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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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Deletion of endothelial enriched Erg in endocardial cells prevents EnMT in vitro. (A) In vitro collagen gel assays performed with AVC and OFT explants isolated from Erg+/+ (i,iii) or ErgΔEx4/ΔEx4 (ii,iv) E10.5 hearts. The number of invading and migrating cells formed in Erg+/+ explants was substantially higher than in ErgΔEx4/ΔEx4 explants. (B) Erg+/+ (i,iii) and ErgΔEx4/ΔEx4 (ii,iv) explant cultures stained with an antibody (green) against the mesenchymal marker α-SMA. Nuclei were visualized by staining with DAPI (blue). (C) The numbers of mesenchymal cells formed in Erg+/+ (n=12) and ErgΔEx4/ΔEx4 (n=7) explants were quantified. The number of migrating and invading mesenchymal cells was 16.66±2% in the AVC and 13.15±2% in the OFT regions in ErgΔEx4/ΔEx4 compared with the Erg+/+ embryos. *P<0.005. Error bars indicate s.d.
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Figure 11: Deletion of endothelial enriched Erg in endocardial cells prevents EnMT in vitro. (A) In vitro collagen gel assays performed with AVC and OFT explants isolated from Erg+/+ (i,iii) or ErgΔEx4/ΔEx4 (ii,iv) E10.5 hearts. The number of invading and migrating cells formed in Erg+/+ explants was substantially higher than in ErgΔEx4/ΔEx4 explants. (B) Erg+/+ (i,iii) and ErgΔEx4/ΔEx4 (ii,iv) explant cultures stained with an antibody (green) against the mesenchymal marker α-SMA. Nuclei were visualized by staining with DAPI (blue). (C) The numbers of mesenchymal cells formed in Erg+/+ (n=12) and ErgΔEx4/ΔEx4 (n=7) explants were quantified. The number of migrating and invading mesenchymal cells was 16.66±2% in the AVC and 13.15±2% in the OFT regions in ErgΔEx4/ΔEx4 compared with the Erg+/+ embryos. *P<0.005. Error bars indicate s.d.

Mentions: We next analyzed the transformation of endocardial cells to mesenchymal cells by performing AVC and OFT explant cultures, using a collagen gel culture system (Runyan and Markwald, 1983), from E9.5 and E10.5 embryos, respectively. Endocardial cells from Erg+/+ AVC and OFT explants were motile and invaded the collagen matrix, whereas the cells from ErgΔEx4/ΔEx4 explants showed a marked reduction in migration and invasion (Fig. 11A). We further characterized EnMT in the ErgΔEx4/ΔEx4 and Erg+/+ AVC and OFT explant cultures by immunofluorescence analysis. The explants were stained for the mesenchymal marker smooth muscle α-actin (α-SMA) and stained cells that had migrated away from the explant into the matrix were counted and quantified (Fig. 11B,C). α-SMA-stained cells were found in abundance in Erg+/+ explants, whereas very few were observed in the explants from ErgΔEx4/ΔEx4 embryos (Fig. 11C).


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)

Deletion of endothelial enriched Erg in endocardial cells prevents EnMT in vitro. (A) In vitro collagen gel assays performed with AVC and OFT explants isolated from Erg+/+ (i,iii) or ErgΔEx4/ΔEx4 (ii,iv) E10.5 hearts. The number of invading and migrating cells formed in Erg+/+ explants was substantially higher than in ErgΔEx4/ΔEx4 explants. (B) Erg+/+ (i,iii) and ErgΔEx4/ΔEx4 (ii,iv) explant cultures stained with an antibody (green) against the mesenchymal marker α-SMA. Nuclei were visualized by staining with DAPI (blue). (C) The numbers of mesenchymal cells formed in Erg+/+ (n=12) and ErgΔEx4/ΔEx4 (n=7) explants were quantified. The number of migrating and invading mesenchymal cells was 16.66±2% in the AVC and 13.15±2% in the OFT regions in ErgΔEx4/ΔEx4 compared with the Erg+/+ embryos. *P<0.005. Error bars indicate s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3472597&req=5

Figure 11: Deletion of endothelial enriched Erg in endocardial cells prevents EnMT in vitro. (A) In vitro collagen gel assays performed with AVC and OFT explants isolated from Erg+/+ (i,iii) or ErgΔEx4/ΔEx4 (ii,iv) E10.5 hearts. The number of invading and migrating cells formed in Erg+/+ explants was substantially higher than in ErgΔEx4/ΔEx4 explants. (B) Erg+/+ (i,iii) and ErgΔEx4/ΔEx4 (ii,iv) explant cultures stained with an antibody (green) against the mesenchymal marker α-SMA. Nuclei were visualized by staining with DAPI (blue). (C) The numbers of mesenchymal cells formed in Erg+/+ (n=12) and ErgΔEx4/ΔEx4 (n=7) explants were quantified. The number of migrating and invading mesenchymal cells was 16.66±2% in the AVC and 13.15±2% in the OFT regions in ErgΔEx4/ΔEx4 compared with the Erg+/+ embryos. *P<0.005. Error bars indicate s.d.
Mentions: We next analyzed the transformation of endocardial cells to mesenchymal cells by performing AVC and OFT explant cultures, using a collagen gel culture system (Runyan and Markwald, 1983), from E9.5 and E10.5 embryos, respectively. Endocardial cells from Erg+/+ AVC and OFT explants were motile and invaded the collagen matrix, whereas the cells from ErgΔEx4/ΔEx4 explants showed a marked reduction in migration and invasion (Fig. 11A). We further characterized EnMT in the ErgΔEx4/ΔEx4 and Erg+/+ AVC and OFT explant cultures by immunofluorescence analysis. The explants were stained for the mesenchymal marker smooth muscle α-actin (α-SMA) and stained cells that had migrated away from the explant into the matrix were counted and quantified (Fig. 11B,C). α-SMA-stained cells were found in abundance in Erg+/+ explants, whereas very few were observed in the explants from ErgΔEx4/ΔEx4 embryos (Fig. 11C).

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