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doi: 10.1242/10.1242/dev.00580

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A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart

Sigolène M. Meilhac, Robert G. Kelly^*, Didier Rocancourt, Sophie Eloy-Trinquet, Jean-François Nicolas and Margaret E. Buckingham

CNRS URA 2578, Département de Biologie du Développement, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France

View larger version (36K): [in a new window]   Fig. 1. Targeted integration of the nlaacZ1.1 reporter gene into the second exon of the mouse -cardiac actin gene. (A) Structure of the targeting vector and the -cardiac actin locus before homologous recombination and after integration and crossing with a PGK-Cre transgenic mouse. Arrows at right angles show the transcriptional start sites of the -cardiac actin promoter and PGK promoters in the selection cassettes. Numbered red boxes represent -cardiac actin exons. Arrows in the nlaacZ1.1 gene (blue box) indicate the position and length of the intragenic duplication (1.1 kb), which introduces a TGA STOP codon at amino acid 665. Black triangles represent loxP target sites for Cre recombinase. The PGK-NeoR (neomycin) cassette for positive selection (speckled box) and the PGK-DTA (diphtheria toxin A-chain) cassette for negative selection (open box) are indicated. The nlaacZ and selection cassettes contain a polyadenylation sequence (pA) at their 3' end. A site for the meganuclease ISceI was introduced immediately after the second loxP site to facilitate future manipulation of the modified locus. Probes derived from the nlaacZ1.1 sequence and 5' or 3' external genomic sequences are shown below as grey boxes. E, EcoRV; H, Hind III; N, NcoI. (B-D) Southern blot analysis of genomic DNA from parental ES cells (wild type) or correctly targeted clones (46, 110). The 5' probe detects 10.7 kb and 7.8 kb bands from the wild-type and targeted alleles, respectively, after digestion with HindIII/EcoRV (B); the probe derived from the laacZ sequence detects 7.8 kb and 1.1 kb bands from the targeted allele and the nlaacZ1.1 intragenic duplication, respectively, after digestion with HindIII/EcoRV (C) and the 3' probe detects 8 kb and 5.5 kb bands from the wild-type and targeted alleles, respectively, after digestion with NcoI (D). (E1) Whole-mount in situ hybridisation with a probe that detects laacZ transcripts on an E10.5 c-actin+/nlaacZ1.1 embryo showing expression of the targeted allele throughout the myocardium (h) and in the skeletal muscle of the myotomes (m) at this stage. (E2) magnification of right lateral view of the heart shown in E1. (F1) Whole-mount in situ hybridisation with an -cardiac actin probe on an E10.5 wild-type embryo showing expression of the endogenous gene in the same domains. (F2) Magnification of right lateral view of the heart shown in F1. Signal intensity is lower in the wild-type compared with the targeted embryo, probably owing to the smaller size of the -cardiac actin specific probe. Signal intensity in the heart appears lower in the outflow tract (O) and atria (A) compared with the ventricles (V) because of differential thickness of the tissue. Scale bars: 500 µm in the detailed heart views; 1 mm in the whole embryo images.

View larger version (35K): [in a new window]   Fig. 2. Examples of hearts with a small number of ß-galactosidase-positive cells. Ventral views of E8.5 (A), E10.5 (B), E14.5 (C) and P7 (D) c-actin+/nlaacZ1.1 hearts coloured by X-gal to reveal the distribution of cells containing a recombined nlacZ allele (first column of panels). Each blue dot represents a single nucleus since the reporter gene contains a nuclear localisation signal. At higher magnification and by changing the focus, it is possible to obtain reliable counts of ß-galactosidase-positive cells at E8.5 and E10.5. Arrowheads show that labelled cells form coherent clusters. Schematic representations of hearts at these stages indicate the cardiac subregions (second column of panels). The numbers in the lower right corner of the panels indicate the stage followed by the identification number of the positive embryos. AP, arterial pole; LA, left atrium; LV, left ventricle; LVP, left venous pole; PV, primitive ventricle; RA, right atrium; RV, right ventricle; RVP, right venous pole; OFT, outflow tract. Scale bars: 500 µm.

