Limits...
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

Mutation of the Tinman consensus site in the -2775/-2432 enhancer results in loss of enhancer activity in cardiac cells.(A-F)-2775/-2432 Mef2-lacZ embryos at stages 14 (A-C) and 16 (D-F). (A,D)Antibody stain against MEF2. MEF2 could be detected in all cells of the heart and throughout the somatic mesoderm. (B,E)Antibody stain against β-Galactosidase. Activity of the late stage enhancer was almost identical to that of Mef2 expression. In (B), the enhancer is just becoming active in the heart cells, therefore a few cells lacked activity at this stage. By stage 16 (E) all cardiac cells showed ßGal accumulation. (C)Merge of (A) and (B); (F)Merge of (D) and (E). (G-L)Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the Tinman consensus site mutated, at stages 14 (G-I) or 16 (J-L). (G,J)Antibody stain against MEF2. MEF2 marks all cells of the heart and the somatic mesoderm. (H,K)Antibody stain against β-Galactosidase. Activity of the mutated enhancer was completely lost from the cardiac cells, and was slightly reduced in the somatic mesoderm. (I)Merge of (G) and (H). (L)Merge of (J) and (K). Arrows point to MEF2 positive cardiac cells, arrowheads point to the same cells lacking β-Galactosidase. (M-R) Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the three Pannier consensus sites mutated, at stages 14 (M-O) or 16 (P-R). (O)Merge of (M) and (N). (R) Merge of (P) and (Q). Arrows point to cardiac cells, arrow heads point to cells that have lost enhancer activity. Asterisk denotes activity in the amnioserosa. Bar, 100μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520567&req=5

pone.0132965.g003: Mutation of the Tinman consensus site in the -2775/-2432 enhancer results in loss of enhancer activity in cardiac cells.(A-F)-2775/-2432 Mef2-lacZ embryos at stages 14 (A-C) and 16 (D-F). (A,D)Antibody stain against MEF2. MEF2 could be detected in all cells of the heart and throughout the somatic mesoderm. (B,E)Antibody stain against β-Galactosidase. Activity of the late stage enhancer was almost identical to that of Mef2 expression. In (B), the enhancer is just becoming active in the heart cells, therefore a few cells lacked activity at this stage. By stage 16 (E) all cardiac cells showed ßGal accumulation. (C)Merge of (A) and (B); (F)Merge of (D) and (E). (G-L)Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the Tinman consensus site mutated, at stages 14 (G-I) or 16 (J-L). (G,J)Antibody stain against MEF2. MEF2 marks all cells of the heart and the somatic mesoderm. (H,K)Antibody stain against β-Galactosidase. Activity of the mutated enhancer was completely lost from the cardiac cells, and was slightly reduced in the somatic mesoderm. (I)Merge of (G) and (H). (L)Merge of (J) and (K). Arrows point to MEF2 positive cardiac cells, arrowheads point to the same cells lacking β-Galactosidase. (M-R) Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the three Pannier consensus sites mutated, at stages 14 (M-O) or 16 (P-R). (O)Merge of (M) and (N). (R) Merge of (P) and (Q). Arrows point to cardiac cells, arrow heads point to cells that have lost enhancer activity. Asterisk denotes activity in the amnioserosa. Bar, 100μm.

Mentions: Having determined that Tin could bind to the consensus site in the enhancer, we next determined if the site was required for enhancer activity. Using site-directed mutagenesis, we mutated the Tin consensus site within the context of the full-length enhancer, fused the mutated enhancer to a lacZ reporter, and generated transgenic flies carrying this construct. We analyzed reporter expression in transgenic embryos of the wild-type construct (Fig 3A–3F) relative to the Tin-mutant construct. We noted that lacZ reporter activity of the mutant construct was slightly reduced in the somatic mesodermal cells, but still present. However, reporter expression was lost from all cells of the heart (Fig 3G–3L). This loss of enhancer activity was apparent at early and late stages of cardiogenesis, suggesting that Tin is a direct and essential activator of this enhancer in vivo during the embryonic stage. In addition, we mutated the three Pnr consensus sites and assessed enhancer-lacZ activity in vivo. Here, we saw a reduction of reporter activity in the heart cells (Fig 3M–3R), which suggested that Pnr does have direct interaction with its consensus sites and contributes to enhancer robustness as seen in our cell culture experiments. We were unable to observe a direct interaction between Pnr protein and the enhancer in vitro; however, the mutation of the consensus binding sites in vivo clearly diminished enhancer activity and the synergistic activation of the enhancer with Tinman was robust, suggesting the Pnr does in fact interact directly. In the Pnr-mutant enhancer-lacZ lines, we also saw an expansion of enhancer activity in the amnioserosa. We hypothesize that while the Pnr consensus sites are required for full activation of the enhancer in cardiac cells, the sites are also required for repression of Mef2 in the amnioserosa, as Pnr has been shown previously to act as a repressor of transcription [46].


