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In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation

¹ Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK
² Institut d’Embryologie Cellulaire et Moleculaire, College de France et CNRS, Nogent-sur-Marne, 94736, France

View larger version (64K): [in a new window]   Fig. 1. Microsurgical grafting procedures. The neural tube, including the neural crest, situated between somites 3-6 was excised from chick embryos at 10 ss. Approximately 1 day later, at 25 ss, in the same NC-ablated chick embryos, the sacral region of the neuraxis was excised. The region involved corresponded to the neural tube caudal to the 25th pair of somites (25, arrowhead) and included the chordoneural hinge and rostral region of the tail bud (TB, arrowhead). After excision of the sacral NC, the vagal NC was removed from donor quail embryos at the 10 ss and grafted into the vacant sacral region of the neuraxis, ensuring that the appropriate rostrocaudal/dorsovental orientation was maintained.

View larger version (90K): [in a new window]   Fig. 2. Vagal-to-sacral heterotopic graft, E3. (A) Vagal NCC migrated away from the neural tube (nt), and entered a lateral pathway, underneath and within the ectoderm (arrowheads), and a dorsoventral pathway, lateral to the dorsal aorta (da) leading to the gut (g). (B) Boxed area in A; occasional QCPN+ cells (arrowheads) were evident within the splanchnopleural mesenchyme of the developing gut (g). Scale bars: 100 µm.

View larger version (133K): [in a new window]   Fig. 3. Vagal-to-sacral heterotopic graft, E4. (A) Many QCPN+ cells entered the dorsoventral pathway. This extended from the dorsal neural tube (nt), lateral to the notochord and dorsal aorta (da), and into the dorsal aspect of the gut (g) (arrows, A and B). Occasional immunopositive cells (arrowheads) were present within the mesonephros (m). (B) At the level of the cecal gut, QCPN+ cells encircled the wall of the intestine, and were present within the outer mesenchymal layers of the cecal buds (c). (C) In the hindgut, transplanted vagal NCC accumulated to form the nerve of Remak (R), adjacent to the gut wall, and were present within the undifferentiated layers of the gut mesenchyme (arrowheads). (D) In control embryos, where isotopic and isochronic sacral NC grafts were performed, although occasional cells accumulated to form the rudiment of the nerve of Remak (arrowheads), no QCPN+ cells were observed within the gut wall. Scale bars: 200 µm in A,B,D; 100 µm in C.

View larger version (112K): [in a new window]   Fig. 4. Vagal-to-sacral heterotopic graft, E7. (A) In the colorectum, QCPN+ cells encircled the gut, within the region external to the circular muscle layer, corresponding to the myenteric plexus (mp) being heavily colonised. Internal to the circular muscle layer, in the region corresponding to the submucosal plexus (smp), QCPN+ cells were more sparsely distributed. (B) In the cecal region, the intestine wall and cecal buds were colonised by transplanted vagal NCC. In the intestine, QCPN+ cells were present on either side of the circular muscle wall, while in the cecal buds, immunopositive cells were only present external to the circular muscle layers (arrowheads). (C) In the post-umbilical intestine, QCPN+ cells were present external to the circular muscle layer (arrowheads). Note that in all regions, the nerve of Remak (R) was poorly developed compared with controls. (A',B',C') In control embryos at E7 no sacral NCC were present within any regions of the gut wall. However, the nerve of Remak was well developed and large ganglia (R) were apparent in transverse sections. Scale bars: 100 µm.

View larger version (122K): [in a new window]   Fig. 5. Vagal-to-sacral heterotopic graft, E10. (A) In the colorectum, the myenteric (mp) and submucosal (smp) plexuses contained numerous QCPN+ cells. The nerve of Remak (R) appeared to be poorly developed in transverse sections. (B) Higher magnification revealed that the vast majority of plexus cells were QCPN+, with only a few exceptions in the submucosal plexus (arrowheads).(C) In the post-umbilical intestine, QCPN+ cells were mainly situated external to the circular muscle layers (arrowheads). (A',B',C') In control gut at E10, only very occasional sacral-derived cells were present with the myenteric and submucosal plexuses of the colorectum (B, arrowheads). No immunopositive cells were present in the post-umbilical intestine at this stage of development. Scale bars: 200 µm in A,A'; 50 µm in B,B'; 100 µm in C,C'.

