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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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Targeting of ErgΔEx3/+ and ErgΔEx4/+ loci. (A) The wild-type murine Erg locus, the targeting construct, and targeted locus with exon 3 replaced by the lacZ-Neo reporter cassette. The numbered boxes represent Erg exons. Bold lines indicate the arms of homology. The positions of Southern blotting probes are indicated. (B) Southern analysis to identify ErgΔEx3/+ ES cell clones using 5′ and 3′ probes that distinguish NheI fragments in the wild-type and targeted alleles. (C) Erg wild-type locus, the targeting construct, and targeted locus with exon 4 replaced by the lacZ-Neo reporter cassette. (D) Southern analysis to identify ErgΔEx4/+ ES cell clones using 5′ and 3′ probes that distinguish BssSI and NheI fragments in the wild-type and targeted allele, respectively.
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Figure 2: Targeting of ErgΔEx3/+ and ErgΔEx4/+ loci. (A) The wild-type murine Erg locus, the targeting construct, and targeted locus with exon 3 replaced by the lacZ-Neo reporter cassette. The numbered boxes represent Erg exons. Bold lines indicate the arms of homology. The positions of Southern blotting probes are indicated. (B) Southern analysis to identify ErgΔEx3/+ ES cell clones using 5′ and 3′ probes that distinguish NheI fragments in the wild-type and targeted alleles. (C) Erg wild-type locus, the targeting construct, and targeted locus with exon 4 replaced by the lacZ-Neo reporter cassette. (D) Southern analysis to identify ErgΔEx4/+ ES cell clones using 5′ and 3′ probes that distinguish BssSI and NheI fragments in the wild-type and targeted allele, respectively.

Mentions: To further define the role of exon 3- and exon 4-specific isoforms during development, we generated ErgΔEx3/ΔEx3 and ErgΔEx4/ΔEx4 knockout mice using standard gene targeting strategies. In brief, we designed a targeting vector to replace either the ATG of exon 3 or exon 4 with a lacZ-Neo cassette (Fig. 2A,C). Targeting of these exons blocks protein expression of Erg isoforms 1-4 and 5-7, respectively, which is replaced by expression of lacZ. Mouse ES cells were transfected with the linearized targeting vectors and neomycin-resistant ES cell clones were picked and screened. Successful homologous recombination was verified by PCR and Southern blot analysis (Fig. 2B,D). ErgΔEx3/+ and ErgΔEx4/+ ES cells were used to produce ErgΔEx3/+ and ErgΔEx4/+ heterozygous knockout mice, respectively. lacZ staining was first observed in the ErgΔEx3/+ mice at E9.5 and in ErgΔEx4/+ mice at E7.5 (Fig. 3A).


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)

Targeting of ErgΔEx3/+ and ErgΔEx4/+ loci. (A) The wild-type murine Erg locus, the targeting construct, and targeted locus with exon 3 replaced by the lacZ-Neo reporter cassette. The numbered boxes represent Erg exons. Bold lines indicate the arms of homology. The positions of Southern blotting probes are indicated. (B) Southern analysis to identify ErgΔEx3/+ ES cell clones using 5′ and 3′ probes that distinguish NheI fragments in the wild-type and targeted alleles. (C) Erg wild-type locus, the targeting construct, and targeted locus with exon 4 replaced by the lacZ-Neo reporter cassette. (D) Southern analysis to identify ErgΔEx4/+ ES cell clones using 5′ and 3′ probes that distinguish BssSI and NheI fragments in the wild-type and targeted allele, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Targeting of ErgΔEx3/+ and ErgΔEx4/+ loci. (A) The wild-type murine Erg locus, the targeting construct, and targeted locus with exon 3 replaced by the lacZ-Neo reporter cassette. The numbered boxes represent Erg exons. Bold lines indicate the arms of homology. The positions of Southern blotting probes are indicated. (B) Southern analysis to identify ErgΔEx3/+ ES cell clones using 5′ and 3′ probes that distinguish NheI fragments in the wild-type and targeted alleles. (C) Erg wild-type locus, the targeting construct, and targeted locus with exon 4 replaced by the lacZ-Neo reporter cassette. (D) Southern analysis to identify ErgΔEx4/+ ES cell clones using 5′ and 3′ probes that distinguish BssSI and NheI fragments in the wild-type and targeted allele, respectively.
Mentions: To further define the role of exon 3- and exon 4-specific isoforms during development, we generated ErgΔEx3/ΔEx3 and ErgΔEx4/ΔEx4 knockout mice using standard gene targeting strategies. In brief, we designed a targeting vector to replace either the ATG of exon 3 or exon 4 with a lacZ-Neo cassette (Fig. 2A,C). Targeting of these exons blocks protein expression of Erg isoforms 1-4 and 5-7, respectively, which is replaced by expression of lacZ. Mouse ES cells were transfected with the linearized targeting vectors and neomycin-resistant ES cell clones were picked and screened. Successful homologous recombination was verified by PCR and Southern blot analysis (Fig. 2B,D). ErgΔEx3/+ and ErgΔEx4/+ ES cells were used to produce ErgΔEx3/+ and ErgΔEx4/+ heterozygous knockout mice, respectively. lacZ staining was first observed in the ErgΔEx3/+ mice at E9.5 and in ErgΔEx4/+ mice at E7.5 (Fig. 3A).

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