Fig. 2

The origins of trunk vSMCs. (A,B) Schematic diagrams illustrating two alternative models for the origin and migration of vSMCs recruited to the dorsal aorta, either from the sclerotome (A) or from the trunk neural crest (B). (C-H) Transverse sections of the trunk from 1 dpf (C-E) or 3 dpf (F-H) animals subjected to whole-mount in situ hybridization expression analysis, probed with tagln2 (C,F), pdgfrb (D,G) or twist1a (E,H). All three markers are expressed more broadly in the sclerotome at 1 dpf (C-E), but by 3 dpf their expression becomes restricted to SMCs of the DA (red arrows) and gut (F,G) or DA alone (H). (I) Confocal lateral view image of the mid-trunk of a 3.5 dpf Tg(tagln:gfp), Tg(sox10:rfp) double transgenic animal. The lack of RFP expression along the dorsal aorta suggests tagln⁺ vSMCs are not of neural crest origin. DA, dorsal aorta. (J) Schematic diagram of the Tg(Ola-twist:gal4) and Tg(uas:kaede) transgenes. In Tg(Ola-twist:gal4), Tg(uas:kaede) double transgenic fish, the medaka (Oryzias latipes) twist promoter (Ola-twist) is used to drive expression of Gal4 from the Ola-twist:gal4 transgene, which then activates expression of green to red photoconvertible kaede from the uas:kaede transgene. (K) Confocal lateral view image of the dorsal aorta in a 4 dpf Tg(Ola-twist:gal4), Tg(uas:kaede), Tg(tagln:nlsGFP-2a-CFP-F) triple-transgenic animal in which sclerotome-expressed kaede was photoconverted to red fluorescence at 4 dpf to mark twist1-positive cells. Numerous double-positive tagln⁺ (green nucleus), twist⁺ (red cytoplasm) cells are clearly evident on the dorsal aorta (arrowheads). A total of five fish were imaged in two rounds of photoconversion experiments with equivalent results. (L) Schematic diagram of the Tg(tagln:nlsgfp-2a-cfp-f) transgene, with the tagln promoter driving expression of nuclear-localized GFP and membrane-localized farnesyl CFP, joined by a 2A peptide linker. (M) Selected frames from a confocal time-lapse image series collected from a Tg(tagln:nlsgfp-2a-cfp-f), Tg(kdrl:mCherryCAAX) double-transgenic fish shows the movement of a tagln⁺ nucleus (yellow) from the adjacent sclerotome to the dorsal aorta (demarcated by dashed lines). The image reconstructions shown are ventral views, with rostral to the top. Confocal images were acquired every 10 min and the displayed stills are shown at approximately 50 min intervals. In total, seven tagln⁺ nuclei investing the DA were traced to the sclerotome in five separate time-lapse experiments. Scale bars: 50 µm.