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First published online January 25, 2006
doi: 10.1242/10.1242/dev.02226

Development 133, 737-749 (2006)
Published by The Company of Biologists 2006
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Smooth muscle of the dorsal aorta shares a common clonal origin with skeletal muscle of the myotome

Milan Esner1, Sigolène M. Meilhac1,*, Frédéric Relaix1, Jean-François Nicolas1, Giulio Cossu2,3,4 and Margaret E. Buckingham1,{dagger}

1 CNRS URA 2578, Department of Developmental Biology, Pasteur Institute, 28 rue du Dr Roux, 75 724 Paris Cedex 15, France.
2 Stem Cell Research Institute, Dibit, H.S. Raffaele, Via Olgettina 58, 20132 Milano, Italy.
3 Department of Biology, University of Milan, Via Celoria 26, 20133 Milano, Italy.
4 Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical Science Park of Rome, Via Castel Romano 100/2, 00128 Rome, Italy.


Figure 1
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  		Fig. 1. Expression of {alpha}-cardiac actin in cells of the dorsal aorta, visualized as ß-gal activity with the T4 transgenic mouse line. (A) Whole-mount X-gal staining of an E10.5 embryo, showing ß-gal labelling (blue) in the heart (H), somites (S) and dorsal aorta (DA). FL, forelimb; HL, hindlimb. (B) A ventral view of A. (C) Immunohistological staining with an {alpha}-smooth muscle actin antibody (red) showing labelled cells in the dorsal aorta (DA) and myotomes (M), on either side of the neural tube (NT). (D) Co-immunohistology of the section shown in C, stained with antibodies to ß-gal (green) and {alpha}-smooth muscle actin (red). (E) An enlargement of the dorsal aorta (DA), showing co-immunohistological staining with antibodies to {alpha}-smooth muscle actin (red) and ß-gal (green). White arrowheads show {alpha}-smooth muscle actin-positive cells that have ß-gal+ nuclei and are in a periendothelial position. The white arrow points to a vascular smooth muscle cell that is also ß-gal+. (F) The same view with DAPI staining (blue) shows the nuclei of ß-gal+ smooth muscle cells (white arrowheads and white arrow) and adjacent endothelial cells (grey arrowheads). (G) Co-immunohistological staining with anti-laminin (red) and anti-ß-gal (green) antibodies shows a cell (white arrowhead) with a ß-gal+ nucleus in the pericyte position within the basal lamina which underlies the endothelial cell layer of the dorsal aorta (DA) (H) The same section with DAPI staining shows the position of this cell (white arrowhead) immediately below an endothelial cell (grey arrowhead) which faces into the lumen of the dorsal aorta (DA). (I) Co-immunohistological staining with antibodies to the endothelial specific marker CD31/PECAM (red) and ß-gal (green). CD31-positive endothelial cells that also express ß-gal are marked by white arrowheads. Other cells with ß-gal+ nuclei (green) in the dorsal aorta (DA) are not endothelial. NT, neural tube. (J) The same view with DAPI staining showing the endothelial position of the nucleus adjacent to the lumen (white arrowheads) of the ß-gal/CD31-positive cells. (K) Whole-mount X-gal staining of an E9.5 embryo, showing ß-gal labelling in the heart (H) and somites (S). FL, forelimb bud. (L) A ventral view of K shows labelled cells in the dorsal aorta (DA). Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Examples of {alpha}-cardiac actinnlaacZ1.1/+ embryos with ß-gal+ cells in the dorsal aorta. (A) The different categories of clones at E10.5 are shown, with right and left lateral views to reveal cells in the myotomes (*) of the somites on either side of the neural tube and a ventral view (centre) to reveal cells (delimited by arrowheads) in the dorsal aorta. An enlargement of this region is shown as an inset (indicated by arrow). The position (not precise number) of labelled cells (black dots) in each clone is shown schematically on the right of the figure. Right (R) and left (L) somites (1-34) are presented on either side of the dorsal aorta. The hypaxial (H) and epaxial (E) extremities of the somites are indicated. (B) The different categories of clones at E9.5 are shown, with ß-gal+ cells in the dorsal aorta. Right and left lateral views reveal cells in the myotome (*) of somites on either side of the neural tube. A ventral view (centre) shows the dorsal aorta where ß-gal+ cells are delimited by black arrowheads. An enlargement of this region is shown as an inset (indicated by arrow). The position (not precise number) of labelled cells is shown schematically on the right of the figure. Right (R) and left (L) somites (1-25) are presented on either side of the dorsal aorta, which has not yet fused anteriorly.

