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First published online 7 January 2004
doi: 10.1242/dev.00953

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Transcriptional regulation of the cardiac-specific MLC2 gene during Xenopus embryonic development

Branko V. Latinki^*, Brian Cooper, Stuart Smith, Surendra Kotecha, Norma Towers, Duncan Sparrow and Timothy J. Mohun

Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (53K): [in a new window]   Fig. 6. The overlapping CArG-like and YY1 sites are required for strong and restricted expression of the XMLC2 promoter in the heart. (A) A binding activity present in tadpole extracts binds the combined CArG-like/YY1 site in proximal XMLC2 promoter (–122/–85). This activity (arrow) was identified as YY1 as it is blocked by a specific antiserum to YY1, but not by anti-SRF antibodies. The same complex is specifically competed by an unlabelled YY1 site, but not by SP1 or GATA sites. (Ab, antibody; Fold, fold molar excess of competitor; wt, unlabelled probe). (B,C) Mutation of theYY1 site alone (B) or in combination with the CArG-like/YY1 site (C) results in variable (though generally weaker) and less uniform expression of GFP in the heart (arrowhead), as well as in ectopic expression (arrows). Comparison of the two panels indicates the variability in expression obtained with these mutations. (D) The sequence of CArG-like/YY1 site and mutated versions tested by transgenesis (see Table 1). The YY1 motif in the wild-type sequence is underlined; mutated residues are shown in red.

View larger version (39K): [in a new window]   Fig. 7. SRF and GATA4 can simultaneously bind XMLC2 promoter in vitro, and can synergistically activate an XMLC2-luciferase reporter in animal pole explants. (A) The CArG-like site in –122/–85 promoter fragment binds SRF only weakly (complex labelled S); GATA4 protein binds efficiently to the GATA#2 site present in the same probe (labelled G); simultaneous binding of SRF and GATA4 can be detected (arrow). (B) Overexpression of SRF activates an XMLC2(–123/–41) cyt.actin-luciferase reporter only weakly in animal pole explants, while ectopic GATA4 gives strong activation. Co-expression of both factors leads to synergistic activation. Animal poles were excised at stage 9 and collected after three hours of incubation at 23°C. Firefly luciferase activity was normalised using coinjected tk-Renilla luciferase as a control and was set at 1 for XMLC2(–123/–41) cyt.actin-luciferase in the absence of SRF or GATA4. Values shown represent a single experiment; a second experiment gave similar results.

View larger version (102K): [in a new window]   Fig. 1. XMLC2 is expressed throughout the myocardium of developing Xenopus tadpole. (A) The myosin light chain 2a gene is expressed throughout the heart of the Xenopus tadpole (stage 42). At higher magnification (B), expression is evident in both atrial and ventricular chambers as well as the outflow tract. Ventral views of the heart-forming region reveal XMLC2 expression in bilateral patches of cardiac mesoderm (C) prior to heart tube formation (stage 28). Subsequent formation of a contractile myocardial tube (D; stage 32), looping of the heart tube (E; stage 35) and differentiation of distinct atrial and ventricular chambers (F; stage 42) is clearly visible. Red arrows indicate direction of blood flow (posterior to anterior). (G-J) Transverse sections through the looped heart tube (stage 35) show expression in the myocardial walls of the outflow tract (ot), ventricular (v) and atrial (a) regions and the sinus venosus (sv).

View larger version (23K): [in a new window]   Fig. 2. Sequence of the XMLC2 promoter. (A) The XMLC2 promoter contains GATA-, MEF2- and CArG-binding motifs (shown in red, green and blue, respectively), which are potential binding sites for GATA, MEF2 and SRF transcription factors. An additional proximal motif (purple) combines a YY1 site with a weak SRF-binding site. The proximal 700 nucleotides of promoter sequence are shown, with the TATA box (bold) and 19 nucleotides of exon1 (lower case) indicated. The minimal promoter fragment capable of supporting heart-specific expression in transgenic studies (–123/–41) is highlighted. (B) Promoter regions used in transgenesis experiments. For some constructs, the promoter fragment was fused to a heterologous minimal promoter from the Xenopus laevis cytoskeletal actin or Herpes simplex thymidine kinase genes (orange box). All other constructs included to XMLC2a basal promoter and transcription start site.

