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Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis.

Vijayaraj P, Le Bras A, Mitchell N, Kondo M, Juliao S, Wasserman M, Beeler D, Spokes K, Aird WC, Baldwin HS, Oettgen P - Development (2012)

Bottom Line: Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes.The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells.We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous Erg(ΔEx3/ΔEx3) knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous Erg(ΔEx4/ΔEx4) knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

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ErgΔEx4/ΔEx4 embryos show changes in genes regulating EnMT. (A) Differential expression of genes that regulate EnMT in ErgΔEx4/ΔEx4 compared with Erg+/+ E10.5 heart samples. For each gene evaluated, control expression was normalized to 1 in order to compare the relative change in expression in the knockout versus control. Error bars indicate s.d. of at least six independent qPCR reactions per genotype. (B,C) Snail1 plus Snail2 staining of sections of Erg+/+ and ErgΔEx4/ΔEx4 AVC regions at E10.5, showing a marked reduction in Snail1/2-positive cells in the mutant. Dashed lines outline the endocardial cells. (D,E) Representative serial sections of B and C, respectively, stained with IgG controls. A, atrium; AVC, atrioventricular canal.
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Figure 12: ErgΔEx4/ΔEx4 embryos show changes in genes regulating EnMT. (A) Differential expression of genes that regulate EnMT in ErgΔEx4/ΔEx4 compared with Erg+/+ E10.5 heart samples. For each gene evaluated, control expression was normalized to 1 in order to compare the relative change in expression in the knockout versus control. Error bars indicate s.d. of at least six independent qPCR reactions per genotype. (B,C) Snail1 plus Snail2 staining of sections of Erg+/+ and ErgΔEx4/ΔEx4 AVC regions at E10.5, showing a marked reduction in Snail1/2-positive cells in the mutant. Dashed lines outline the endocardial cells. (D,E) Representative serial sections of B and C, respectively, stained with IgG controls. A, atrium; AVC, atrioventricular canal.

Mentions: In an attempt to identify regulators and downstream targets of Erg in endocardial cushion formation, we collected RNA from Erg+/+ and ErgΔEx4/ΔEx4 hearts at E10.5 and isolated RNA for qRT-PCR analysis of several genes involved in EnMT in the developing heart (Fig. 12A; supplementary material Table S1). Of the genes analyzed, Snail1 (also known as Snail or Snai1) and Snail2 (Slug or Snai2) were significantly downregulated in ErgΔEx4/ΔEx4 heart. Snail1 and Snail2 encode zinc-finger transcriptional repressors that have been reported to induce EnMT during embryonic development. Deficiency of Snail2 is partially rescued by the expression of Snail1 (Niessen et al., 2008). Accordingly, deficiency of both Snail1 and Snail2 leads to lethality at E10.5 from EnMT defects (Niessen and Karsan, 2008). This helped us identify Snail1 and Snail2 as potential downstream targets of Erg. Snail2 has been reported to be a direct target of Notch during EnMT and cardiac cushion cellularization (Timmerman et al., 2004; Grego-Bessa et al., 2007; Niessen et al., 2008). Interestingly, there were no significant changes in the expression of Notch1/4, Bmp2/4 or Tgfb1/2, which are the main pathways involved during valve morphogenesis. Consistently, hearts stained with Snail1 plus Snail2 antibody revealed a drastic reduction in the number of stained cells in the AVC region (Fig. 12C) and OFT (data not shown) of ErgΔEx4/ΔEx4 embryos compared with Erg+/+ controls (Fig. 12B). These data suggest that Erg acts downstream of crucial regulators such as Notch, BMP and Tgfβ and upstream of Snail1 and Snail2.


Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis.

Vijayaraj P, Le Bras A, Mitchell N, Kondo M, Juliao S, Wasserman M, Beeler D, Spokes K, Aird WC, Baldwin HS, Oettgen P - Development (2012)

ErgΔEx4/ΔEx4 embryos show changes in genes regulating EnMT. (A) Differential expression of genes that regulate EnMT in ErgΔEx4/ΔEx4 compared with Erg+/+ E10.5 heart samples. For each gene evaluated, control expression was normalized to 1 in order to compare the relative change in expression in the knockout versus control. Error bars indicate s.d. of at least six independent qPCR reactions per genotype. (B,C) Snail1 plus Snail2 staining of sections of Erg+/+ and ErgΔEx4/ΔEx4 AVC regions at E10.5, showing a marked reduction in Snail1/2-positive cells in the mutant. Dashed lines outline the endocardial cells. (D,E) Representative serial sections of B and C, respectively, stained with IgG controls. A, atrium; AVC, atrioventricular canal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 12: ErgΔEx4/ΔEx4 embryos show changes in genes regulating EnMT. (A) Differential expression of genes that regulate EnMT in ErgΔEx4/ΔEx4 compared with Erg+/+ E10.5 heart samples. For each gene evaluated, control expression was normalized to 1 in order to compare the relative change in expression in the knockout versus control. Error bars indicate s.d. of at least six independent qPCR reactions per genotype. (B,C) Snail1 plus Snail2 staining of sections of Erg+/+ and ErgΔEx4/ΔEx4 AVC regions at E10.5, showing a marked reduction in Snail1/2-positive cells in the mutant. Dashed lines outline the endocardial cells. (D,E) Representative serial sections of B and C, respectively, stained with IgG controls. A, atrium; AVC, atrioventricular canal.
Mentions: In an attempt to identify regulators and downstream targets of Erg in endocardial cushion formation, we collected RNA from Erg+/+ and ErgΔEx4/ΔEx4 hearts at E10.5 and isolated RNA for qRT-PCR analysis of several genes involved in EnMT in the developing heart (Fig. 12A; supplementary material Table S1). Of the genes analyzed, Snail1 (also known as Snail or Snai1) and Snail2 (Slug or Snai2) were significantly downregulated in ErgΔEx4/ΔEx4 heart. Snail1 and Snail2 encode zinc-finger transcriptional repressors that have been reported to induce EnMT during embryonic development. Deficiency of Snail2 is partially rescued by the expression of Snail1 (Niessen et al., 2008). Accordingly, deficiency of both Snail1 and Snail2 leads to lethality at E10.5 from EnMT defects (Niessen and Karsan, 2008). This helped us identify Snail1 and Snail2 as potential downstream targets of Erg. Snail2 has been reported to be a direct target of Notch during EnMT and cardiac cushion cellularization (Timmerman et al., 2004; Grego-Bessa et al., 2007; Niessen et al., 2008). Interestingly, there were no significant changes in the expression of Notch1/4, Bmp2/4 or Tgfb1/2, which are the main pathways involved during valve morphogenesis. Consistently, hearts stained with Snail1 plus Snail2 antibody revealed a drastic reduction in the number of stained cells in the AVC region (Fig. 12C) and OFT (data not shown) of ErgΔEx4/ΔEx4 embryos compared with Erg+/+ controls (Fig. 12B). These data suggest that Erg acts downstream of crucial regulators such as Notch, BMP and Tgfβ and upstream of Snail1 and Snail2.

Bottom Line: Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes.The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells.We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous Erg(ΔEx3/ΔEx3) knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous Erg(ΔEx4/ΔEx4) knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

Show MeSH
Related in: MedlinePlus