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)

Mutation of the Tinman consensus site in the -2775/-2432 enhancer results in loss of enhancer activity in cardiac cells.(A-F)-2775/-2432 Mef2-lacZ embryos at stages 14 (A-C) and 16 (D-F). (A,D)Antibody stain against MEF2. MEF2 could be detected in all cells of the heart and throughout the somatic mesoderm. (B,E)Antibody stain against β-Galactosidase. Activity of the late stage enhancer was almost identical to that of Mef2 expression. In (B), the enhancer is just becoming active in the heart cells, therefore a few cells lacked activity at this stage. By stage 16 (E) all cardiac cells showed ßGal accumulation. (C)Merge of (A) and (B); (F)Merge of (D) and (E). (G-L)Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the Tinman consensus site mutated, at stages 14 (G-I) or 16 (J-L). (G,J)Antibody stain against MEF2. MEF2 marks all cells of the heart and the somatic mesoderm. (H,K)Antibody stain against β-Galactosidase. Activity of the mutated enhancer was completely lost from the cardiac cells, and was slightly reduced in the somatic mesoderm. (I)Merge of (G) and (H). (L)Merge of (J) and (K). Arrows point to MEF2 positive cardiac cells, arrowheads point to the same cells lacking β-Galactosidase. (M-R) Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the three Pannier consensus sites mutated, at stages 14 (M-O) or 16 (P-R). (O)Merge of (M) and (N). (R) Merge of (P) and (Q). Arrows point to cardiac cells, arrow heads point to cells that have lost enhancer activity. Asterisk denotes activity in the amnioserosa. Bar, 100μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520567&req=5

pone.0132965.g003: Mutation of the Tinman consensus site in the -2775/-2432 enhancer results in loss of enhancer activity in cardiac cells.(A-F)-2775/-2432 Mef2-lacZ embryos at stages 14 (A-C) and 16 (D-F). (A,D)Antibody stain against MEF2. MEF2 could be detected in all cells of the heart and throughout the somatic mesoderm. (B,E)Antibody stain against β-Galactosidase. Activity of the late stage enhancer was almost identical to that of Mef2 expression. In (B), the enhancer is just becoming active in the heart cells, therefore a few cells lacked activity at this stage. By stage 16 (E) all cardiac cells showed ßGal accumulation. (C)Merge of (A) and (B); (F)Merge of (D) and (E). (G-L)Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the Tinman consensus site mutated, at stages 14 (G-I) or 16 (J-L). (G,J)Antibody stain against MEF2. MEF2 marks all cells of the heart and the somatic mesoderm. (H,K)Antibody stain against β-Galactosidase. Activity of the mutated enhancer was completely lost from the cardiac cells, and was slightly reduced in the somatic mesoderm. (I)Merge of (G) and (H). (L)Merge of (J) and (K). Arrows point to MEF2 positive cardiac cells, arrowheads point to the same cells lacking β-Galactosidase. (M-R) Embryos carrying the -2775/-2432 Mef2-lacZ enhancer with the three Pannier consensus sites mutated, at stages 14 (M-O) or 16 (P-R). (O)Merge of (M) and (N). (R) Merge of (P) and (Q). Arrows point to cardiac cells, arrow heads point to cells that have lost enhancer activity. Asterisk denotes activity in the amnioserosa. Bar, 100μm.
Mentions: Having determined that Tin could bind to the consensus site in the enhancer, we next determined if the site was required for enhancer activity. Using site-directed mutagenesis, we mutated the Tin consensus site within the context of the full-length enhancer, fused the mutated enhancer to a lacZ reporter, and generated transgenic flies carrying this construct. We analyzed reporter expression in transgenic embryos of the wild-type construct (Fig 3A–3F) relative to the Tin-mutant construct. We noted that lacZ reporter activity of the mutant construct was slightly reduced in the somatic mesodermal cells, but still present. However, reporter expression was lost from all cells of the heart (Fig 3G–3L). This loss of enhancer activity was apparent at early and late stages of cardiogenesis, suggesting that Tin is a direct and essential activator of this enhancer in vivo during the embryonic stage. In addition, we mutated the three Pnr consensus sites and assessed enhancer-lacZ activity in vivo. Here, we saw a reduction of reporter activity in the heart cells (Fig 3M–3R), which suggested that Pnr does have direct interaction with its consensus sites and contributes to enhancer robustness as seen in our cell culture experiments. We were unable to observe a direct interaction between Pnr protein and the enhancer in vitro; however, the mutation of the consensus binding sites in vivo clearly diminished enhancer activity and the synergistic activation of the enhancer with Tinman was robust, suggesting the Pnr does in fact interact directly. In the Pnr-mutant enhancer-lacZ lines, we also saw an expansion of enhancer activity in the amnioserosa. We hypothesize that while the Pnr consensus sites are required for full activation of the enhancer in cardiac cells, the sites are also required for repression of Mef2 in the amnioserosa, as Pnr has been shown previously to act as a repressor of transcription [46].

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