolishing both Slug and Snail expression results in EMT defects at E10.5. Consistent with a requirement for both Snail and Slug during cardiac EMT, both members are expressed during mouse and human heart development with similar localization in a subset of endothelial cells and the mesenchymal cells of the AV canal and OFT. It is of interest that deletion of Slug results in up-regulation of Snail. This finding suggests that Slug may act to repress Snail, either by directly targeting the Snail promoter or through the repression of elements of the TGF-β–related or Notch pathways. Consistent with the latter hypothesis, we have seen that both Hey2 and Smad7 are up-regulated in Slug-deficient hearts at E11.5 (unpublished data). Additionally, it has been demonstrated that Slug does not affect Snail-promoter activity (Peiro et al., 2006), which we have verified (unpublished data). Our findings are concordant with a recent study showing that Snail heterozygosity increases the penetrance of palate defects in Slug-deficient mice, suggesting that Snail also compensates for Slug deficiency during palate development (Murray et al., 2007). In addition, the finding that there is increased Slug expression in the developing palate in Snail-deficient embryos (Murray et al., 2007) suggests reciprocal regulation of gene expression between Slug and Snail.
The interaction between the Notch and TGF-β pathways likely occurs at multiple levels and may be context-dependent. Targeting of CSL results in reduced TGF-β2 and its receptor TBRII in the mouse heart (Timmerman et al., 2004). In contrast, the Notch-ligand Jagged1 has been shown to be induced by TGF-β at the onset of EMT in epithelial cells (Zavadil et al., 2004). Despite our evidence showing cooperation of the TGF-β and Notch pathways in cardiac cushion development, several studies have suggested that constitutively active NotchICD inhibits TGF-β signaling through the sequestration of Smad3 or the coactivator p300 (Masuda et al., 2005; Sun et al., 2005). However, overexpression of NotchICD may have resulted in artifactual sequestering of TGF-β signaling components. Alternatively, the outcome of Notch–TGF-β cross talk may be dependent on the context. Indeed, in mouse embryonic endothelial cells, BMP signaling synergizes with NotchICD through a ternary interaction between Smad5, NotchICD, and p/CAF (Itoh et al., 2004).
Combining the findings in this paper with published data, one can propose a model where endothelial Notch activation induces Slug and release of TGF-β and BMPs from the cushion myocardium activates the cardiac endothelium to up-regulate Snail, which is enhanced in Notch-activated cells. Based on the Slug-deficient hearts and RNAi studies in the AV explants, a minimal total dose of Snail/Slug is required in order for EMT to be initiated at E9.5, with a diminishing requirement at E10.5 as cushion development proceeds.
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–smooth muscle actin antibody was obtained from Thermo Fisher Scientific.
Cell culture and gene transfer
The HMEC-1 human microvascular endothelial cell line, HUVEC, and human aortic endothelial cells were obtained and cultured as previously described (Noseda et al., 2004). Endothelial cells were transduced using the retroviral vectors pLNCX, pLNC-Slug-FLAG, pLNC-FLAG-CSL, MIY, MIY-Slug-FLAG, MIY-Notch4IC-HA, MIY-Notch1IC, and MIY-CSL-VP16 as previously described (Karsan et al., 1996). pcDNA3-Slug-FLAG cDNA was a gift from E.R. Fearon (The University of Michigan Heath Systems, Ann Arbor, MI).
RNA collection and RT-PCR
RNA was isolated and cDNA was made as previously described (Noseda et al., 2004). PCR was performed on a PCR cycler (PTC-200 [Bio-Rad Laboratories] or 7900HT [Applied Biosystems]) with primers listed in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1).
Luciferase reporter assay
8 x 104 HMEC was plated 24 h before transfection in 24-well plates. HMEC were transfected using SuperFect (QIAGEN) reagent, with 0.3125 µg of total plasmid DNA as per the manufacturer's recommendations. Each well was transfected with 0.3 µg of the VE-cadherin promoter plasmid or mutant VE-cadherin promoter constructs, 5 ng pcDNA3 or pcDNA3-Slug-FLAG, and 7.5 ng pRL-CMV (Promega). Luminescence was measured on a Lumat LB 9507 (EG&G Berthold) 24 h after transfection using dual luciferase reporter assays according to the manufacturer's recommendations (Promega).
