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First published online 2 April 2008
doi: 10.1242/dev.017418

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Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation

Amanda J. Barlow^*, Adam S. Wallace, Nikhil Thapar and Alan J. Burns

Neural Development Unit, UCL Institute of Child Health, London, UK.

View larger version (81K): [in this window] [in a new window]   Fig. 1. HNK-1 immunohistochemistry (red) and DAPI staining (blue) of 11-34ss chick embryos. (A,A') NCC located on the dorsal aspect of the embryo, adjacent to somites 1-2 (bracketed). (B,B') NCC grouped at the base of somites 1-3, adjacent to an accumulation of cells arising from the post-otic region (dashed line). (C,C') NCC concentrated at the base of somites 3-5 (bracketed). (D,D') NCC ventral to somites 1-5. Arrow indicates cells advancing towards the foregut from the base of somite 3. (E,E') NCC are absent from somites 3-6, and reduced in numbers in somites 1, 2 and 7. (F,F') A small stream of NCC progressing towards the foregut (arrow). OV, otic vesicle. Scale bars: 100 µm in A, 50 µm in D and F.

View larger version (103K): [in this window] [in a new window]   Fig. 2. TuJ1 immunostaining of E4, E7 and E10 control and 3-6 ablated whole-mount gut preparations. (A-D) E4 control gut showing the ENS network in (A) the stomach, where nerve fibres from the vagus nerve are apparent (arrows), and (B) the duodenum. (C) NC-derived cells are not present at the level of the umbilicus. (D) The migration wave-front contains mostly fasciculated axons and no isolated cells. (E-H) 3-6 ablated gut showing numerous vagus nerve fibre projections (arrows) and a reduced ENS network in (E) the stomach and (F) the duodenum. (G) NC-derived cells are not present at the level of the umbilicus and only the NoR is TuJ1+. (H) The migration wave-front has loosely organised TuJ1+ cells and more isolated cells (arrowheads). (I-L) Control E7 gut with characteristic TuJ1+ ENS network in (I) the stomach and (J) the duodenum. (K) At the level of the umbilicus, the NoR projects fibres into the gut wall. (L) TuJ1+ cells have migrated through the cecal buds and extend into the hindgut. NoR fibres project into the gut co-incident with the most advanced cells at the migration wave-front (dashed line). (M-P) 3-6 ablated gut. The TuJ1+ migration wave-front terminates at the caudal extent of the duodenum (N), which represents the anterior extent of the NoR (arrow). The umbilicus level of the intestine (O), the cecal buds and hindgut (P) do not contain NC-derived cells. (Q-T) E10 control gut showing characteristic ENS networks. (U-X) 3-6 ablated gut. There are fewer perivascular extensions (arrow) within the stomach compared with controls. (V) The caudal extent of the migration wave-front in the duodenum is co-incident with the anterior extent of the NoR. Nerve fibres project from the NoR into the gut (V, arrowheads; W,X). NoR, Nerve of Remak; umb, umbilicus; cb, cecal buds; hg, hindgut. Scale bars: 300 µm in A; 100 µm in B.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Schematic showing extent of gut colonisation by vagal NC-derived cells in control (green shading) and 3-6 ablated (blue shading) E4-E10 chick embryos. Table shows percentage of small intestine (SI) length colonised in control and ablated embryos. n3 at all stages examined.

View larger version (61K): [in this window] [in a new window]   Fig. 4. TuJ1 (green) and QCPN (red) immunostaining of E7 3-6 ablated + Q3 whole-mount gut preparations. (A-D) TuJ1 immunolabelling of (A) stomach, (B) duodenum, (C) umbilicus and (D) the migration wave-front at the end of the cecal buds (arrow). (E-I) Confocal images showing the extensive contribution of quail NCC in the gut regions. The majority of TuJ1+ cells are stained with QCPN in the (E) stomach, (F) duodenum, (G) umbilicus and (H) cecal buds. Arrowheads indicate TuJ1+QCPN-cells. (I) TuJ1 immunostaining in the NoR in the hindgut. NoR, Nerve of Remak; umb, umbilicus; cb, cecal buds; hg, hindgut. Scale bar in E: 50 µm.

