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First published online 13 February 2008
doi: 10.1242/dev.014563

Development 135, 1157-1167 (2008)
Published by The Company of Biologists 2008
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Atrial myocardium derives from the posterior region of the second heart field, which acquires left-right identity as Pitx2c is expressed

Daniela Galli1,*, Jorge N. Domínguez2, Stephane Zaffran1,{dagger}, Andrew Munk1,{ddagger}, Nigel A. Brown2 and Margaret E. Buckingham1,§

1 Department of Developmental Biology, URA 2578 CNRS, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, France.
2 Division of Basic Medical Sciences, St George's, University of London, London, UK.


Figure 1
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  		Fig. 1. X-Gal staining and Islet1 in situ hybridisation or immunofluorescence in Mlc1v-nlacZ-24 and Mlc3f-nlacZ-2E mouse embryos between the 4- and 7-somite stages. (A-F) X-Gal staining for β-gal (blue) and whole-mount in situ hybridisation for Islet1 (Isl1) transcripts (purple) on Mlc1v-nlacZ-24 (A,C,E) and Mlc3f-nlacZ-2E (B,D,F) embryos. β-gal activity in the Mlc1v-nlacZ-24 line marks the AHF and its myocardial derivatives, whereas in the Mlc3f-nlacZ-2E line it marks differentiated myocardial cells. Islet1 is transcribed in splanchnic mesoderm. Labelling by the Islet1 probe, rostral to the heart-forming region, under the head folds, marks neurectoderm of the future central nervous system. Islet1 is also expressed in endoderm. Embryos at the 4-(A,B), 6-(C,D) and 7-(E,F) somite stages. Lines in A,C,E indicate plane of sections in A' and A'', C' and C'', E'. (A'') Merge of Hoechst and Islet1 immunofluorescence. (C',C'') Merge of Hoechst, β-gal and Islet1 immunofluorescence. The arrow in C' points to a double-positive cell co-expressing Mlc1v-nlacZ-24/Fgf10 and Islet1. PC, pharyngeal cavity. The arrows in C'' indicate the Islet1-positive mesoderm near the intraembryonic coelomic cavity. The arrows in E indicate the sinus venosus (sv) and the underlying endoderm (En). The arrowhead in E' indicates the heart tube that is negative for β-gal, and the arrow points to the endoderm adjacent to the foregut, which is also positive for Islet1 transcripts. CC, cardiac crescent; HT, heart tube; NT, neural tube. Black rectangles in A,B,C,D indicate the regions used for explants. L, left side of embryo; R, right side of embryo. Beneath is shown a schematic summary of expression of Islet1 and the Mlc1v-nlacZ-24 (Fgf10) transgene in the second heart field (SHF) and anterior heart field (AHF), respectively, and of myosin in cardiomyocytes of the cardiac crescent (CC) and heart tube (HT); p, posterior.

 

Figure 2
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  		Fig. 2. Analysis of progenitor cell and myocardial markers in explants after 12 and 72 hours of culture. (A-F) Posterior (p) SHF explants at the 4- to 6-somite stages after 12 (A-C) and 72 (D-F) hours of culture. (A,D) Hoechst staining; (B,E) Islet1 (Isl1) immunofluorescence; (C,F) myosin heavy chain (MHC) immunofluorescence. The analysis has been repeated on five explants for each time point. (G) RT-PCR analysis on right (R) and left (L) explants (five pooled explants for each side) at the 4- to 6-somite stage after 72 hours of culture, with primers for Mlc2a, COUP-TFII (Nr2f2) and Mlc2v mRNAs. cDNA from the common atrium (A) or from the ventricular region (V) of five hearts from E9 embryos. Controls (-) contained RNA without reverse transcription. β-actin transcripts indicate similar quantities of RNA. (H-K) An explant of the pSHF from Mlc1v-nlacZ-24 embryos at the 4- to 6-somite stage after 72 hours of culture. (H) Hoechst staining; (I) MHC immunofluorescence; (J) β-gal immunofluorescence; (K) merge of H,I,J. Scale bars: 10 µm in F for A-F; 30 µm in H-K.

 

Figure 3
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  		Fig. 3. DiI injection in the posterior SHF of embryos at the 4- to 6-somite stage. (A,B) DiI injection (at sites arrowed) in the right (A) and left (B) pSHF of mouse embryos at the 4-somite stage. The squares indicate the pSHF region (see also Fig. 1). (A',B') DiI labelling (red) in the same embryos after 40 hours of culture, focussing on the heart (dorsal view). (C) DiI (red) injection in the left side and DiR (blue) injection in the right side of an embryo at the 7-somite stage. (C') The same embryo after 40 hours of culture (dorsal view). AVC, atrioventricular canal; LCA, left common atrium; OFT, outflow tract; RCA, right common atrium; RV, right ventricle.

 

Figure 4
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  		Fig. 4. Atrial progenitors are present in the posterior SHF on right and left sides of Mlc3f-nlacZ-2E embryos at the 4-somite stage. (A-H) Left (L) (A-D) and right (R) (E-H) explants of the pSHF from Mlc3f-nlacZ-2E mouse embryos at the 4-somite stage, after 72 hours of culture. (A,E) Hoechst staining; (B,F) myosin heavy chain (MHC) immunofluorescence; (C,G) β-gal immunofluorescence. (D) Merge of B and C; (H) merge of F and G. Scale bar: 10 µm in H for A-H. (I) Quantitative analysis of β-gal and MHC double-positive cells, as a percentage of the total number of cells, in 30 explants from each side of the pSHF. White, percentage in left explants; black, percentage in right explants. (J) qRT-PCR analysis on ten pooled explants from left (l) and right (r) sides. lacZ (arrowheads) and Mlc2a transcript abundance is expressed as a percentage of that of Gapdh transcripts.

