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Cues from neuroepithelium and surface ectoderm maintain neural crest-free regions within cranial mesenchyme of the developing chick

Division of Neurobiology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK

View larger version (85K): [in a new window]   Fig. 1. Altered pattern of cranial NCC migration 20 hours after unilateral r3 removal. (A-C) Whole-mount HNK-1 immunostaining of NCCs showing the operated (A) and unoperated (B) sides of the same embryo. (C) Dorsal view. (D-F) Sox10 in situ hybridisation to show migrating NCCs on the operated (D) and unoperated (E) sides of the same embryo. (F) A transverse section through the embryo at the level of r3 that corresponds to the broken line in D,E. On the operated side, in addition to the normal pattern of NCC migration (r2 crest migrates into ba1; r4 crest migrates into ba2), a stream of NCCs (arrow in A,C,D,F) migrates aberrantly through the mesenchyme adjacent to the removed r3 (r3*). This ectopic NCC stream is more clearly defined by Sox10 mRNA expression than by HNK1 immunoreactivity. In addition, HNK1+ cells are detected within the space previously occupied by r3 (arrowhead in A). ba1 and ba2, branchial arches 1 and 2; OV, otic vesicle.

View larger version (63K): [in a new window]   Fig. 2. Time-course and rhombomeric origin of aberrantly migrating NCCs. Before left-side r3 removal, cells in r4 or r2 were marked in one of two ways. In some embryos, cells within dorsal r4 or r2 were labelled by focal DiI injection. These embryos were allowed to develop for a further 5, 10, 20 or 30 hours and processed for Sox10 in situ hybridisation. In other embryos, r4 or r2 were replaced homotopically with quail rhombomeres. These embryos were allowed to develop for a further 72 hours and processed for anti-Q¢PN immunohistochemistry. (A-C) Dorsal views 5 hours after r3 removal. r4 cells have migrated appropriately towards ba2, while very few cells migrate aberrantly into mesenchyme adjacent to the removed r3 (r3*) (arrow) or directly into the space left by removing r3 (arrowhead). Appropriately migrating r4 cells express Sox10 (a marker of migrating NCCs), but Sox10 expression is not detected in aberrantly migrating r4 cells. (D-F) Dorsal views 10 hours after r3 removal. Several r4 cells have now migrated aberrantly into r3* mesenchyme (arrow) and Sox10 is expressed within proximal r3* mesenchyme. However, some aberrantly migrating r4 cells, more distal to the neuroepithelium, do not express Sox10 (arrowhead). (G-I) Dorsal views 20 hours after r3 removal. A robust stream of aberrantly migrating r4 cells is present within r3* mesenchyme (arrow) and intersects the stream of neural crest cells from r2. All of the aberrantly migrating r4 cells within r3* mesenchyme now fall within the region of Sox10 expression. (J-L) Lateral views of operated side 30 hours after r3 removal. Aberrantly migrating r4 cells are within r3* mesenchyme and can be detected within the developing trigeminal ganglion (arrowheads). (M-O) Dorsal views 20 hours after r3 removal. Many r2 cells are migrating appropriately towards ba1, but DiI labelled r2 cells seldom migrate aberrantly into r3* mesenchyme (arrow), even though a robust stream of Sox10 expressing, aberrantly migrating cells was detected within r3* mesenchyme in these embryos (arrowhead). (P-R) Dorsal views. By 30 hours after r3 removal, r2 cells had migrated into r3* mesenchyme (arrow) and were occasionally seen within the developing facial/acoustic ganglion (arrowhead). (S-X) Sagittal sections of quail-to-chick homotopic r2 or r4 grafts, stained with Q¢PN antibody 72 hours after r3 removal. (S) r2-derived quail cells were located appropriately within the trigeminal ganglion (gV) and ectopically within the facial ganglion (gVII) and the ectopic cranial nerve (arrow). (T-U) Quail r2 cells were not found within ba2 on either the operated (T) or control (U) sides. (V) Quail r4-derived cells were located appropriately within the facial ganglion and ectopically within the trigeminal ganglion and the ectopic cranial nerve (arrow). (W) Quail r4 cells were found ectopically within ba1 (arrow), but were not seen in ba1 on the unoperated side (X). ba1, branchial arch 1; ba2, branchial arch 2.

