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First published online May 17, 2004
doi: 10.1242/10.1242/dev.01155

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Cell lineage tracing during Xenopus tail regeneration

Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK

View larger version (116K): [in a new window]   Fig. 1. Three-day regenerating tails in longitudinal (A-C,H) and transverse (D-G) sections immunostained with 12/101 mAb and counterstained with Haematoxylin. (A) Low-power magnification showing neural ampulla (NAmp), regenerating notochord (R.Not) and blastema (i.e. visibly undifferentiated cells) formation close to the cut surface. (B) Enlarged view of boxed area in A, showing in detail the NAmp. (C) Enlarged view of boxed area in A showing R.Not. (D) Stump transversal section, proximal to amputation surface, showing notochord (Not), spinal cord (SC) and muscle (Mus). (E) Detail of the enlargement of the ependymal layer in the NAmp. (F) Transverse section distal to amputation level showing R.Not and the surrounding undifferentiated blastema cells. (G,H) Degenerating muscle (D.Mus) (black arrows) near the amputation level. Scale bars: 100 µm.

View larger version (52K): [in a new window]   Fig. 2. Transgenic tadpoles expressing GFP under control of the CMV promoter. (A) Five days after tail amputation, the regenerate keeps expressing GFP. (B) Ten-day regenerating tissue. (C) Complete tail regeneration, all the re-formed tissues still express GFP. White line indicates amputation level. Scale bars: 500 µm.

View larger version (46K): [in a new window]   Fig. 3. The grafting technique for the three tail main tissues performed at neurula stage (stage 14/15 NF). (A) CMV-GFP donor embryo. (B) Normal host showing retraction of posterior neural plate section.

View larger version (63K): [in a new window]   Fig. 4. GFP-labelled grafts of the three embryonic tissues to the presumptive tail region. (A-C) Embryo stage NF37. (D-F) Tadpole stage NF 49. (G-I) Transverse sections of caudal region of host at tailbud embryo stage NF 37. (L-N) sections of tail at tadpole stage NF 49. The sections of the three different graft types are immunostained with anti GFP (red/brown) and counterstained with Haematoxylin (blue) showing specific labelling of each of the three tissue types. Scale bars: 100 µm.

View larger version (99K): [in a new window]   Fig. 5. (A-C) Spinal cord-labelled regenerating tadpole tails showing localisation of the GFP exclusively in the spinal cord of the stump and the regenerate. (A) Five days after amputation. (B) Ten days after amputation. (C) Twenty days after amputation. (D-F) Notochord-labelled regenerating tadpole tails showing localization of the GFP exclusively in the notochord of the stump and the regenerate. (D) Five days post-amputation. (E) Ten days post-amputation. (F) Twenty days post-amputation. White bar indicates the amputation level. Scale bars: 500 µm.

View larger version (73K): [in a new window]   Fig. 6. (A-D) Car-Cre/Lox-GFP transgenics expressing GFP in the myofibres after intrachromosomal recombination. (E-H) Car-GFP transgenics expressing GFP in all myofibres. (A) Stage 28 NF embryo showing a few somitic cells starting to express GFP. (B) Stage 49 tadpole, before tail amputation, expressing GFP in numerous myofibres. (C) Regenerating tail 3 days after amputation showing no GFP-labelled cells in the blastema. (D) Five-day regenerating tail starting to express GFP in a few new myofibres. (E,F) Stage 28 embryo and stage 49 tadpole expressing GFP in the muscle under control of the cardiac actin promoter (Car). (C) Three-day regenerating tail showing no GFP-labelled cells in the blastema. (D) Five-day regenerating tail showing substantial new formation of labelled myofibres. White bar indicates amputation level. Scale bars: 200 µm.

View larger version (60K): [in a new window]   Fig. 7. Presomite mesoderm (PSM) grafts from different stages and positions. In all cases, GFP expression is visible in the somites then in the myotomes. (A-C) Transplanted embryos at stage 28 NF. (D-F) Transplanted larvae stage 49 NF.

View larger version (64K): [in a new window]   Fig. 8. Regenerating tadpoles labelled in the myotomes by early medial PSM graft. (A) Three days post-amputation. (B) Five days post-amputation. (C) Ten days post-amputation. (D) Twenty days post-amputation. White line indicates amputation level. There is no labelling of myofibres, or any other cells, in the regenerate. Scale bars: 200 µm.

View larger version (84K): [in a new window]   Fig. 9. Regenerating tadpoles labelled in the muscles by late medial PSM graft. (A) Three days post-amputation. (B) Five days post-amputation. (C) Ten days post-amputation (D) Twenty days post-amputation. White line indicates amputation level. There is substantial labelling of myofibres in the regenerate. Scale bars: 200 µm.

View larger version (66K): [in a new window]   Fig. 10. Regenerating tadpoles labelled in the muscles by early lateral PSM graft. (A) Three days post-amputation. (B) Five days post-amputation. (C) Ten days post-amputation (D) Twenty days post-amputation. White line indicates amputation level. There is slight labelling of myofibres in the regenerate. Scale bars: 200 µm.

View larger version (126K): [in a new window]   Fig. 11. (A-C) GFP expression in myotomes after PSM grafts performed at different stages and level. Transverse sections were immunostained with antibody against GFP (brown) and counterstained with Haematoxylin (blue). (A) Early medial PSM grafted muscles expressing GFP in the myofibre nuclei (black arrows), but not in the satellite cell nuclei (red arrows). (B) Late medial PSM grafted muscles showing GFP expression in both myofibre nuclei and in satellite cells. (C) Early lateral PSM grafted muscle showing GFP expressed in myofibre nuclei and in a few satellite cells. (D) Muscle transverse section immunostained with Pax7 antibody (brown) (not counterstained). Pax7 expression is detected in the flat nuclei (red arrows) and not in the large round myofibre nuclei (black arrows), suggesting that the flat nuclei are those of satellite cells. Scale bars: 10 µm.

View larger version (86K): [in a new window]   Fig. 12. BrdU incorporation in Xenopus tadpole muscle shown by immunostaining and counterstained with Haematoxylin (BrdU in red). (A) Transverse section. (B) Longitudinal section. Positive cells are indicated by red arrows, and are mostly the flat nuclei at the edges of myofibres, indicative of satellite cells. Scale bars: 5 µm.

View larger version (95K): [in a new window]   Fig. 13. Immunofluorescence with anti-Pax7 (red) and anti-GFP (green) on transverse sections of tadpole myotomes. (A) Pax7 in ungrafted tadpole. (B) GFP immunoreaction on myotome in PSM grafted embryo. (C) Double immunostaining of Pax7 and GFP on muscles from late medial PSM graft. (D) Double immunostaining of Pax7 and GFP on muscles from early medial PSM graft. Co-labelling is seen only in the late grafts. Scale bars: 5 µm.

View larger version (29K): [in a new window]   Fig. 14. The composition of the regeneration bud during Xenopus tadpole tail regeneration.

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