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First published online 28 May 2008
doi: 10.1242/dev.022020

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Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity

Michael J. McGrew¹, Adrian Sherman¹, Simon G. Lillico¹, Fiona M. Ellard², Pippa A. Radcliffe², Hazel J. Gilhooley¹, Kyriacos A. Mitrophanous², Noemí Cambray³, Valerie Wilson^3,* and Helen Sang^1,*,

¹ The Roslin Institute and Royal Dick School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, UK.
² Oxford Biomedica (UK) Ltd, Medawar Centre, Oxford Science Park, Oxford, UK.
³ Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.

View larger version (79K): [in this window] [in a new window]   Fig. 1. GFP fluorescence in CAG-GFP transgenic chickens. (A) The provirus is flanked by self-inactivating LTRs (LTR), and contains the virus packaging site () and a neomycin resistance open reading frame 5' to the CAG-eGFP transgene. (B-E) GFP fluorescence in transgenic embryos at (B) new laid egg stage, (C) stage 4 HH, (D) stage 15 HH and (E) 5 days (transgenic on left). (F,G) Transverse sections of (F) stage 15 HH GFP+ and control embryos, and (G) day 5 GFP+ and control embryos. (H) Stage 13 HH embryos hybridised for GFP (transgenic on left). (I) Flow cytometric analysis of GFP fluorescence (stage 11 HH embryos). Black line, non-transgenic embryo; blue and green lines, transgenic lines 158 and 205, respectively. Scale bars: 0.5 mm.

View larger version (130K): [in this window] [in a new window]   Fig. 2. The CNH region of the tail bud. (A) Caudal region of a stage 15 HH (26 somite) chicken embryo with the paraxial mesoderm removed and grafted regions indicated: Nc, notochord; CNH, chordoneural hinge; TBM, tail bud mesoderm. (B,B') Mid-sagittal section of a same stage chicken embryo (B) immunostained for laminin (red in B'). Arrow, cloacal membrane; arrowhead, posterior surface ectoderm. The three grafted regions are indicated: 1, CNH; 2, dpTB; 3, ventral TB. (C,C') Mid-sagittal section of a corresponding stage mouse embryo (10.5 dpc, C) immunostained for laminin (red in C'). Arrowhead, posterior surface ectoderm. Regions equivalent to those in B' are indicated. Blue, nuclear stain. Scale bars: 0.1 mm.

View larger version (118K): [in this window] [in a new window]   Fig. 3. Homotopic grafts in the chicken tail bud. (A) CNH, dpTB or vTB regions of stage 15 HH GFP+ embryos were grafted homotopically to host embryos and incubated for 48 hours. (A') Embryo immediately after a CNH graft. (A'') Mid-sagittal section of a similarly grafted embryo after 48 hours (stage 24 HH). Arrow, CNH. (B) Mid-sagittal section of the embryo in A' two hours after grafting. (B',B'') Embryo (B') and transverse section (B'') with a CNH graft at 48 hours. (C) Mid-sagittal section of an embryo with a dpTB graft two hours after grafting. (C',C'') Embryo (C') and transverse section (C'') with a dpTB graft after 48 hours. (D) Mid-sagittal section of an embryo with a vTB graft two hours after grafting. (D',D'') Embryo (D') and transverse section (D'') with a vTB graft after 48 hours. Blue, nuclear stain. Scale bars: A',B,B',C,C',D,D', 0.5 mm; A'',B'',C'',D'', 0.1 mm.

View larger version (148K): [in this window] [in a new window]   Fig. 4. Heterotopic grafts in the chicken tail bud. (A-F) Transverse sections of host embryos 48 hours after grafting of the CNH to (A) the dpTB or (B) the vTB; the dpTB to (C) the CNH or (D) the vTB; and the vTB to (E) the CNH or (F) the dpTB. Blue, nuclear stain. Scale bars: 0.1 mm.

View larger version (105K): [in this window] [in a new window]   Fig. 5. Heterochronic transplantation of tail bud regions to early embryos. (A) Tissue caudal to the node or tail bud regions from stage 15 HH embryos were grafted into 6-somite (stage 8 HH) hosts and incubated for 48 hours. (B) An embryo immediately after grafting. Line indicates level of the section. (B') Mid-sagittal section of host embryo two hours after grafting. Arrow, Hensen's node. (C-F') Embryos and transverse sections 48 hours after (C,C') homotopic graft of caudal node tissue; (D,D') heterochronic graft of CNH; (E,E') heterochronic graft of the dpTB; (F,F') heterochronic graft of the vTB. Blue, nuclear stain. Scale bars: B,C,D,E, 0.5 mm; C',D',E', 0.1 mm.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Serial transplantation of the CNH. (A) Serial grafting experiments between stage 15 HH and stage 20 HH embryos. (B-D) Embryos immediately after grafting (left) and after incubation (right). (B) CNH from a stage 15 HH GFP+ embryo grafted to a host embryo and incubated for 20 hours. (C) CNH region from the first host was grafted to a stage 15 HH host and incubated for 20 hours. (D) CNH region from the second host was grafted to a stage 15 HH host and incubated for 48 hours. (E) Transverse section of the third host embryo immunostained for the neuronal marker TUJ1 (red), which labels some GFP+ cells (arrow). Blue, nuclear stain. Scale bars: B-D, 0.5 mm; E, 0.1 mm.

View larger version (128K): [in this window] [in a new window]   Fig. 7. Hox identity is not determined in tail bud progenitor cells. (A) Tail bud (stage 15 HH) hybridised for Hoxa10. (A') Mid-sagittal section (stage 16 HH) immunostained for Hoxc10. Arrow indicates the CNH. (B-D') CNH or dpTB regions (stage 15 HH) grafted into stage 8 HH hosts. In situ hybridisations were carried out for Hoxa10. (B,B') One hour incubation. GFP+ tissue expresses Hoxa10. Eight-(C,C') or 48 (D,D')-hour incubations. Anterior GFP+ tissue is negative for Hoxa10. (E,E') Anterior transverse section from a similarly grafted embryo after 48 hours, immunostained for Hoxc10. Arrows indicate Hoxc10 positive GFP+ cells. (F-J') Whole-mount (F) and transverse sections (F'-J') of an embryo grafted as in B-D and incubated for four days (stage 25 HH). Transverse sections at the level of the anterior arrow were immunostained for Hoxc8 (F'-H'), and those at the level of the posterior arrow with a Hoxc10 antibody (I-J'). Anterior sections from embryos after (G,G') a homotopic caudal node graft or (H,H') a heterochronic CNH graft, immunostained for Hoxc8. Arrows indicate Hoxc8-positive GFP+ cells; asterisks indicate Hoxc8-positive host cells. Posterior sections after (I,I') a homotopic caudal node graft or (J,J') a heterochronic CNH graft, immunostained for Hoxc10. Arrows indicate Hoxc10-positive GFP+ cells. Blue, nuclear stain. Scale bars: A,B,B'-D', 0.5 mm; A', 0.1 mm; F', 250 µm; E,E',G-I', 30 µm.

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