View larger version (99K): [in a new window]   Fig. 3. The XMLC2 promoter recapitulates expression of the endogenous gene in transgenic embryos. Expression of XMLC2-GFP transgenes, assayed by fluorescence (A,E-G) or whole-mount in situ hybridisation for GFP RNA (B-D). Arrowheads indicate the tadpole heart. A GFP transgene containing 2990 bp of promoter sequence directs strong, consistent and uniform expression of GFP protein in the tadpole heart (A). Variation in fluorescence intensity reflects differences in myocardial wall thickness between heart chambers and the stage of contraction. (B) Transverse section through the forming heart tube heart (stage 30), showing that staining for GFP RNA is restricted to the myocardium. Heart-specific expression is retained after 5' truncation of the promoter to –675 (C) or –249 (D); truncation to –159 retains heart-specific expression, although the level is much reduced (E). Further truncation to –127 yields occasional, weak expression of GFP to the heart and more frequently in ectopic locations (e.g. branchial arches, pronephros, blood island; small arrows). GFP activity in the eyes and hindbrain (F,G) is from the -crystallin co-transgene.

View larger version (115K): [in a new window]   Fig. 4. –123/–41 is sufficient for strong heart-specific expression. Strong, heart-specific expression is obtained with transgenes comprising the XMLC2 promoter region from –1558 to –48 (A) or –249 to –36 (B) fused to the minimal TK promoter. Two copies of an oligonucleotide comprising the sequence from –123 to –41 direct cardiac-specific expression of GFP from the minimal promoter of the cytoskeletal actin gene (C). By contrast, a similar chimeric promoter containing two copies of the promoter region –121 to –85 is insufficient for cardiac expression, giving only ectopic expression in the branchial arches (D). The tadpole heart region is indicated (white arrowhead). GFP activity in the eyes and hindbrain (C,D; black arrowheads) is from the -crystallin co-transgene. Intense endogenous fluorescence adjacent to the heart in B comes from the gall bladder (arrowed) and gut (g).

View larger version (79K): [in a new window]   Fig. 5. Proximal, high-affinity GATA sites are critical for expression of the XMLC2 promoter. Heart-specific expression is unaffected by targeted mutation of individual GATA sites, such as GATA#2 (A). When both GATA#2 and GATA#3 sites are inactivated, expression in the heart is maintained, but it is weak (B; ventral view). Mutation of both GATA#1 and GATA#2 abolishes activity of the promoter (C). Note that transgenesis in this case is confirmed by expression of the co-transgene gamma-crystallin in the eye and hindbrain. (D) GATA#1 and GATA#2 have greater affinity for GATA4 than the more distal GATA#3. S, specific competitor (unlabelled probe); N, non-specific competitor (both used at 100x molar excess). (E) Competition assay, using 20, 50 and 100x molar excess of competitors confirms this result.

View larger version (71K): [in a new window]   Fig. 8. Pan-myocardial expression of XMLC2-lacZ in transgenic mice. (A,B) E7.5-8.0 (early and late respectively) showing nuclear localised staining in the cardiac crescent; (C,D) E8.0-8.5 (early and late respectively) showing staining throughout the linear heart tube; (E) E9.5; (F) E10.5; (G) isolated heart tube from E9.5. The atrial (A), ventricular (V) and outflow tract (OFT) regions are indicated; (H) oblique frontal view of isolated heart from E10.5. The right atrium (RA) and right ventricle (RV) are marked. Note the abrupt boundary in staining in the outflow tract (arrowheads); (I) neonatal heart with attached thymus. (J) Transverse wax section (6 µm) through the newly formed linear heart tube (E8.0); (K) Cryostat section (10 µm) through the E10.5 heart. In each case, expression is confined to nuclei of the myocardium (mc) and is entirely absent from the endocardium (ec). (L) Cryostat section through the E16.5 heart (8 µm). High levels of expression are detected throughout the myocardium of all four heart chambers but absent from the walls of the great vessels, such as the aorta (arrowheads). (M) Nuclear staining is detected in the myocardium of the right atrial appendage (RAp) and right ventricle (RV). (N) A noticeably higher density of stained nuclei is evident in the myocardium of the left ventricle (LV) but absent from the coronary arteries (inset; ca). (O) Staining is also absent from tissue of the aortic valve (av) and the wall of the aorta (arrowheads).