View larger version (76K): [in a new window]   Fig. 3. Heterotopic NCC grafting. Cartoons on the left summarise each experiment, and panels show dorsal views of the distribution of grafted cells 10 hours after surgery. Phase, combined phase/DiI, DiI and Hoxb1 in situ images. Broken lines mark the neuroepithelial outlines. (A-D) Unilaterally, r4 was replaced by DiI-labelled r2, and then r3 was removed. Ectopic r2 cells migrate along the normal r4 NCC pathway (arrowheads), but few cells migrate into r3* mesenchyme (arrow). Ectopic r2 did not express the r4 marker, Hoxb1 (D). (E-H) Unilaterally, r2 was replaced by DiI-labelled r4, and then r3 was removed. Ectopic r4 cells migrate along the normal r2 NCC pathway (arrowhead) and many cells migrate caudally into r3* mesenchyme (arrow), with some entering the normal r4 NCC pathway (small arrow). Ectopic r4 maintains Hoxb1 expression (H). (I-P) In similar experiments, small clusters of neuroepithelial cells were heterotopically grafted between r2 and r4. (I-L) Ectopically grafted r2 cells migrate out of r4 within the r4 NCC stream (arrowheads) and several cells migrate rostrally into r3* mesenchyme (arrow). Hoxb1 expression within r4 appears unaltered. (M-P) Ectopically grafted r4 cells migrate within the r2 NCC stream (arrowhead), but cells rarely deviate caudally into r3* mesenchyme. (P) Grafted and control sides of host hindbrain flatmount, stripped of mesenchyme to aid viewing. Within r2 neuroepithelium, very few grafted r4 cells maintain Hoxb1 expression (arrows), suggesting that in small clusters, ectopic NCCs lose their original positional identity.

View larger version (52K): [in a new window]   Fig. 4. After r3 removal, r3* mesenchyme gradually loses its NCC repulsive character. Experiments were performed to determine whether maturational changes intrinsic to NCCs (A,C-E), or surgically induced changes within the mesenchyme (B,F-K) were responsible for the observed delay between r3 removal and the onset of the aberrant NCC migration phenotype. Broken lines mark the neuroepithelial outlines. (A) r3 was removed at 10ss, clusters of donor DiI-labelled 16ss r4 cells were grafted homotopically into r4 and the embryos incubated for a further 5 hours. (C-E) Phase, combined phase/DiI, and DiI images, respectively, reveal that grafted cells seldom migrate into r3* mesenchyme, indicating that the r3 mesenchymal repulsive activity persists for up to 5 hours after r3 removal and affects late-migrating NCCs (cluster of cells within r2 in D,E is a cell injection artefact and does not represent migration from r4). (B) r3 was removed at 10ss and embryos were incubated for 5 hours before either directly labelling host r4 cells with DiI (I; F-H) or homotopically grafting age-matched DiI labelled r4 cell clusters (II; I-K). Embryos were then incubated for a further 3 hours. (F-K) Phase (F,I), combined phase/DiI (G,J) and DiI (H,K) images reveal that several r4 cells rapidly migrate (within 3 hours) into rostral r3* mesenchyme (arrows) following this additional post-operative period. This indicates that the mesenchymal repulsive activity is absent within 8 hours of r3 removal.

View larger version (81K): [in a new window]   Fig. 5. Following r3 removal, positional identity marker expression is unchanged within the neuroepithelium, but depends on r4 NCC density within r3* mesenchyme. (A-F) Dorsal views of two embryos (A-C and D-F) in which r4 cells were labelled with DiI at the time of r3 removal and the distribution of migrating r4 cells was examined 20 hours later. These embryos were subsequently labelled with Hoxa2 riboprobe. NCCs from r4 express Hoxa2 as they migrate along their normal pathway towards ba2 (C,F). (A-C) Under conditions of high density, aberrantly migrating r4 NCCs continue to express Hoxa2 within r3* mesenchyme (arrows) and even within ba1 (arrowheads). (D-F) However, when relatively small numbers of DiI-labelled r4 cells enter r3* mesenchyme (arrow in D) they no longer maintain Hoxa2 expression in ectopic locations (arrow in F). (G) Hoxb1 expression within r4 is unaffected 20 hours after r3 removal and although our previous dye-labelling experiments show that some r4 cells repopulate r3*, we found no evidence of Hoxb1-expressing cells within r3*. (H,I) EphA4 is normally expressed by r3 and r5. Some embryos were processed for EphA4 in situ hybridisation immediately after r3 removal (0 hours) to demonstrate the clean removal of r3 (H). Other embryos were processed for EphA4 in situ 20 hours after surgery and demonstrate that none of the cells repopulating r3* express EphA4 (I). ba1, branchial arch 1; ba2 branchial arch 2.

