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Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways

¹ Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway Mill Hill, London NW7 1AA, UK
² Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA

View larger version (71K): [in a new window]   Fig. 1. Migration patterns of mouse cranial neural crest cells (ncc). (A,B) The entire proximodistal extent of the first branchial arch (ba1) is extensively populated by laterally migrating DiI-labelled, neural crest cells derived from r1 (A) and r2 (B). (C) r3 produces relatively few ncc, which do not migrate laterally. Rather they move anteriorly (arrowhead) and posteriorly (arrow) to contribute to the most proximal regions only of ba1 and ba2, respectively. (D) The second branchial arch (ba2) is composed primarily of laterally migrating r4-derived ncc. (E) r5 produces more ncc than r3, which contribute to ba2 and also ba3. r5 ncc migrate anteriorly (arrowhead) and posteriorly (arrow) around the developing otic vesicle. (F) The third branchial arch (ba3) is composed primarily of ncc derived from r6. (G) Summary of ncc migration patterns [adapted from fig. 1a in Trainor and Krumlauf (Trainor and Krumlauf, 2000a)]. ht, heart; ov, otic vesicle; V, trigeminal; VII, facial; IX, glossopharyngeal motor nerves.

View larger version (50K): [in a new window]   Fig. 2. The patterns of cell death during ncc migration. (A) Acridine Orange stain of a 6-somite stage embryo showing different patterns of cell death in adjacent neural folds (arrowheads). (B-D) Nile Blue staining of 7s (B), 9s (C) and 9.5 d.p.c. (D) embryos showing temporally and spatially dynamic patterns of cell death and the absence of rhombomere-specific death.

View larger version (106K): [in a new window]   Fig. 3. Bmp4 and Msx2 are not segmentally expressed. (A-C) Bmp4 expression is confined to ventral tissues such as the heart (ht) at 8.5 d.p.c. (A) and to Rathke’s pouch and the anterior ectoderm of ba1 in 9.5 d.p.c. embryos (B, arrow). Bmp4 is not expressed in the dorsal neural tube in 8.5-9.5 d.p.c. embryos (A-C). (D-F) Msx2 is initially expressed uniformly in the dorsal neural plate of 8.5 d.p.c. embryos (D), but by 9.5 d.p.c. becomes restricted to the distal halves of ba1 and ba2 (E) and also to the roof plate (F).

View larger version (68K): [in a new window]   Fig. 4. Odd rhombomeres have the capacity to generate ncc. (A) DiI-labelled r3 cells transplanted back into r3 generate small numbers of ncc, which migrate primarily into the proximal region of ba2 (arrows). (B) r3 cells transplanted into r4 generate elevated numbers of ncc, which colonise the entire length of ba2. (C) r3 cells transplanted into r2 also generate increased amounts of ncc, which colonise the entire length of ba1. (D) r5 cells produce ncc, which extensively populate ba1 when grafted into r2. Ht, heart. (E) Bright-field image of 9.5 d.p.c. embryo post grafting of r3 cells into r4 and 24 hours culture. (F) Confocal image showing that r3 cells, taken from an EphA4-GFP transgenic embryo and transposed into r4, maintain their identity in the hindbrain environment. In contrast, graft-derived neural crest cells have reduced reporter expression (GFP), showing that they do alter their identity. (G) Confocal image of DiI-labelled r3 cells grafted into r4, showing increased levels of ncc generation that populate ba2. (H) Confocal overlay of F and G, highlighting the maintenance of identity of the grafted rhombomeric tissue and plasticity of the graft-derived ncc.

View larger version (110K): [in a new window]   Fig. 5. The capacity of r4 to generate ncc is governed by the environment. (A) Control graft of DiI-labelled r4 cells back into r4, showing that r4 generates ncc, which colonise ba2. (B) Heterotopic graft of r4 cells into r2, showing that r4 can generate ncc in this ectopic environment, and populate ba1. (C) Heterotopic graft of r4 cells into r3, showing that the capacity of r4 to generate ncc is repressed in this ectopic environment. (D,E) Lateral (D) and dorsal (E) views of an embryo in which r4 cells transposed into r3 fail to generate crest. (F) Overlay of TUNEL (green) and DiI (red) staining in the same embryo showing that the grafted rhombomeric cells are not being eliminated by cell death.

View larger version (43K): [in a new window]   Fig. 6. Environmental influence of mesoderm and surface ectoderm on ncc migration. (A) Control of r4 DiI-labelled ncc migrating into ba2. (B-E) DiO-labelled mesoderm (B) and surface ectoderm (D) isolated from adjacent to r3 and transplanted next to r4 were unable to block the migration of r4-derived ncc (labelled with DiI), which populated ba2 (C and E, respectively).

View larger version (66K): [in a new window]   Fig. 7. Conserved repression of crest migration in mouse/chick grafts. (A) DiI-labelled mouse r3 cells transplanted into r4 of the chick generate numerous ncc, which colonise ba2. Ov, otic vesicle. (B) DiI-labelled r4 cells derived from the Hoxb1-lacZ transgenic line and transplanted into r3 of the chick are unable to generate ncc. (C) This is not due to a change in their A-P identity or cell death, as grafted mouse cells continue to strongly express the reporter gene.

View larger version (39K): [in a new window]   Fig. 8. Model of the mechanisms patterning the pathways of mouse ncc migration. Each rhombomere has the capacity to generate ncc. From r2 and r4, ncc migrate laterally into ba1 and ba2, respectively. r3 and r5 both generate neural crest that migrates anteriorly and posteriorly to join the laterally migrating even-rhombomere ncc streams. Note that r3 produces fewer ncc (small green circles) than r5 (yellow curves). This creates crest-free zones adjacent to the odd-numbered rhombomeres. Neural crest generation from r3 is regulated by inhibitory signals (?) in the adjacent environment (green bar). This crest-free zone adjacent to r3 is maintained by multiple mechanisms. ErbB4 signalling (black arrows and bar) from the hindbrain to the adjacent mesenchyme keeps even-rhombomere ncc streams segregated dorsally, and Eph/ephrin signalling (grey bars) maintains the segregation ventrally. This prevents the infilling of neural crest from flanking regions. In the case of the neural crest-free zone adjacent to r5 this pattern is generated by the physical inhibition created by induction and formation of the otic vesicle. Adapted from fig. 6F in Golding et al. (Golding et al., 2000).