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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 -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 Pax3^GFP/+ (D,E) and Pax3^GFP/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 Pax3^GFP/+ 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 Pax3^GFP/+ 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 Pax3^GFP/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 -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 -smooth muscle actin antibody (red), as well as the antibody to GFP (green). A white arrowhead indicates an X-gal-stained nucleus of an -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 Pax3^GFP/+ 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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