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The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development

Da-Zhi Wang^1,*, M. Renee Valdez^1,*, John McAnally¹, James Richardson^1,2 and Eric N. Olson^1,

¹ Department of Molecular Biology,
² Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9148, USA
^* These authors contributed equally to this work

View larger version (25K): [in a new window]   Fig. 1. Schematic diagram of the mouse Mef2c gene. (A) The genomic structure of the mouse Mef2c gene is shown. Open boxes represent UTR. Closed boxes represent translated sequence. Skeletal muscle specific-transcripts contain exon 1 spliced directly to exon 4. Exons 2 and 3 are specific to heart and brain, respectively. The translational start site for Mef2c is located in exon 4 (ATG) and the stop codon (TGA) is located in exon 14. The contributions of exons 4-14 to the translated MEF2C protein are detailed in the black boxes below the genomic structure. Numbers above the boxes indicate the number of amino acids in each exon. The MADS/MEF2 domain is encoded by exons 4 and 5. Exons 6, 7, 9, 11 and 14 are used alternatively (see text). Exons 14a and 14b are generated by alternate splice acceptor sites that maintain the same open reading frame. However exon 14a is longer than exon 14b by 32 amino acids. (B) The skeletal muscle-specific 5'-untranslated sequence of Mef2c from the beginning of exon 1 to the translational start site (boxed) is given. The junction between exons 1 and 4 is located in the region between the arrowheads.

View larger version (22K): [in a new window]   Fig. 2. Transgenes used to identify the Mef2c skeletal muscle control region. Regions of Mef2c 5' flanking DNA used in lacZ transgenes are shown. (Top) The region containing endogenous skeletal-muscle specific promoter activity is indicated in yellow. The position of the Hand2 neural crest enhancer (NCE), which was used as an internal control for lacZ expression with construct 1, is shown. (Bottom) +1 indicates the transcriptional start site for skeletal muscle-specific transcripts. The minimal skeletal muscle regulatory region is indicated in pale blue. The location of the MEF2 binding site at -64 bp and the E-box at -38 bp are indicated in the enlarged construct 7. x, mutation; >, sense orientation; <, antisense orientation; < >, sense or antisense orientation. A minimum of two transgenic F0 embryos were analyzed for each construct. Multiple independent stable transgenic lines were also generated with constructs 7 and 11; they showed the same expression patterns seen in F0 transgenic mice. Broken lines indicate deleted regions.

View larger version (36K): [in a new window]   Fig. 3. ß-galactosidase staining of mouse embryos harboring the Mef2c-lacZ transgene. F0 embryos harboring construct 1, containing the region from -6.6 kb to +77 bp (see Fig. 2), were stained for lacZ expression. A neural crest enhancer (NCE) was also included in this construct as a positive control for lacZ activity. (A) An E9.5 transgenic embryo shows lacZ expression in rostral somites and weaker activity in more caudal somites. (B) Dorsal view of an E10.5 embryo shows expression in rostral and caudal somites. (C) Transverse section of an E10.5 embryo at the level of thoracic somites demonstrates lacZ staining in the myotome. (D) Dorsal view of an E11.5 embryo shows staining in somites and ventral myoblasts (vm). (E) At E14.5, transgene expression is evident throughout much of the embryonic musculature. Also evident is pharyngeal arch expression driven by the NCE (A,D), which is indicated by arrowheads. Arrows, somites; d, deltoid; ld, latissimus dorsi; m, myotome; t, trapezius; vm, ventral myoblasts.

View larger version (31K): [in a new window]   Fig. 4. Expression of construct 7 during embryogenesis. The nucleotide region from -1.1 kb to +77 bp was fused to hsp68-lacZ and used to create transgenic mice (see Fig. 2). (A) At E9.0, lacZ expression is detected in rostral somites. (B) An E9.5 embryo shows lacZ reporter expression from the most rostral somites, to some somites in the region of the hind limb. (C) At E11.5, transgene expression is evident throughout the somites and in ventral myoblasts. (D) Transverse section through somites at the level of the fore limb shows lacZ expression in the myotome. Neural tube staining is not reproducible. (E) At E16.5, expression of the transgene is seen throughout embryonic musculature, including facial, epaxial, hypaxial and limb muscle. Arrows, somites; arrowheads, ventral myoblasts; d, deltoid; m, myotome; q, quadriceps; t, trapezius.