View larger version (100K): [in a new window]   Fig. 6. Vagal-to-sacral heterotopic graft, E16. (A) In the colorectum, transplanted vagal NCC colonised the myenteric plexus (mp), the submucosal plexus (smp) and were also present (arrowheads) within the circular muscle layer (cm). No immunopositive cells were observed within the mucosa. (B) Using the pan-neuronal marker ANNA-1, neurons were labelled within the submucosal plexus (smp) and myenteric plexus (mp). (C,C',C'') Double labelling with QCPN and ANNA-1 revealed that all ANNA-1-positive neurons carried the QCPN+ quail nucleus, indicating that these neurons were derived from transplanted vagal NCC. Scale bars: 100 µm in A,B; 10 µm in C''.

View larger version (98K): [in a new window]   Fig. 7. NADPH-diaphorase staining, myenteric plexus, chick colorectum, E16. (A) The myenteric plexus in control (non-operated) animals consisted of interconnected ganglia containing NADPH+ neurons and nerve fibres. (B) In vagal NC-ablated animals, few ganglia were observed in either the myenteric or submucosal plexus. However, numerous nerve fibres and occasional NADPH+ neurons were evident. (C) Following vagal NC ablation/vagal-to-sacral transplantation, numerous NADPH+ neurons were evident within interconnected ganglia in both the myenteric and submucosal plexuses. Scale bar: 100 µm.

View larger version (121K): [in a new window]   Fig. 8. Ret antibody labelling, chick hindgut, E16. (A) Widespread Ret staining (blue) was present within the myenteric plexus (mp), submucosal plexus (smp) and interconnecting nerve fibres spanning the circular muscular layer (arrowheads). (B,B') Following orthotopic grafts of the sacral NC, double stained Ret (blue) and QCPN (brown) cells were present within the nerve of Remak (B), myenteric ganglia (B') and submucosal ganglia (not shown). Double stained cells contained a brown nucleus and blue cytoplasm (arrowheads). Cells immunopositive for Ret and negative for QCPN were also observed (double arrowhead). (C,C') Vagal-to-sacral heterotopic graft. Ret/QCPN double stained cells were present within the nerve of Remak (C), and within submucosal (arrowheads) and myenteric (not shown) ganglia. Scale bars: 50 µm in A; 25 µm in B,C; 10 µm in B',C'.

View larger version (37K): [in a new window]   Fig. 9. The spatiotemporal progression of vagal NCC migration fronts within the developing chick gut. Normal vagal NCC (red dots) migrated rostrocaudally and transplanted vagal-to-sacral NCC (blue check) migrated caudorostrally. Intermingling of these two NCC populations occurred after E5.5.

View larger version (82K): [in a new window]   Fig. 10. Vagal-to-sacral heterotopic graft, E16 gut wholemount. (A) The nerve of Remak (arrowheads) extended from the cloaca, along the gut wall to the duodeno-jejunal junction, where the bile and pancreatic ducts (d) entered the gut. (B) Adjacent to the colorectum, the nerve was large in diameter and distinct ganglia were evident (B, double arrowheads). (A') After vagal-to-sacral grafting, the nerve of Remak extended from the cloaca to the duodeno-jejunal junction, as in controls. However, the nerve was much finer in diameter. (B') Adjacent to the colorectum, although extremely fine in diameter, the nerve of Remak contained small ganglia (double arrowheads). Scale bars: 10 mm in A; 2 mm in B.

View larger version (95K): [in a new window]   Fig. 11. Sacral-to-vagal heterotopic graft, E8. (A) Numerous transplanted sacral cells (arrowheads) were present external to the circular muscle layers, in the region corresponding to the myenteric plexus. (B) In the intestine, sacral cells were evident within the myenteric plexus (mp) external to the circular muscle. Occasional QCPN+ cells were present in the submucosal plexus region (smp) (arrowheads). (C) No transplanted sacral NC-derived cells were observed in the colorectum at this stage of development. Scale bars: 100 µm.