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

Ectopic expression of tinman and pannier results in expansion of enhancer activity.All embryos are stage 13–14 embryos carrying the -2775/-2432 Mef2-lacZ with the Lame duck consensus site mutated. (Left column) accumulation of MEF2; (center column) accumulation of ß-Gal; (right column) merge of prior two channels. Embryos have ectopic ectodermal expression of the following genes: (A-C)no additional genes expressed, note that activity of the enhancer can be seen in all cells of the heart (arrows), but the somatic mesodermal stain is reduced; (D-F)ectopic pnr expression, stains are similar to (A-C); (G-I) ectopic tin expression, note that activity of the enhancer is expanded in the ectoderm and amnioserosa (arrowheads). Arrow indicates cardiac cells. (J-L) ectopic tin and pnr expression, note that the enhancer is more robustly activated in the ectoderm and amnioserosa when compared to (H), which can been seen more dramatically in the inset, and ß-Gal accumulation co-localizes with the MEF2 expression seen in (J). Arrow indicates to cardiac cells. Scale bar, 100μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132965.g004: Ectopic expression of tinman and pannier results in expansion of enhancer activity.All embryos are stage 13–14 embryos carrying the -2775/-2432 Mef2-lacZ with the Lame duck consensus site mutated. (Left column) accumulation of MEF2; (center column) accumulation of ß-Gal; (right column) merge of prior two channels. Embryos have ectopic ectodermal expression of the following genes: (A-C)no additional genes expressed, note that activity of the enhancer can be seen in all cells of the heart (arrows), but the somatic mesodermal stain is reduced; (D-F)ectopic pnr expression, stains are similar to (A-C); (G-I) ectopic tin expression, note that activity of the enhancer is expanded in the ectoderm and amnioserosa (arrowheads). Arrow indicates cardiac cells. (J-L) ectopic tin and pnr expression, note that the enhancer is more robustly activated in the ectoderm and amnioserosa when compared to (H), which can been seen more dramatically in the inset, and ß-Gal accumulation co-localizes with the MEF2 expression seen in (J). Arrow indicates to cardiac cells. Scale bar, 100μm.

Mentions: We generated transgenic flies carrying this mutated construct, and found that LacZ expression was reduced in the somatic mesoderm, but not completely lost. This result was consistent with the identification by Busser et al [34] of the Lmd binding sites, since the most promoter-distal Lmd site is retained in the -2432/-2775 enhancer, and could contribute to enhancer activity in the somatic mesoderm. However, the activity of the mutated enhancer was sufficiently reduced to enable clear visualization of cardiac enhancer-lacZ expression. Interestingly, at stage 16, the cardiac cell activity of the enhancer became inconsistent, with random groups of cells losing expression (Fig 4A–4C compared to Fig 3B). This patchy enhancer activity was consistent with an experiment where we tried to remove the 3’ 170 bp of sequence from the enhancer, that included the site mutated here, and we lost all enhancer activity (data not shown), suggesting that 3’ sequences are critical to enhancer activity.


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)

Ectopic expression of tinman and pannier results in expansion of enhancer activity.All embryos are stage 13–14 embryos carrying the -2775/-2432 Mef2-lacZ with the Lame duck consensus site mutated. (Left column) accumulation of MEF2; (center column) accumulation of ß-Gal; (right column) merge of prior two channels. Embryos have ectopic ectodermal expression of the following genes: (A-C)no additional genes expressed, note that activity of the enhancer can be seen in all cells of the heart (arrows), but the somatic mesodermal stain is reduced; (D-F)ectopic pnr expression, stains are similar to (A-C); (G-I) ectopic tin expression, note that activity of the enhancer is expanded in the ectoderm and amnioserosa (arrowheads). Arrow indicates cardiac cells. (J-L) ectopic tin and pnr expression, note that the enhancer is more robustly activated in the ectoderm and amnioserosa when compared to (H), which can been seen more dramatically in the inset, and ß-Gal accumulation co-localizes with the MEF2 expression seen in (J). Arrow indicates to cardiac cells. Scale bar, 100μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132965.g004: Ectopic expression of tinman and pannier results in expansion of enhancer activity.All embryos are stage 13–14 embryos carrying the -2775/-2432 Mef2-lacZ with the Lame duck consensus site mutated. (Left column) accumulation of MEF2; (center column) accumulation of ß-Gal; (right column) merge of prior two channels. Embryos have ectopic ectodermal expression of the following genes: (A-C)no additional genes expressed, note that activity of the enhancer can be seen in all cells of the heart (arrows), but the somatic mesodermal stain is reduced; (D-F)ectopic pnr expression, stains are similar to (A-C); (G-I) ectopic tin expression, note that activity of the enhancer is expanded in the ectoderm and amnioserosa (arrowheads). Arrow indicates cardiac cells. (J-L) ectopic tin and pnr expression, note that the enhancer is more robustly activated in the ectoderm and amnioserosa when compared to (H), which can been seen more dramatically in the inset, and ß-Gal accumulation co-localizes with the MEF2 expression seen in (J). Arrow indicates to cardiac cells. Scale bar, 100μm.
Mentions: We generated transgenic flies carrying this mutated construct, and found that LacZ expression was reduced in the somatic mesoderm, but not completely lost. This result was consistent with the identification by Busser et al [34] of the Lmd binding sites, since the most promoter-distal Lmd site is retained in the -2432/-2775 enhancer, and could contribute to enhancer activity in the somatic mesoderm. However, the activity of the mutated enhancer was sufficiently reduced to enable clear visualization of cardiac enhancer-lacZ expression. Interestingly, at stage 16, the cardiac cell activity of the enhancer became inconsistent, with random groups of cells losing expression (Fig 4A–4C compared to Fig 3B). This patchy enhancer activity was consistent with an experiment where we tried to remove the 3’ 170 bp of sequence from the enhancer, that included the site mutated here, and we lost all enhancer activity (data not shown), suggesting that 3’ sequences are critical to enhancer activity.

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