View larger version (73K): [in a new window]   Fig. 3. Examples of hearts with multiple clusters. ß-galactosidase-positive hearts at E8.5 with at least three ß-galactosidase-positive clusters (black arrowheads), which are clonally related (see text), are shown in the arterial pole (AP) of the cardiac tube (A), in the primitive ventricle (PV) (B), in the venous pole (VP) (C) or throughout the cardiac tube (D). The white arrowhead in D1 shows two isolated cells that may not be clonally related to the others. D2 is a schematic representation of the clusters (blue open circles show location along the axis of the tube) in the heart at E8.5 with an exceptionally large number of labelled cells (82) presented in D1. Later, large and closely associated clusters, which are clonally related (see text), are shown in the outflow region at E10.5 (E), and E14.5 (F). A heart at E10.5, with an exceptionally large number of labelled cells (420), contains seven clusters of more than 20 cells (G1) and is schematised in G2. Dispersion of the clusters is parallel to the venous-arterial axis of the cardiac tube (broken red lines). Note the increase in the relative sizes of the clusters with age. In all panels, the orientation is cranial (upwards) to caudal (downwards). (A) Right lateral view; (B,D-F) Ventral views; (C) Left lateral view; (G) Dorsal view. AVC, atrioventricular canal; IFT, inflow tract; LA, left atrium; LV, left ventricle; RV, right ventricle. Scale bars: 200 µm.

View larger version (75K): [in a new window]   Fig. 4. Organisation of coherent clusters observed on the surface of the heart. Examples of clusters viewed at the surface of the heart are shown at stages E8.5 (first line of panels), E10.5 (second and third lines of panels), E14.5 (fourth line of panels) and P7 (fifth line of panels). Low level of intermingling between unlabelled (nlaacZ genotype) and labelled (nlacZ genotype) cells is observed. The main axis of the clusters is represented in three examples (I,L,N) by a broken red line. The axes of rows of cells within a cluster are represented in the same examples by unbroken red lines. In M and O, the unbroken red line outlines the orientation of cardiac myofibres at P7, which lie parallel to the rows of labelled cells. At P7, the staining often appears as an elongated unit (M-N), in contrast to the dotted nuclei in other panels, probably reflecting the leakage of ß-galactosidase to the cytoplasm, either during the binucleation of cardiomyocytes or because of a saturation of the staining. (A,C) The primitive ventricle; (B) the venous pole; (D,K,N) the atria; (E) the atrioventricular canal; (F,I,L,M,O) the ventricles; (G,J) the inflow tract; (H) the outflow tract. (A,B) Hearts with a single cluster. Scale bars: 200 µm.

View larger version (53K): [in a new window]   Fig. 5. Organisation of coherent clusters observed through the thickness of the ventricular wall from E10.5. Examples of large clusters viewed at the surface (A1-E1,F,G) and on the transverse plane of bisected hearts (A2-E2,C3-D3) are shown at stages E10.5 (A), E14.5 (B-D) and P7 (E-G). C2-C3 and D2-D3 each show the two sides of a transverse cut. In C1, the projection on the surface of the main transmural axis of the cluster is represented by a broken red line. In F,G, the axes of the rows of cells within a cluster are represented by red continuous lines, numbered from the outside inwards (the inner rows appear increasingly out of focus and lightly stained). (E1) Right lateral view. (C1) Ventral view. (A1,B1,D1) Dorsal views. In all sections, the epicardial (outer) side is at the top of the panel and the endocardial (inner) side is at the bottom. Broken black lines indicate the outline of epicardial and endocardial surfaces, when these were not too folded. At E14.5, these contours correspond also to the separation between compact and trabeculated ventricular myocardium. G is in the right ventricle. IVS, interventricular septum; cLV, compact left ventricle; cRV, compact right ventricle; tLV, trabeculated left ventricle; tRV, trabeculated right ventricle; VC, ventricular cavity. Scale bars: 500 µm in A1-E1; 250 µm in the other panels.

View larger version (20K): [in a new window]   Fig. 6. Mode of growth of myocardial cells. (A) Distribution of the number of ß-galactosidase-positive clusters per heart at E8.5, as an indication of the mode of growth of the dispersive phase, i.e. of the precursors of the clusters. Theoretical models of growth (adapted from Nicolas et al., 1996) are presented in the inset, including lineage relationships between cells and the predicted distribution of clone size. In a stem cell growth mode, the pool of precursor cells (in black in this example), which generate large clones (five to eight cells) is bigger than in a proliferative mode, and therefore larger clones are much more frequent. All hearts from embryos of 9-17 somites, in which clusters were clearly distinguishable, are taken into account (n=84). (B) Distribution of cluster size in hearts with a single cluster at E10.5, as an indication of the mode of growth of the coherent phase (compare with inset in A). All hearts with a single cluster at E10.5 are taken into account (n=215). (C) Distribution of cluster size in hearts with multiple clusters at E8.5, as an indication of the number of cell divisions from the initiation of the coherent growth phase to E8.5. Theoretical models of growth are presented in the inset. All hearts with multiple clusters from embryos of 9-17 somites, in which clusters were clearly distinguishable, are taken into account (69 clusters).

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