View larger version (89K): [in a new window]   Fig. 5. Expression of construct 7 in postnatal skeletal muscle. Expression of construct 7 was analyzed in F0 neonatal transgenic mice. High expression was observed in muscles of the (A) face, (B) neck, (C) fore limb, (D) rib cage, (E) back and (F) hindlimb. ei, external intercostals; ms, masseter; q, quadriceps; st, spinalis thoracis; t, trapezius; tb, triceps brachii.

View larger version (57K): [in a new window]   Fig. 6. Delineation of the minimal Mef2c skeletal muscle regulatory region. Whole-mount E11.5 embryos expressing constructs 14, 8, 9 and 11 are shown. (A) The nucleotide region from -1058 to +27 (construct 14) was fused directly upstream of the promoterless lacZ cassette and was used to generate F0 transgenic embryos. At E11.5, ß-gal staining is evident from rostral somites through somites at the level of the hind limb. This expression is weaker and less extensive than that of approximately the same nucleotide region fused to hsp68-lacZ (see Fig. 4C). (B) The nucleotide region from -1058 bp to -507 bp (construct 8) was fused to hsp68-lacZ and used to create F0 transgenic mice. Small, discrete regions of ß-galactosidase staining are seen in a metameric pattern throughout rostral and caudal somites. (C,D) Transverse sections at the level of forelimb somites of the embryo in B demonstrate that the ß-galactosidase staining marks the extreme dorsomedial aspect of the myotome and ventrolateral myoblasts in the limb. (E) The nucleotide region from -512 bp to +41 bp (construct 9) was fused to hsp68-lacZ and used to create F0 transgenic mice. Strong lacZ expression is evident throughout the somites and in ventral myoblasts. (F) Transverse section through the somites of an E11.5 embryo expressing construct 9. Expression is evident throughout the myotome. (G) The nucleotide region from -158 bp to +4 bp (construct 11) was fused to hsp68-lacZ and used to create F0 transgenic mice. Somites and ventral myoblasts show strong lacZ expression. (H) Transverse section through the somites of an E11.5 embryo expressing construct 11. Section shows lacZ expression throughout the myotome. Arrows, somites; m, myotome; arrowheads, dorsomedial myotome; *, limb myoblasts.

View larger version (44K): [in a new window]   Fig. 7. Binding of MyoD and MEF2 to the Mef2c skeletal muscle regulatory region. (A) Sequence of the minimal skeletal muscle regulatory region is shown. The MEF2-binding site extends from nucleotides -64 to -56. The E-box includes nucleotides -38 through -33. (B) Schematic representation of the 1.1 kb Mef2c skeletal muscle control region. The region encompassing nucleotides -512 to +41, with respect to the transcriptional start site of Mef2c (see Fig. 1). The specific MEF2 site and E-box nucleotides mutated for the experiments shown in Fig. 8 are indicated beneath the wild-type sequence. -32P-labeled oligonucleotides for the (C) MEF2 site and (D) E-box of the Mef2c skeletal muscle regulatory region were used as probes in gel mobility shift assays with the in vitro translated (C) Myc-tagged MEF2C or (D) MyoD and E12 proteins. Gel shift reactions using unprogrammed reticulocyte lysate are included in parallel lanes. A 100-fold excess of unlabeled (C) MEF2 site or (D) E-box oligonucleotides was used as competitor. (C) Anti-Myc and (D) anti-MyoD antibodies were used for supershifts. Labeled arrows indicate the positions of the various protein-DNA complexes.

View larger version (77K): [in a new window]   Fig. 8. Inactivation of the Mef2c skeletal muscle enhancer by individual mutations in the MEF2 binding site and E-box. Mutations of the (A-C) MEF2 site (MEF2 mut) and (D-F) E-box (E-box mut), shown in Fig. 7B, were introduced individually in the context of the nucleotide region from -512 bp to +41 bp fused to hsp68-lacZ and F0 transgenic embryos were generated. Embryos were analyzed for ß-galactosidase expression at E9.5 (A,B,D,E) and E11.5 (C,F). (B,E) Enlargements of the regions indicated by the boxes in A,D, respectively. (A,B) Only the MEF2 mut-hsp68-lacZ construct was weakly active in somites at E9.5. Constructs were inactive at all other time points. Arrows, somitic expression.

View larger version (15K): [in a new window]   Fig. 9. A model for the regulation of Mef2c transcription during skeletal muscle development. (A) Regulation of Mef2c expression during myogenesis. The skeletal muscle regulatory region of Mef2c contains binding sites for myogenic bHLH and MEF2 proteins. The E-box is required for initiation and maintenance of Mef2c expression. By contrast, the MEF2 site is required for amplification and maintenance of Mef2c expression. (B) A model for the genetic network of myogenic bHLH and MEF2 factors during myogenesis.