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The Drosophila Transcription Factors Tinman and Pannier Activate and Collaborate with Myocyte Enhancer Factor-2 to Promote Heart Cell Fate.

Lovato TL, Sensibaugh CA, Swingle KL, Martinez MM, Cripps RM - PLoS ONE (2015)

Bottom Line: We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression.By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed.Two additional cardiac markers were also expanded in their expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, United States of America.

ABSTRACT
Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.

No MeSH data available.


Related in: MedlinePlus

Identification of a proximal Mef2 Cardiac Enhancer.(A)Diagram of the Mef2 gene and its cardiac enhancers. The most distal enhancer (-6877/-6388) refers to the Seven-up cell enhancer. -5903/-5667 represents the Tinman-dependent enhancer and the red box (-2775/-2432) is the late stage cardiac and somatic mesodermal enhancer characterized here. (B) Activity of Mef2 cardiac enhancers fused to lacZ reporters. The embryos were stained for β-Gal accumulation. Top row, the -5903/-5667 enhancer was active earliest in development, and reporter activity was detected until stage 16. Middle row, the -2775/-2432 enhancer became active at stage 13 in cardiac cells (arrow) and skeletal myoblasts, and was active in all cardiac cells by stage 16. Bottom row, when the two enhancers were fused, there was reporter expression in all cardiac cells during embryogenesis. Arrows indicate heart cells. Bar, 100μm. (C)Alignment of the proximal enhancer sequence with four Drosophila species. A conserved Tinman binding site is marked by a blue box, Pannier sites are marked with red boxes, and the proposed Lame duck binding site identified by Duan et al [33] is marked with a green box.
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pone.0132965.g001: Identification of a proximal Mef2 Cardiac Enhancer.(A)Diagram of the Mef2 gene and its cardiac enhancers. The most distal enhancer (-6877/-6388) refers to the Seven-up cell enhancer. -5903/-5667 represents the Tinman-dependent enhancer and the red box (-2775/-2432) is the late stage cardiac and somatic mesodermal enhancer characterized here. (B) Activity of Mef2 cardiac enhancers fused to lacZ reporters. The embryos were stained for β-Gal accumulation. Top row, the -5903/-5667 enhancer was active earliest in development, and reporter activity was detected until stage 16. Middle row, the -2775/-2432 enhancer became active at stage 13 in cardiac cells (arrow) and skeletal myoblasts, and was active in all cardiac cells by stage 16. Bottom row, when the two enhancers were fused, there was reporter expression in all cardiac cells during embryogenesis. Arrows indicate heart cells. Bar, 100μm. (C)Alignment of the proximal enhancer sequence with four Drosophila species. A conserved Tinman binding site is marked by a blue box, Pannier sites are marked with red boxes, and the proposed Lame duck binding site identified by Duan et al [33] is marked with a green box.

Mentions: We have worked to isolate the cardiac specific portion of the large late stage enhancer first identified by Nguyen and Xu [32], which lies more proximal to the transcription start site of the Mef2 gene than the previously identified cardiac enhancers (Fig 1A and S1 Fig). We generated PCR fragments from this region, cloned them into a plasmid containing a lacZ reporter gene and generated transgenic flies that contained the Mef2-lacZ constructs. Embryos from these lines were stained with an antibody against β-Galactosidase to visualize activity of the enhancer. The smallest enhancer fragment with complete activity in both cardiac cell types lies at -2432/-2775 relative to the Mef2 transcription start site. This 345-bp fragment also contained the 170bp mesodermal enhancer characterized by Duan et al [33] and a portion of the mesodermal enhancer characterized by Busser et al ([34] that had additional regulatory regions lying downstream of our enhancer. Attempts to separate the cardiac and somatic enhancer activities by 5’ or 3’ deletions resulted in loss of activity from both the cardiac cells and somatic mesoderm (data not shown). This observation indicated that certain enhancer sequences are utilized in both cardiac and skeletal muscle tissues.