View larger version (128K): [in this window] [in a new window]   Fig. 5. TuJ1 (green) and QCPN (red) immunostaining of E10 3-6 ablated + Q3, 3-6 ablated + Q sacral and 3-6 ablated + Q trunk whole-mount gut preparations. (A) Characteristic ENS network and perivascular projections in the stomach. (B,C) The duodenum (B) and the umbilicus level (C) contain extensive interconnected ganglia. (D) The cecal buds and hindgut are completely colonised by NC-derived cells. Similar immunostaining was also observed in 3-6 ablated + Q sacral and 3-6 ablated + Q trunk guts. (E-I) 3-6 ablated + Q3 guts showing extensive colonisation by quail NCC within (E) the duodenum, (F) small intestine, (G) umbilicus, (H) cecal buds and (I) hindgut. (J-N) 3-6 ablated + Q sacral gut. The coincidence of TuJ1 and QCPN within (J) the duodenum and (K) small intestine. Fewer quail NCC were observed within the TuJ1+ ENS network. Only occasional QCPN+ cells were present within the umbilicus region (L). No quail cells were present in the cecal bud region (M) or hindgut (N). (O-S) Immunolabelling of 3-6 ablated + Q trunk guts reveals that there are no quail NCC throughout the entire gut. Scale bar in E: 100 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 6. TuJ1 immunohistochemistry of E10 1-7 ablated, 1-7 ablated + Q1 and 1-7 ablated + Q3 guts. (A-D) 1-7 ablated gut. ENS network in (A) the stomach and (B) the duodenum, which is mainly innervated by NoR fibres. The umbilicus (C) and cecal buds to the end of the terminal hindgut (D) show TuJ1 immunolabelling of the NoR and associated fibres. (E-H) 1-7 ablated + Q1 gut. Vagal NC-derived cells are apparent in the duodenum (F). (G,H) The umbilicus and cecal buds have immunolabelling of the NoR and associated fibres. (I-L) 1-7 ablated + Q3. The gut is completely colonised by vagal NC-derived cells. NoR, Nerve of Remak; umb, umbilicus; cb, cecal buds; hg, hindgut.

View larger version (87K): [in this window] [in a new window]   Fig. 7. Migration and proliferation of different populations of vagal NCC grown in culture for 18 hours. (A-C) Migration of NCC from the neural tube adjacent to (A) somites 1+2, (B) somites 3+4 and (C) somites 5+6. (D-F) BrdU labelling of NCC from (D) somites 1+2, (E) somites 3+4 and (F) somites 5+6. (G,H) Bar charts showing (G) the distance of migration (µm) and (H) the percentage of proliferating NCC from different somite regions. NT, neural tube. Scale bar in A: 100 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 8. QCPN (red) and BrdU (green) immunolabelling with DAPI staining (blue) of E4.5 1-7 ablated + Q3 embryos. (A,D) QCPN staining of NCC that have migrated from the grafted neural tube (not shown) towards the developing foregut. (B,E) BrdU detects proliferating cells within the embryo and within the NCC adjacent to the foregut, respectively. Inset in E shows QCPN and BrdU labelled cells. (C,F) Merged images of (C) A,B and (F) D,E with DAPI staining. Bracketed region in C shows the remnants of the neural tube following ablation. Arrowheads (E,F) indicate proliferating quail NCC. fg, foregut. Scale bars: 200 µm in A; 50 µm in D.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Schematic showing the migration of vagal NC-derived cells (green dots and shading) and quail NCC (red dots and shading) from the neural tube towards and along the gut. (A) In normal embryos NCC migrate from somites 1-7, `fill' the precursor pool, and thus have sufficient population pressure to colonise the entire length of the gut. (B) In 3-6 ablated embryos, the numbers of NCC in the precursor pool are reduced, such that there is insufficient population pressure to advance their migration into and along the gut. (C) Addition of the quail neural tube from somite 3 to 3-6 ablated embryos provides the critical number of cells to the precursor pool to allow complete colonisation of the gut. (D,E) 3-6 ablated + Q sacral (D) and 3-6 ablated + Q trunk (E) show that the sacral and trunk NCC can contribute sufficient cells to the precursor pool to enable full colonisation of the gut. The sacral NCC maintain their intrinsic identity and only contribute to the ENS in the proximal gut. Trunk NCC do not contribute any cells to the ENS, thus their role is restricted to providing critical numbers to the precursor pool to allow the remaining host vagal NCC to colonise the gut. (F) Grafting of somite 3 neural tube into the position of the third somite in 1-7 ablated embryos results in complete colonisation of the gut since these cells, which are more proliferative than their rostral neighbours, are placed directly into the optimal pathway to the gut.

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