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First published online 14 July 2004
doi: 10.1242/dev.01256

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Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development

¹ Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0130, USA
² Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0130, USA

View larger version (40K): [in a new window]   Fig. 3. Deletional analysis of the Mef2c anterior heart field enhancer identifies an endogenous promoter and a minimal evolutionarily conserved enhancer that is necessary and sufficient for expression in vivo. (A) Genomic organization and deletion constructs of the Mef2c anterior heart field enhancer. The organization of the enhancer is denoted at the top. The black box denotes the third 5' untranslated exon; the red boxes indicate regions of mouse to human homology (>75%). Construct fragment number and nucleotides are indicated on the left. Anterior heart field expression of lacZ at 9.5 and 11.5 days post coitum (dpc) is denoted in the column to the right. The column on the far right indicates the number of independent transgenic lines or F0 embryos that expressed lacZ in the anterior heart field as a fraction of the total number of transgene positive F0 embryos or lines examined. The green box denotes the presence of the heterologous HSP68 promoter. (B-G) Representative embryos collected at 9.5 dpc for selected Mef2c anterior heart field enhancer deletion constructs. Fragment 2 (B), Fragment 3 (C), Fragment 6 (D) and Fragment 10 (G) directed strong expression to the pharyngeal mesoderm/anterior heart field (arrowheads), outflow tract and right ventricle (RV). Fragment 10 (G) represents a minimal sufficiency enhancer construct required for lacZ expression in the anterior heart field. Fragment 3 (C) utilizes an endogenous promoter from Mef2c.

View larger version (23K): [in a new window]   Fig. 1. A Mef2c-lacZ bacterial artificial chromosome (BAC) transgene is sufficient to direct the complete pattern of Mef2c expression. (A) Schematic representation of the mouse Mef2c locus contained within BAC clone GS133. The BAC contains the first six known exons from Mef2c (vertical black lines), including the first two coding exons, which encode the MADS and MEF2 domains of Mef2c. The red box represents a region of high evolutionary conservation that functions as an anterior heart field specific transcriptional enhancer. (B) Schematic representation of Mef2c-F6-lacZ, which contains the Mef2c anterior heart field enhancer subcloned into the transgenic reporter plasmid HSP68-lacZ. Red box, Mef2c noncoding sequences representing the anterior heart field enhancer; black box, Mef2c untranslated exon 3; green box, HSP68 promoter (pr); blue box, lacZ gene; yellow box, SV40 splice and polyadenylation (polyA) sequence. (C) Expression of the Mef2c BAC transgene, Mef2c-L8-lacZ, at 7.5 days post coitum (dpc) shown in frontal view. The Mef2c-L8-lacZ BAC transgene directs expression exclusively to the anterior heart field at this stage, recapitulating the earliest expression of endogenous Mef2c. (D) Lateral view of a transgenic embryo collected at 8.0 dpc showing expression of Mef2c-L8-lacZ. Strong expression is evident in the anterior heart field at the arterial pole of the linear heart tube. (E) Ventral view of an 8.5 dpc Mef2c-L8-lacZ transgenic embryo. Strong expression remains evident in the anterior heart field at the arterial pole of the linear heart tube. Transgene expression is also evident in the neural crest (NC) at this stage and expression can begin to be seen in vascular endothelial cells within the dorsal aortae and in the yolk sac (not shown). (F) Lateral view of a 9.5 dpc Mef2c-L8-lacZ transgenic embryo. At this stage the expression directed by the BAC transgene completely recapitulated the pattern of endogenous Mef2c expression during embryonic development at this stage. Expression was present in vascular endothelium (V), neural crest, somites (S), branchial arches (BA), pharyngeal mesoderm, outflow tract and heart. RV, right ventricle. (C-F) Representative X-gal stained transgenic embryos at each stage. Arrowheads point to pharyngeal mesoderm/anterior heart field. Two F0 transgenic embryos and embryos collected from an independent BAC transgenic line displayed identical patterns of expression at all stages.

View larger version (52K): [in a new window]   Fig. 2. A transcriptional enhancer from Mef2c is sufficient to direct expression to the anterior heart field in vivo. A 6142 bp fragment of the mouse Mef2c gene was cloned into the lacZ reporter plasmid HSP68-lacZ and used to generate transgenic mice. Representative X-gal stained, transgenic embryos are shown at 7.5 days post coitum (dpc) (A), 8.0 dpc (B), 9.5 dpc (C,D), and 11.5 dpc (F,G). X-gal stained hearts are shown from a transgenic fetus collected at 16.5 dpc (I) and a transgenic adult dissected at 12 weeks of age (J). (E,H) Transverse sections of transgenic embryos collected and X-gal stained at 9.5 dpc and 11.5 dpc, respectively. No expression was observed outside the anterior heart field at any stage. (A) Parafrontal view; (B,C,F) lateral views from the right; (D,G,I,J) ventral views. BA, branchial arches; LA, left atrium; LV, left ventricle; NT, neural tube; OFT, outflow tract; PA, pulmonary artery; RA, right atrium; RV, right ventricle. Arrowheads denote expression in the pharyngeal mesoderm/anterior heart field. Five independent transgenic lines all displayed nearly identical patterns of expression.

