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First published online May 12, 2005
doi: 10.1242/10.1242/dev.01849

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BOP, a regulator of right ventricular heart development, is a direct transcriptional target of MEF2C in the developing heart

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
² Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
³ Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA

View larger version (41K): [in a new window]   Fig. 1. Downregulation of cardiac Bop expression in Mef2c mutant embryos. (A) Southern blot of PCR-amplified cDNA derived from hearts of wild-type and Mef2c mutant embryos at E9.0 demonstrated a decrease of Bop expression in hearts from Mef2c mutant embryos. The -enolase transcript, which is expressed ubiquitously and is independent of Mef2c expression, was used as an internal control. (B) Differential Bop expression is detected by in situ hybridization in sections of wild-type and Mef2c mutant embryos at E9.0. Bop transcripts are significantly downregulated in the Mef2c mutant. a, atrium; lv, left ventricle; rv, right ventricle; v, ventricle.

View larger version (14K): [in a new window]   Fig. 2. Transgene constructs used to localize cardiac and skeletal muscle regulatory elements associated with the mouse Bop gene. (A) Structure of the 5' region of the mouse Bop gene. The Bop gene contains two first exons that code for the amino termini of Bop gene products expressed in T lymphocytes (tBOP) and striated muscle (mBOP). These exons, which are separated by an intron of 70 kb, are spliced to a common second exon located 22 kb downstream of mBop exon 1. Other more 3' protein coding exons are not shown. White boxes denote 5' untranslated exon sequence and black boxes denote protein-coding exon sequence. The position of the muscle regulatory region upstream of mBop exon 1 is shown in red. (B) Regions of 5' flanking DNA immediately upstream of mBop exon 1 used to create lacZ transgenes are shown. Nucleotides are numbered relative to the transcriptional start site, which is designated `0'. Construct number is indicated on the left, and the corresponding expression pattern is summarized on the right. All constructs, except construct 4, contain the indicated upstream genomic regions fused to the hsp68 basal promoter and the lacZ gene. Construct 4 contains the region from –986 to +75 bp fused directly to promoterless lacZ. The MEF2 site was mutated in constructs 5 and 6, and the three E-boxes were mutated in construct 7. Asterisk denotes constructs that were also used to generate stable transgenic lines. The numbers of F0 transgenic embryos compared with the total number of transgene-positive embryos are shown.

View larger version (53K): [in a new window]   Fig. 3. Expression patterns of transgenes directed by distal (–3303/+196) and proximal (–637/+196) regulatory regions of Bop at E11.5. Whole-mount views of F0 transgenic embryos at E11.5 with construct 1 (–3304/+196; A-D) and construct 2 (–637/+196; E-H) are shown (see constructs in Fig. 2). (A,B,E,F) Left and right sides of embryos are shown. (C,D,G,H) Histological sections. (C,D) Robust expression of construct 1 is seen in the ventricular and atrial chambers of the heart and somite myotomes. Identical expression patterns were seen in 6 out of 7 embryos with this construct (data not shown). (G,H) Construct 2 is expressed specifically in the right ventricular chamber and somite myotomes. Identical expression patterns were seen in 5 out of 5 embryos with this construct (data not shown). m, myotomes; oft, outflow tract; ra, right atrium; rv, right ventricle; lv, left ventricle; la, left atrium.

View larger version (91K): [in a new window]   Fig. 4. Expression patterns of transgenes directed by –637/+196 and –986/+75 regulatory regions of Bop at different developmental stages. (A-H) Transgenic embryos of the indicated ages from a stable transgenic line harboring construct 2 (–637/+196; see Fig. 2). (A-D,G,H) Frontal views; (E) right-side view; (F) transverse section. Two independent stable transgenic lines displayed identical patterns of lacZ expression. (I-P) Transgenic embryos of the indicated ages from a stable transgenic line harboring construct 4 (–986/+75; see Fig. 2). (J,K) Enlarged regions of the embryo shown in I. (M,N) Transverse sections at different levels of the embryo shown in L. (P) Transverse section of the embryo shown in O. rv, right ventricle; lv, left ventricle; oft, outflow tract; vc, common ventricular chamber; m, myotome;

View larger version (45K): [in a new window]   Fig. 5. A MEF2-binding site is required for cardiac expression of Bop (A) The sequence of the mouse Bop gene containing the essential MEF2 site and the E-boxes is shown along with homology to the corresponding regions from other species. (B) 5' flanking sequences of mouse Bop genes showing conserved MEF2 site at –329/-320. (C) A gel mobility shift assay was performed using nuclear extracts from COS-1 cells transfected with a MYC-MEF2C expression plasmid and the radiolabeled MEF2 site shown in panel A as probe. Specific and nonspecific competitors were used at 50-fold molar excess. Antibody supershift used 1 µg of polyclonal anti-MYC antibody. (D,E) MEF2 site is essential for Bop expression in the anterior heart field in vivo. F0 transgenic mouse embryos were generated using constructs 1, 2, 5 and 6 (see Fig. 2B), and stained for expression of ß-galactosidase at E11.5. Mutation of the MEF2 site abolished expression in the anterior heart field without affecting expression in skeletal muscle. Four independent F0 transgenic embryos were analyzed with construct 5 (D) and five independent embryos were analyzed for construct 6 (E). All embryos with each transgene showed comparable expression patterns.

View larger version (60K): [in a new window]   Fig. 6. E-boxes are required for Bop expression in developing skeletal muscle. Mutation of the three E-boxes in the Bop upstream region (construct 7 in Fig. 2B) caused a loss of skeletal muscle expression without affecting expression in the heart, as seen in a whole-mount lateral view (A,B) and transverse sections (C) of five independent F0 transgenic embryos at E11.5. a, atrium; rv, right ventricle; lv, left ventricle; oft, outflow tract.

View larger version (18K): [in a new window]   Fig. 7. Putative network of transcription factors involved in ventricular development. Direct interactions of transcription factors with regulatory sequences associated with downstream target genes are shown by solid lines; steps in which transcription factors have been implicated, but direct target genes have not been identified, are shown by broken lines.

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