 

Figure 3
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  		Fig. 2B. See previous page for legend.

 

Figure 4
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  		Fig. 3. Distribution of ß-gal+ cells in the dorsal aorta and myotomes, classified according to their myotomal labelling. (A) The position of labelled cells in the dorsal aorta for clones within each class is represented by grey boxes, `segments', at the corresponding somite (S) level, for somites 1-34 at E10.5. Asterisks (*) indicate the position of somites with ß-gal+ cells in the myotome. The figure below each column indicates the total number of ß-gal+ cells in the dorsal aorta (DA) for each clone (Total DA). The average number of ß-gal+ cells in the dorsal aorta per `segment' (DA/segment) is indicated at the bottom. s.d., standard deviation, is shown in parentheses. The number above each column identifies individual clones. The category of clone is indicated above each group. (B) Comparison between ß-gal+ cells per `segment' of the dorsal aorta in different categories of clone with labelled cells in the myotome. Student's t-test compares average values and the Snedecor F-test compares distributions. Numbers in brackets indicate the threshold figure for a significant statistical difference. In all cases, with a probability of P=0.05, there is no significant difference. (C) Representation of clones at E9.5, with a similar presentation to A.

 

Figure 5
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  		Fig. 4. Localization and tissue identity of ß-gal+ cells in the dorsal aorta in {alpha}-cardiac actinnlaacZ/+ embryos. (A) A section from the long clone, E10.5-788, stained with X-gal shows a ß-gal+ cell (arrowhead) in the dorsal wall of the dorsal aorta in a periendothelial position. The dorsal/ventral orientation of the dorsal aorta (DA) is indicated. (B) Immunohistochemistry on the same section using an {alpha}-smooth muscle antibody (red) shows that the ß-gal+ nucleus is in a cell expressing {alpha}-smooth muscle actin (white arrowhead). DAPI staining shows cell nuclei. (C) A section from the short clone E10.5-5, stained with X-gal, shows labelled cells in the ventral wall of the dorsal aorta in a periendothelial position (black arrowheads). (D) The same section treated with an {alpha}-smooth muscle actin antibody (red) shows that these cells are positive (white arrowheads). DAPI staining shows the nuclei. When nuclei are strongly ß-gal+ they appear dark blue after DAPI staining. (E) Another section of the same clone as in C shows that ß-gal+ cells (black arrowheads) are also present immediately adjacent to the lumen of the dorsal aorta in an endothelial cell position. (F) Immunohistochemistry on the same section as in E with an {alpha}-smooth muscle actin antibody shows that these cells (white arrowheads) are not smooth muscle positive, consistent with an endothelial identity. (G) A section from the single somite labelled clone E10.5-168, where a periendothelial cell (brown arrowhead), and vascular smooth muscle cells (white arrowheads) are stained by X-gal. (H) The same section as in G shows that all X-gal stained cells (brown and white arrowheads) are co-stained with an {alpha}-smooth muscle actin antibody (red). Scale bars: 20 µm.

 

Figure 6
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  		Fig. 5. Distribution of different cell types in the dorsal aorta in different categories of clone. (A) The numbers of labelled endothelial (E), periendothelial (P) and vascular smooth muscle (vSM) cells in different categories of clones, at E10.5. (B) Schematic diagram showing progenitors for the myotome (My) and dorsal aorta (summarizing all cell types observed) for each category of clone. The progenitors of long clones and short clones give rise to endothelial cells (E), as well as mural smooth muscle cells. The predominant cell type - periendothelial (P) or vascular smooth muscle (vSM) - in the dorsal aorta is indicated in heavy type. (C) The same as A, at E9.5. DA, dorsal aorta only. (D) At E9.5, a similar lineage tree to those in B, is shown for the category of single somite clones.