View larger version (23K): [in a new window]   Fig. 4. Alignment of conserved mouse and human sequence in the necessary and sufficient region of the Mef2c anterior heart field enhancer. Three conserved candidate GATA sites (red shading), two conserved ISL sites (green shading), two candidate E boxes (yellow shading) and a conserved NKE (black box) are noted. The three GATA sites are contained within Fragment 9, while the two ISL sites and the GATA sites are included in Fragment 10. Numbers at the right represent the nucleotide positions within the mouse Mef2c-F6/Frag2 sequence depicted in Fig. 3 and deposited in GenBank (accession number AY324098).

View larger version (60K): [in a new window]   Fig. 5. Two evolutionarily conserved GATA sites in the Mef2c anterior heart field enhancer are bound by GATA4. GATA4 was transcribed and translated in vitro and incubated with radiolabeled, double-stranded oligonucleotides spanning the Mef2c GATA-p site (lanes 9-14), the Mef2c GATA-d site (lanes 15-20) or the gs1 GATA site from the Nkx2.5 gene as a control (lanes 1-8). GATA4 efficiently bound to all three GATA sites (lanes 2, 10 and 16). Binding of GATA4 to the GATA-p site was specifically competed by excess unlabeled Mef2c GATA-p (Gp) (lane 13) and by excess unlabeled Nkx2.5 gs1 (lane 11) but not by a 100-fold excess of a mutant GATA-p site (Mp) or by a 100-fold excess of a mutant Nkx2.5 gs1 site (M1) (lanes 12 and 14). Likewise, the binding of GATA4 to the GATA-d site was specifically competed by excess unlabeled GATA-d (Gd) (lane 19) and by excess unlabeled Nkx2.5 gs1 (lane 17) but not by a 100-fold excess of either mutant GATA-d (Md) or mutant gs1 (M1) (lanes 18 and 20). The Mef2c GATA-p (Gp) and GATA-d (Gd) sites also efficiently competed for GATA4 binding to the control gs1 GATA site from the Nkx2.5 enhancer (lanes 5 and 7), but mutant versions of the GATA-p (Mp) and GATA-d (Md) sites were unable to compete for binding to the Nkx2.5 gs1 site (lanes 6 and 8). In samples where in vitro translated GATA4 protein was not included (denoted by a minus sign in lanes 1, 9 and 15), an equal amount of unprogrammed reticulocyte lysate was included (lysate-derived bands are noted).

View larger version (42K): [in a new window]   Fig. 6. The ISL sites in the Mef2c anterior heart field enhancer are specifically bound by ISL1. Isl1 was transcribed and translated in vitro and incubated with radiolabeled, double-stranded oligonucleotides spanning the Mef2c ISL-p site (lanes 9-14), the Mef2c ISL-d site (lanes 15-20) or a consensus ISL1 binding site from the rat Insulin I gene as a control (lanes 1-8). ISL1 efficiently bound to all three ISL sites (lanes 2, 10 and 16). Binding of ISL1 to the ISL-p site was specifically competed by excess unlabeled Mef2c ISL-p (I-p) (lane 11) and by excess unlabeled Insulin I ISL1 site (In) (lane 13) but not by a 100-fold excess of mutant ISL-p site (mI-p) (lane 12) or by a 100-fold excess of a mutant Insulin I site (mIn) (lane 14). Likewise, the binding of ISL1 to the ISL-d site was specifically competed by excess unlabeled ISL-d (I-d) (lane 17) and by excess unlabeled Insulin I site (lane 19) but not by a 100-fold excess of either mutant ISL-d (mI-d) (lane 18) or mIn control (lane 20). The Mef2c ISL-p and ISL-d sites also efficiently competed for ISL1 binding to the control ISL1 site from the Insulin I promoter (lanes 5 and 7), but mutant versions of the ISL-p and ISL-d sites were unable to compete for binding to the Insulin I site (lanes 6 and 8). In samples where in vitro translated ISL1 protein was not included (denoted by a minus sign in lanes 1, 9 and 15), an equal amount of unprogrammed reticulocyte lysate was included.

View larger version (52K): [in a new window]   Fig. 7. The Mef2c anterior heart field enhancer is dependent on GATA and ISL sites for function in vivo. The wild-type Mef2c enhancer transgene construct, Mef2c-F6/Frag2-lacZ, or transgenes carrying mutations in either the two GATA sites or the two ISL sites in that context, were used to generate transgenic mice. Representative transgenic embryos at 9.5 days post coitum (dpc) are shown. The wild-type fragment directed strong expression to the pharyngeal mesoderm, outflow tract and right ventricle (A). Mutation of the GATA-p element nearly completely eliminated transgene expression in vivo, although very weak expression could sometimes be seen in the pharyngeal mesoderm. Mutation of the GATA-d site had a dramatic impact on the level of transgene expression, although the pattern of expression remained unchanged. Mutation of both GATA sites in the enhancer (mGATA) completely eliminated lacZ expression (as shown in B). For the double GATA mutant, eight independent transgenic events were analyzed, and none showed any expression in the heart. Mutation of both ISL sites (mISL) in the enhancer completely eliminated all transgene expression (C) in all six independent transgenic events analyzed. The wild-type 3970 bp enhancer construct directed robust lacZ expression, as shown in (A), in 16 of 17 transgenic lines. Arrowheads denote expression in the pharyngeal mesoderm/anterior heart field. RV, right ventricle.

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