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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00824

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Cell history determines the maintenance of transcriptional differences between left and right ventricular cardiomyocytes in the developing mouse heart

Robert G. Kelly*,{ddagger}, Marguerite Lemonnier, Stephane Zaffran, Andrew Munk and Margaret E. Buckingham{ddagger}

CNRS URA 2578, Department of Developmental Biology, Pasteur Institute, 25 Rue du Dr Roux, Paris 75015, France



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  		Fig. 1. Mlc3f-nlacZ transgene expression status is maintained ex vivo. (A) Expression pattern of the Mlc3f-nlacZ-2 transgene in a neonatal heart, showing ß-galactosidase activity restricted to the right atrial and left ventricular compartments. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (B) Primary ventricular culture following immunocytochemistry to detect {alpha}-actinin (red) and Hoechst staining to detect nuclei (blue). Most cells in the cultures are {alpha}-actinin positive cardiomyocytes; a minority of nonmyogenic cells are also present (arrowheads). (C) Co-immunohistochemistry of primary left ventricular cardiomyocytes showing that ß-galactosidase activity (green) is specific to cardiomyocyte nuclei (arrowhead), identified by {alpha}-actinin expression (red). (D) Phase contrast image of X-gal-stained primary ventricular cultures showing that few ß-galactosidase-positive cardiomyocytes (arrowhead) are observed in right ventricular free-wall cultures, whereas (E) many ß-galactosidase-positive cardiomyocytes are observed in left ventricular free-wall cultures. (F, G) Low magnification bright-field view of right (F) and left (G) primary ventricular cultures after X-gal staining. Scale bars: 10 µm (B-C), 25 µm (D-G).

 


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  		Fig. 2. Nontransgenic left and right primary ventricular cultures after transient transfection. (A,B) Phase contrast image of X-gal-stained rat primary cultures prepared from right (A) and left (B) free ventricular walls after transfection with the Mlc3f-nlacZ-2 construct. Similar numbers of ß-galactosidase positive nuclei are observed in left and right cultures. (C,D) Low magnification bright-field view of right (C) and left (D) rat primary cultures after transfection with the Mlc3f-nlacZ-2 construct and X-gal staining. Scale bars: 25 µm.

 


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  		Fig. 3. ß-galactosidase activity in primary ventricular myocyte cultures. (A) The ratio of ß-galactosidase activity in left over right cultures is approximately 1 for different Mlc3f constructs and RSV-lacZ reporter genes after transfection into either rat (black circles) or nontransgenic mouse (open circles) primary ventricular free-wall cultures. (B) Decreasing the amount of Mlc3f-nlacZ-2 reporter gene does not change the ratio of ß-galactosidase activity in left or right nontransgenic cultures after transient transfection. (C) ß-galactosidase activity in transgenic cultures is cell autonomous. Co-culture with increasing amounts of rat cardiomyocytes from the opposite ventricle does not change the number of ß-galactosidase-positive cells in either left or right ventricular cultures. The average number of ß-galactosidase-positive nuclei over five fields plus s.e.m. is shown for each co-culture.

 


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  		Fig. 4. Mlc3f-nlacZ transgenes are unmethylated in all compartments of the heart. (A) Ethidium bromide-stained gel showing restriction endonuclease digested Mlc3f-nlacZ-2E DNA isolated from different regions of the heart or fast skeletal muscle with HpaII (H) or MspI (M) endonucleases. (B) Southern blot hybridisation of the gel in A using a 2 kb lacZ probe, showing that the reporter gene is unmethylated to an equivalent degree in all samples. EDL, extensor digitorum longus (fast skeletal muscle); LA, left atrium; LV, left ventricle; OFT, right ventricular outlet; RA, right atrium; RV, right ventricle.

 


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  		Fig. 5. Trichostatin A treatment does not modify Mlc3f-nlacZ-2 transgene expression status. Co-immunohistochemistry of primary ventricular cardiomyocyte cultures following treatment with trichostatin A and sodium butyrate for 3 days, showing Hoechst staining to identify all nuclei per field, anti-ß-galactosidase (red), anti-acetylated histone H4 (green) and the overlay of anti-ß-galactosidase and anti-acetylated histone H4 (merged). Note that although the number of nuclei testing positive for acetylated histone H4 increases after trichostatin A treatment, no change in transgene expression status is observed (see also Table 1). Scale bar: 25 µm.

 


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  		Fig. 6. Overexpression of Hand1 and Hand2 in primary ventricular cultures. (A) Anti-ß-galactosidase immunocytochemistry of right ventricular transgenic cardiomyoytes after transfection with a CMV-Hand1 expression vector showing a ß-galactosidase-positive nucleus (white arrowhead). (B) Co-immunocytochemistry with an anti-myc tag antibody showing three positive nuclei (green arrowheads). (C) Hoechst staining reveals all nuclei in the field. (D) Overexpression of Hand1 and Hand2 under the control of a CMV promoter following adenoviral infection of primary cardiomyocytes prepared from left and right free ventricular walls of Mlc3f-nlacZ-2 hearts. Cells were stained with X-gal (dark nuclei) and photographed under a fluorescent filter. All adenoviral-infected cells express green fluorescent protein. Despite a high degree of infection, Mlc3f-nlacZ-2 transgene expression status is unaffected by overexpression of either Hand gene or of CMV-GFP alone. Control shows mock-infected X-gal-stained cells. White arrowheads indicate right ventricular cells infected with a Hand1 encoding adenovirus that do not express the transgene and left ventricular cells infected with a Hand2 encoding adenovirus that continue to express the transgene. (E) Overexpression of Tbx5 under the control of a CMV promoter following adenoviral infection of right or left ventricular cardiomyocte cultures does not modify Mlc3f-nlacZ-2 expression status in the presence or absence of trichostatin A (TSA), and co-infection with a Hand1 encoding adenovirus. Cells were stained with X-gal (dark nuclei). Scale bars: 25 µm.

 

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© The Company of Biologists Ltd 2003