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First published online 4 October 2006
doi: 10.1242/dev.02610

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Myocardin is a direct transcriptional target of Mef2, Tead and Foxo proteins during cardiovascular development

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
² Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.

View larger version (52K): [in a new window]   Fig. 1. Identification of cis-acting cardiovascular enhancer regions in the myocardin locus. (A) Schematic representation of the genomic organization of the myocardin locus and the constructs used for the creation of transgenic mice (see Materials and methods for construct details). The enhancer element that drives expression in heart and smooth muscle (fragment 5) is located 20-30 kb upstream of myocardin's translational start in exon 1, and is shown in red. The number of genotype-positive E12.5 embryos obtained from a total of three injections per construct are indicated between brackets. Shown at the top are the first three exons of myocardin (in black) and the exon organization of an upstream gene (mRNA AK042647) in gray. (B) Expresssion of the myocardin enhancer (MyE) throughout embryonic development was determined using a stable transgenic line bearing fragment 5, linked to Hsp68lacZ. Embryos harvested from staged matings demonstrated that the enhancer recapitulates the expression pattern of myocardin. Note strong lacZ expression in the cardiac crescent (a,b). As the linear heart tube forms (c) and loops (d,f), lacZ staining is detected at high levels throughout the entire heart. In the vasculature we detected lacZ expression in the developing dorsal aorta as early as E9.0-9.5 (f,g), in branchial arch arteries at E10.5 (h,i) and carotid arteries at E12.5 (k,l). In adult tissues, lacZ expression was detected in the heart (e) and in the vasculature of the lung (j) and aorta (o).

View larger version (112K): [in a new window]   Fig. 2. Expression of the myocardin cardiac and smooth muscle enhancer. Embryos and adult tissues from the stable transgenic line harboring construct 5 (MyE1) were stained for ß-galactosidase activity, sectioned and counterstained with Nuclear Fast Red. Note intense staining in the cardiogenic plate at E7.5 (A) and in the bilaterally symmetric cardiac mesoderm at E7.75 (B). At E8.5, expression is detected throughout the primitive myocardium, including the endocardial tissue lining the primitive heart (C). At E12.5 expression is seen in the ventricles and atria (D,E). Note that the endocardial cushion is devoid of lacZ expression. Using dark-field microscopy (E,F) lacZ expression could clearly be observed in the dorsal aorta at E12.5 (E,F). In adult vascular tissues (G-J), lacZ expression was detected in smooth muscle cells of the lung arteries (G) and aorta (H). A cross-section through the esophagus revealed strong expression in the vessel wall of small arteries but not veins (I). A high magnification of adult heart shows strong expression in cardiomyocytes (J). a, artery; ao, aorta; b, bronchus; cm, cardiac mesoderm; cp, cardiogenic plate; ec, endocardial cushion; ra, right atria; rv, right ventricle; sm, splanchnic mesoderm.

View larger version (57K): [in a new window]   Fig. 3. Identification of an evolutionarily conserved minimal enhancer element sufficient to direct expression in heart and smooth muscle. (A) Representative expression pattern of F0 transgenic embryos with the two minimal transcriptional enhancers (MyE6 and MyE8). Whereas MyE6 directed lacZ expression to the heart, the 350 bp MyE8 fragment was sufficient to direct expression in heart and developing dorsal aorta and head vessels at E12.5. (B) Transgenic construct used to identify minimal elements of the myocardin enhancer (MyE). The construct numbers (MyE1-MyE9) are indicated on the left and fractions of F0 transgenic embryos showing cardiac expression at E12.5 are indicated in the right column. The 10 kb enhancer fragment MyE1 is identical to construct 5 in Fig. 1. An evolutionary conservation track shows the overall conservation score of MyE1 across all species, as well as pairwise alignment of rat, human, dog and chicken, each aligned to the mouse genome (Markov model, http://genome.ucsc.edu) (Kent et al., 2002). By transgenic analysis two evolutionarily conserved minimal enhancer elements, indicated in red, were identified (MyE6 and MyE8; 2 kb and 350 bp, respectively). Note that MyE8, but not MyE6, is conserved in the chicken. (C) Alignment of conserved mouse and human sequences in the myocardin enhancer MyE8. The conserved Mef2-binding site (blue shading), five candidate Foxo sites (yellow shading) and a conserved Tead-binding site (green shading) are noted.