EMSA
For Slug EMSA assays, in vitro–translated (TNT; Promega) Slug-FLAG or control luciferase protein was preincubated with FLAG-M2 antibody overnight at 4°C in 12 mM Hepes, pH 7.9, 4 mM Tris, pH 7.9, 133 mM KCl, 10% Glycerol, and 2 µg PolydI-dC binding buffer. 50-fold excess nonradioactive duplex oligos were preincubated for 15 min on ice, and then 150,000-cpm 32P-labeled double-stranded oligo nucleotides were added and incubated for 30 min at room temperature. Binding reactions were run on 5% Tris-borate EDTA gels and exposed to a phosphorimager plate for 12–16 h. For CSL EMSA assays, nuclear lysates were collected from FLAG-CSL–overexpressing HMEC cells. Binding reaction and detection were the same as used for Slug-FLAG EMSA assays.
ChIP
HMEC were transduced with pLNCX or pLNC-FLAG-CSL, and ChIP assay was performed as previously described (Noseda et al., 2006). ChIP DNA was amplified for the ZNF3 promoter or the two CSL binding sites in the human Slug promoter using primers listed in Table S1.
Mice and AV explant assay analysis
Slug-lacZ mice were provided by T. Gridley (Jackson Laboratories, Bar Harbor, ME). Slug-lacZ+/– mice were crossed to C57BL/6J mice for embryo collection. Embryos were assayed for β-galactosidase activity in situ using published protocols (Nagy et al., 2003). AV canal explants were performed as previously described (Camenisch et al., 2002a). Explants were cultured for 48 h and analyzed for the number and distance of migrating cells.
RNA interference
shRNAs targeting human CSL, Snail, and Slug were cloned into the HpaI–XhoI sites of the pLentilox3.7 vector (gift from L. Van Parijs, Massachusetts Institute of Technology, Cambridge, MA; Table S1). Constructs were sequence verified and validated for efficient knockdown.
Collection of human tissues
Human embryonic hearts were collected, after institutionally approved protocols and informed consent, at the Children's and Women's Health Sciences Centre (Vancouver, Canada). Tissue was fixed in 4% PFA overnight, embedded in OCT, and cryosectioned.
In situ hybridization
In situ hybridization was performed as previously described (Wilkinson, 1992). Mouse Snail probe (–55 to +454) was cloned into pBluescript. Human Snail and human Slug probes were comprised of the entire ORF cloned into pCDNA3.
BrdU analysis
Slug-lacZ+/– male and female mice were crossed and pregnant females were injected with 1,500 mg/ml BrdU (Sigma-Aldrich) 2 h before killing. Embryos were collected, paraffin embedded, and sectioned (6 µm) onto Histobond slides (Marienfeld Laboratory Glassware). Slides were boiled for 30 min in 0.1M citrate buffer, rinsed in water, and then denatured in 2N HCl for 45 min at 37°C. Slides were then rinsed in PBS, and BrdU staining was performed using mouse anti-BrdU (BU33; Sigma-Aldrich) and goat anti–mouse Alexa 488 (Invitrogen).
Online supplemental material
Fig. S1 shows that the ankyrin repeats of NotchICD are required for induction of Slug expression. Fig. S2 shows the Slug-lacZ expression in the 18 to 29 somite stage heart and that the AV canal EMT defect observed in Slug-deficient embryos at E9.5 is no longer present at E10.5. Fig. S3 shows that Slug represses endothelial cell phenotype in HMEC and HUVEC, that the knockdown of Slug expression in Notch-activated cells restores VE-cadherin and CD31 expression, and that the ectopic expression of Hey1 or Hey2 does not induce Slug expression or repress VE-cadherin expression. Fig. S4 shows that the induction of Snail by TGF-β1 is synergistically enhanced in Notch-activated endothelial cells. Table S1 is a list of primers used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1.
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Acknowledgments |
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This work was supported by grants from the Canadian Institutes of Health Research (MOP 64354), the Heart and Stroke Foundation of British Columbia and the Yukon, Genome Canada, and Genome British Columbia. K. Niessen and L. Chang are supported by Doctoral Research Awards and Y. Fu by a Postdoctoral Award from the Michael Smith Foundation for Health Research. A. Karsan and P.A. Hoodless are Senior Scholars of the Michael Smith Foundation for Health Research, and P.A. Hoodless is a Canadian Institutes of Health Research New Investigator.
Submitted: 11 October 2007
Accepted: 26 June 2008
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