 

Figure 5
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  		Fig. 5. Left-right asymmetry of posterior SHF explants from Mlc3f-nlacZ-2E embryos at the 6-somite stage. (A-H) Left (L) (A-D) and right (R) (E-H) explants from the pSHF of Mlc3f-nlacZ-2E mouse embryos at the 6-somite stage: (A,E) Hoechst staining; (B,F) myosin heavy chain (MHC) immunofluorescence; (C,G) β-gal immunofluorescence. (D) Merge of B and C; (H) merge of F and G. (I-P) Left (I-L) and right (M-P) explants from Mlc3f-nlacZ-9 embryos at the 6-somite stage. (I,M) Hoechst staining; (J,N) MHC immunofluorescence; (K,O) β-gal immunofluorescence. (L) Merge of J and K; (P) merge of N and O. Scale bar: 10 µm in P for A-P. (Q) The number of β-gal and MHC double-positive cells as a percentage of the total number of cells. White and black columns show the percentage in left and right pSHF explants, respectively, from Mlc3f-nlacZ-2E (2E) embryos at the 6-somite stage (50 explants from each side) (P≤0.03). Grey and dark-grey columns show the percentage in left and right explants, respectively, from Mlc3f-nlacZ-9 (9) embryos at the same stage (25 explants for each side). (R) qRT-PCR on ten pooled left (l) or right (r) explants of pSHF from Mlc3f-nlacZ-2E embryos at the 6-somite stage after 72 hours of culture. lacZ (arrowheads) and Mlc2a transcript abundance is expressed as a percentage of that of Gapdh transcripts.

 

Figure 6
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  		Fig. 6. Forced Pitx2c expression in posterior SHF explants from Mlc3f-nlacZ-2E embryos at the 5-somite stage. (A-H) Control GFP adenovirus infection on left (L) (A-D) and right (R) (E-H) explants from the pSHF of Mlc3f-nlacZ-2E mouse embryos. (A,E) Merge of phase-contrast and GFP fluorescence on the live explants; (B,F) GFP fluorescence on the live explants; (C,G) Hoechst staining; (D,H) β-gal immunofluorescence on explants now spread under a coverslip. (I-P) Pitx2c/GFP adenovirus infection on left (L) (I-L) and right (R) (M-P) explants of the pSHF from Mlc3f-nlacZ-2E embryos. (I,M) Merge of phase-contrast and GFP fluorescence on live explants; (J,N) GFP fluorescence on the live explants; (K,O) Hoechst staining; (L,P) β-gal immunofluorescence on explants now spread under a coverslip. Scale bar: 10 µm in P for A-P. (Q) Quantitative analysis of the percentage of β-gal-positive cells in the total cell population, after infection with the control GFP adenovirus (white and black; P≤0.025) or with the Pitx2c/GFP-expressing adenovirus (grey and dark grey). The P-value for left-GFP compared with left-Pitx2c is ≤0.01 and for right GFP compared with right Pitx2c is ≤0.005. White and grey, left explants; black and dark grey, right explants. The quantification was performed on ten explants for each side and for each adenovirus. (R) qRT-PCR analysis after GFP and Pitx2c/GFP infection on five pooled left explants (l) and five pooled right explants (r) after 72 hours of culture for each adenovirus. lacZ (arrowheads point to the difference in rlacZ), GFP and Mlc2a transcript abundance is expressed as a percentage of that of Gapdh transcripts.

 

Figure 7
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  		Fig. 7. Analysis of phosphohistone H3 and Islet1 double-positive cells in mouse embryos at the 5- to 7-somite stage, in left and right horns of the sinus venosus of embryos at the 7-somite stage and in 6-somite-stage posterior SHF explants. (A) Quantitative analysis of the percentage of proliferating cells in right (R) and left (L) sides of the SHF with respect to the total number of Islet1 (Isl1)-positive cells in this mesoderm at the 5-, 6- and 7-somite stages (s). (A') Percentage of proliferating cells with respect to the total number of cells in the right and left sides of the sinus venosus (sv) at the 7-somite stage both for wild-type and for Pitx2c-/- embryos. The values are calculated from ten sections, with two embryos analysed for each stage. Red arrowheads, P≤0.03; black arrowheads, P≤0.025; asterisks, P≤0.01. (B-E) Examples of immunofluorescence analysis on a section of a wild-type (B,C) and a Pitx2c-/- (D,E) embryo at the 7-somite stage. (B,D) Merge of phosphohistone H3 (PHH3) and Hoechst staining on a wild-type (B) and on a Pitx2c-/- (D) embryo section (the arrows indicate PHH3-positive cells within the sinus venosus). NT, neural tube. (C,E) Merge of PHH3 and Islet1 immunofluorescence (the same sections as shown in B and D). The arrowheads indicate Islet1 and PHH3 double-positive cells. En, endoderm; SHF, second heart field. The white lines in B and D indicate the separation of the right (R) and left (L) sides of the sinus venosus. (F-I) Explants of the left (F,G) and right (H,I) pSHF from a Pitx2c-/- embryo after 72 hours of culture. (F,H) Hoechst staining; (G,I) PHH3 immunofluorescence. (J) Quantitative analysis of PHH3-positive cells as a percentage of the total number of cells in ten explants from left (L) and right (R) sides of Pitx2c+/- and Pitx2c-/- embryos. White, percentage in left Pitx2c+/- explants; black, percentage in right Pitx2c+/- explants; grey, percentage in left Pitx2c-/- explants; dark grey, percentage in right Pitx2c-/- explants. Arrowheads, P≤0.03; asterisks, P≤0.02.

 

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© The Company of Biologists Ltd 2008