View larger version (35K): [in a new window]   Fig. 4. Requirement of an Mef2-binding site for activity in heart and smooth muscle of the distal myocardin enhancer. (A) Binding of Mef2c to the Mef2-binding site in the minimal enhancer element MyE8. A 32P-labeled oligonucleotide containing the conserved Mef2 site and Mef2c translated in reticulocyte lysate was used for electrophoretic mobility shift assays. DNA binding was seen only in reactions containing lysates with Mef2c. The DNA-Mef2c complex was supershifted using a Mef2c-specific antibody and unlabeled wild-type (WT) oligonucleotide efficiently competed for DNA binding, whereas unlabeled mutant (Mut) oligonucleotide did not. (B) Mutation of the Mef2-binding site in MyE4 (3 kb) enhancer completely abolishes transgenic expression at E12.5 in heart and smooth muscle. (C) Myocardin activates its own enhancer via Mef2. COS cells (24-well plates, 5x104 cells/well) were transfected with 100 ng of the indicated MyE8-luciferase reporters (wild-type and Mef2 mutant), 50 ng expression vectors encoding Mef2c and myocardin proteins and 30 ng of pCMV-lacZ. Activation of the MyE8 reporter by Mef2c was stimulated by the addition of cardiac isoform of myocardin and required the Mef2-binding site. The empty expression vector (pcDNA3.1) had no effect on the wild-type and mutated MyE8-luciferase reporter (not shown); therefore, the results are expressed as fold activation over empty pcDNA.

View larger version (47K): [in a new window]   Fig. 5. Identification of Foxo proteins as mediators of myocardin expression. (A) Results from transfection assays in the 96-well plate that identified Foxo4 as an activator of the distal myocardin enhancer MyE8. In each well, we transfected the MyE8-luciferase reporter into COS cells along with pools of 100 cDNA clones from a mouse E10.5 expression library. The MyE8-luciferase plasmid was specifically activated in well A10. Sibselection from this pool identified Foxo4 as the activating cDNA. (B) Transient transfections in COS cells show that Foxo4 activates the MyE8 reporter, which contains five predicted Foxo-binding sites, with the consensus sequence AAAC/TA (see Fig. 3C). Foxo4 was less effective in activating the MyE8 reporter in which all five Foxo-binding sites were mutated. (C) DNA binding of Foxo4 to the five predicted Foxo-binding sites (see Fig. 3C) in minimal enhancer element MyE8 is shown. Five 32P-labeled oligonucleotides containing the predicted Foxo-binding sites and Foxo4 translated in reticulocyte lysate were used in electrophoretic mobility shift assays. The strongest binding was seen for Foxo sites 3 and 4, weaker binding for sites 2 and 5, whereas Foxo4 did not bind to the first site. The 32P-DNA-Foxo4 complexes were supershifted using a Foxo4-specific antibody and unlabeled, wild-type oligonucleotides efficiently competed for DNA binding. (D) Requirement of Foxo sites for activity of the 3 kb myocardin enhancer in vivo. F0 transgenic embryos generated with construct MyE4, (see Fig. 3A) containing mutations of the five Foxo-binding sites abolishes transgenic expression at E10.5 in heart and smooth muscle.

View larger version (58K): [in a new window]   Fig. 6. Identification of Tead proteins as specific regulators of myocardin expression in branchial arch arteries and aorta. (A) A eukaryotic expression screen, in which we co-transfected MyE8 luciferase along with pools of 100 cDNA clones from a mouse E10.5 expression library revealed Tead2 as an activating cDNA of the minimal myocardin enhancer (well C12). (B) Transient transfections in COS cells show that Tead2 activates the MyE8-luciferase reporter but was less effective in activating the MyE8 reporter in which the predicted Tead-binding site (see Fig. 3C) was mutated. (C) Binding of Tead1 to the Tead-binding site in minimal enhancer element MyE8. A 32P-labeled oligonucleotide containing the conserved Tead site and Tead1 translated in reticulocyte lysate was used for electrophoretic mobility shift assays. DNA binding was seen only in reactions containing lysates with Tead1. The DNA-Tead complex was supershifted using a Tead1-specific antibody and unlabeled wild-type (WT) oligonucleotide efficiently competed for DNA binding, whereas unlabeled mutant (Mut) oligonucleotide did not. (D) Requirement of the Tead-binding site for activity of the myocardin enhancer (MyE4) specifically in branchial arch arteries at E10.5. Two independent F0 transgenic embryos generated with construct MyE4, and two independent F0 embryos with MyE4 in which the Tead-binding site was mutated, are shown. Note that lacZ expression is absent in the branchial arch arteries of the MyE4-Tead mutant embryos indicated by arrows. (E) Transverse cross-sections of E10.5 transgenic embryos show that cardiac lacZ expression, which is highest in the ventricles, is very similar in wild-type and Tead mutants. LV, left ventricle; RV, right ventricle. (F) A cross-section through the outflow tract region shows lacZ expression in cells (asterisk) surrounding the aortic sac (as) and third branchial arch artery (baa3) of wild-type but not Tead mutant embryos. (G) lacZ expression can be appreciated in the dorsal aorta of wild-type embryos but not in the Tead mutant embryos.

View larger version (35K): [in a new window]   Fig. 7. Schematic diagram of the regulation of the myocardin enhancer.

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