The Drosophila Transcription Factors Tinman and Pannier Activate and Collaborate with Myocyte Enhancer Factor-2 to Promote Heart Cell Fate.

Lovato TL, Sensibaugh CA, Swingle KL, Martinez MM, Cripps RM - PLoS ONE (2015)

Identification of a proximal Mef2 Cardiac Enhancer.(A)Diagram of the Mef2 gene and its cardiac enhancers. The most distal enhancer (-6877/-6388) refers to the Seven-up cell enhancer. -5903/-5667 represents the Tinman-dependent enhancer and the red box (-2775/-2432) is the late stage cardiac and somatic mesodermal enhancer characterized here. (B) Activity of Mef2 cardiac enhancers fused to lacZ reporters. The embryos were stained for β-Gal accumulation. Top row, the -5903/-5667 enhancer was active earliest in development, and reporter activity was detected until stage 16. Middle row, the -2775/-2432 enhancer became active at stage 13 in cardiac cells (arrow) and skeletal myoblasts, and was active in all cardiac cells by stage 16. Bottom row, when the two enhancers were fused, there was reporter expression in all cardiac cells during embryogenesis. Arrows indicate heart cells. Bar, 100μm. (C)Alignment of the proximal enhancer sequence with four Drosophila species. A conserved Tinman binding site is marked by a blue box, Pannier sites are marked with red boxes, and the proposed Lame duck binding site identified by Duan et al [33] is marked with a green box.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520567&req=5

pone.0132965.g001: Identification of a proximal Mef2 Cardiac Enhancer.(A)Diagram of the Mef2 gene and its cardiac enhancers. The most distal enhancer (-6877/-6388) refers to the Seven-up cell enhancer. -5903/-5667 represents the Tinman-dependent enhancer and the red box (-2775/-2432) is the late stage cardiac and somatic mesodermal enhancer characterized here. (B) Activity of Mef2 cardiac enhancers fused to lacZ reporters. The embryos were stained for β-Gal accumulation. Top row, the -5903/-5667 enhancer was active earliest in development, and reporter activity was detected until stage 16. Middle row, the -2775/-2432 enhancer became active at stage 13 in cardiac cells (arrow) and skeletal myoblasts, and was active in all cardiac cells by stage 16. Bottom row, when the two enhancers were fused, there was reporter expression in all cardiac cells during embryogenesis. Arrows indicate heart cells. Bar, 100μm. (C)Alignment of the proximal enhancer sequence with four Drosophila species. A conserved Tinman binding site is marked by a blue box, Pannier sites are marked with red boxes, and the proposed Lame duck binding site identified by Duan et al [33] is marked with a green box.
Mentions: We have worked to isolate the cardiac specific portion of the large late stage enhancer first identified by Nguyen and Xu [32], which lies more proximal to the transcription start site of the Mef2 gene than the previously identified cardiac enhancers (Fig 1A and S1 Fig). We generated PCR fragments from this region, cloned them into a plasmid containing a lacZ reporter gene and generated transgenic flies that contained the Mef2-lacZ constructs. Embryos from these lines were stained with an antibody against β-Galactosidase to visualize activity of the enhancer. The smallest enhancer fragment with complete activity in both cardiac cell types lies at -2432/-2775 relative to the Mef2 transcription start site. This 345-bp fragment also contained the 170bp mesodermal enhancer characterized by Duan et al [33] and a portion of the mesodermal enhancer characterized by Busser et al ([34] that had additional regulatory regions lying downstream of our enhancer. Attempts to separate the cardiac and somatic enhancer activities by 5’ or 3’ deletions resulted in loss of activity from both the cardiac cells and somatic mesoderm (data not shown). This observation indicated that certain enhancer sequences are utilized in both cardiac and skeletal muscle tissues.

Bottom Line: We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression.By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed.Two additional cardiac markers were also expanded in their expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, United States of America.

ABSTRACT
Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.

No MeSH data available.


Related in: MedlinePlus