View larger version (102K): [in a new window]   Fig. 6. Aberrant axon pathfinding following r3 removal. Whole-mount anti-neurofilament staining at 20 hours (A-C), 30 hours (D-F), 45 hours (G) or 72 hours (H) after unilateral r3 removal. (A,D) dorsal views, (B,E,G,H) Operated side; (C,F) unoperated side. In each case, an aberrant axon projection was detected within r3* mesenchyme, between the trigeminal (gV) and facial (gVII) ganglia (arrow in A,B,D,E,G,H) and this ectopic cranial nerve grew thicker over time. (I) DiI injection into the ectopic nerve (45 hours after r3 removal) retrogradely labelled sensory neurones within the trigeminal ganglion (arrow) and facial ganglion (arrowhead), while within the hindbrain (J,K) longitudinal sensory axons were labelled, although motor cell bodies in r1/r2 and r4/r5 were not always labelled (J shows combined phase/DiI view of flatmounted hindbrain, K shows DiI view only). Lateral views of embryos after bilateral DiI injections into medial r2 (L,M) or medial r4 (N,O) 45 hours after r3 removal. Anterogradely labelled axons were rarely detected within the ectopic cranial nerve. f, facial nerve; m, mandibular branch of trigeminal nerve; OV, otic vesicle.

View larger version (64K): [in a new window]   Fig. 7. Altered pattern of cranial NCC migration after surface ectoderm removal. The surface ectoderm overlying r3 was removed unilaterally (left side) and embryos were allowed to develop for a further 20 hours in ovo before Sox10 in situ hybridisation. (A) A dorsolateral view of the operated side after 20 hours. Sox10-expressing NCCs form a robust aberrant projection between the r2 and r4 NCC streams, similar to that seen in r3 removal experiments. (B-D) Serial, slightly oblique, transverse sections through this embryo (broken lines in A show the planes of section in B-D). Arrow in C shows the aberrant NCC projection, while the broken line delineates the dorsoventral extent of surface ectoderm removal. The progression of the phenotype was determined by DiO labelling of dorsal r2 and by DiI labelling of dorsal r4, prior to removal of the r3 surface ectoderm. (E) Combined phase/DiO/DiI dorsal view after 5 hours; (F) DiO/DiI only. Broken lines delineate rhombomere boundaries and outline the neural tube. Some r4 cells (red) deviate rostrally into r3 mesenchyme. (G) A different embryo after 5 hours, revealing some aberrant rostral migration of Sox10-expressing r4 NCCs (arrow). (H) Combined phase/DiO/DiI dorsal view after 10 hours; (I) DiO/DiI only. Predominantly, r4 cells (red) enter r3 mesenchyme. (J) A different embryo after 10 hours, showing a bridge of Sox10-expressing NCC traversing r3 mesenchyme. (K,L) DiI labelling of surface ectoderm prior to r3 surface ectoderm removal revealed that labelled ectodermal cells had only occasionally re-grown into r3 mesenchyme after 10 hours (L shows combined phase/DiI; broken lines delineate the rostrocaudal extent of surface ectoderm removal). (M-T) Separate r2 DiI labelling and r4 DiI labelling experiments were performed to examine cell migration after 20 hours. (M,N) Several r2 cells migrate aberrantly into r3 mesenchyme (arrow in N). (O) The same embryo processed for Sox10 in situ reveals a more sharply defined bridge of Sox10-expressing NCCs through r3 mesenchyme. (P,Q) Several r4 cells also migrate aberrantly into r3 mesenchyme after 20 hours (arrow in Q). (R) Same embryo processed for Sox10 in situ reveals a band of NCCs through r3 mesenchyme. (S,T) DiI labelling of surface ectoderm before r3 surface ectoderm removal revealed that several labelled ectodermal cells had re-grown into r3 mesenchyme after 20 hours (arrow), suggesting that the more disperse Sox10-negative cells within r3 mesenchyme may be of ectodermal origin (T shows combined phase/DiI). ba1 and ba2, branchial arches 1 and 2.