 

Figure 7
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  		Fig. 6. The hypaxial location of clones in the myotome and the distribution of (Pax3)GFP-labelled cells in the somite, dorsal aorta and intervening tissues. (A-C) Examples of clones, at E10.5, with ß-gal+ cells in the dorsal aorta and hypaxial myotome. (A) Lateral view of an X-gal-stained embryo showing labelled cells in the hypaxial myotome (*). The full extension of an adjacent myotome is indicated by a double-headed arrow. (B) Ventral view of the same embryo showing ß-gal+ cells in the dorsal aorta. The extent of labelled cells is indicated by the black arrowheads. On the right side (*) ß-gal+ cells are visible in the hypaxial myotome. (C) A section of a single somite clone, E10.5-804, showing cells stained with X-gal in the hypaxial myotome (HM) lying under the epithelium of the dermomyotome (marked with a dotted line) and cells in the adjacent lateral wall of the dorsal aorta (DA). The section was stained with an {alpha}-smooth muscle actin antibody (red). Labelling is also seen in the mesonephric duct (MD). (D-F) Immunohistochemistry, using an anti-GFP antibody (green) on sections from Pax3GFP/+ (D,E) and Pax3GFP/GFP (F) embryos at E10.5. Labelled cells in the dermomyotome (DM) and between this part of the somite and the dorsal aorta (DA), as well as in the latter, are seen in E, and in the enlargement of the more hypaxial region shown in D, where myogenic progenitor cells migrating to the hindlimb (white arrowheads) are indicated. The dorsal root ganglia (DRG) are also labelled in D and E, but not in F, where neural crest cells are compromised. (G) Immunohistochemistry with a Pax3 antibody (red) of a section from a Pax3GFP/+ embryo at E10.5, showing staining in the dermomyotome and in the myogenic progenitor cells migrating to the hindlimb (black arrowheads). No staining is observed in the region between the somite and the dorsal aorta. (H) Immunohistochemistry with an anti-GFP antibody (green) of a section from a Pax3GFP/+ embryo carrying the T4 transgene, at E10.5, shows staining of cells in the wall of the dorsal aorta, some of which (black arrowheads) are also stained with X-gal. (I) Immunohistochemistry using an anti-GFP antibody (green) of a section from a Pax3GFP/GFP embryo, carrying the T4 transgene, which has also been treated with X-gal. An X-gal positive cell (black arrowhead) is labelled. (J) The same section as is shown in G, with DAPI staining, together with immunohistochemistry with the Pax3 antibody (red). (K) Co-immunohistochemistry of the section shown in H, using an {alpha}-smooth muscle actin (red) antibody, as well as the antibody against GFP (green). White arrowheads indicate X-gal-stained nuclei in mural smooth muscle cells of the dorsal aorta. The highly fluorescent cells of an adjacent sympathetic ganglion (SG) are also observed. (L) Co-immunohistochemistry of the section shown in I, using an {alpha}-smooth muscle actin antibody (red), as well as the antibody to GFP (green). A white arrowhead indicates an X-gal-stained nucleus of an {alpha}-smooth muscle actin-positive periendothelial cell in the dorsal aorta. Note that in the absence of Pax3, the sympathetic ganglion is absent, although GFP-positive cells are still present outside the wall of the aorta. (M-O) Co-immunohistochemistry with an anti-GFP antibody (green) and a CD31/PECAM antibody (red) on sections from a Pax3GFP/+ embryo at E8.5. (M) The dorsal extremity of the non-fused neural tube (NT) is already GFP positive, whereas presomitic mesoderm (PSM) at the posterior part of the embryo is not. The endothelial cells of the dorsal aorta, stained with the CD31/PECAM antibody (red), are GFP negative. (N) GFP-positive cells are detected within the first epithelial somite, but not all somitic cells are positive. The dorsal part of the fused neural tube (NT) is GFP positive. Endothelial cells of the dorsal aorta (DA), stained with a CD31/PECAM antibody (red), are GFP negative. (O) In this more mature epithelial somite, most cells are GFP positive. The endothelial cells of the dorsal aorta (DA), stained with a CD31/PECAM antibody (red) do not co-localize with GFP. Scale bars: in C, 100 µm; in E,F, 50 µm; in D,G-O, 20 µm.

 

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© The Company of Biologists Ltd 2006