Division of Neurobiology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK
*Authors for correspondence (e-mail: jgoldin{at}nimr.mrc.ac.uk and martin.gassman{at}unibas.ch)
Accepted 14 December 2001
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SUMMARY |
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We have used surgical manipulations in the chick to demonstrate that r3 neuroepithelium and its overlying surface ectoderm independently help maintain the NCC-free zone within r3 mesenchyme. In the absence of r3, subpopulations of NCCs enter r3 mesenchyme in a dorsolateral stream and an ectopic cranial nerve forms between the trigeminal and facial ganglia. The NCC-repulsive activity dissipates/degrades within 5-10 hours of r3 removal. Initially, r4 NCCs more readily enter the altered mesenchyme than r2 NCCs, irrespective of their maturational stage. Following surface ectoderm removal, mainly r4 NCCs enter r3 mesenchyme within 5 hours, but after 20 hours the proportions of r2 NCCs and r4 NCCs ectopically within r3 mesenchyme appear similar.
Key words: Chick, Neural crest cells, Mesenchyme, Surface ectoderm, Migration, Patterning
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INTRODUCTION |
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Although the origins, migration pathways and destinations of cranial NCCs are well documented (Koentges and Lumsden, 1996; Lumsden et al., 1991
), relatively few molecules have been found that influence their pathfinding. These include specific ephrins and their Eph receptors (Adams et al., 2001
; Helbling et al., 1998
; Holder and Klein, 1999
; Robinson et al., 1997
; Smith et al., 1997
), Collapsin 1 (Eickholt et al., 1999
), FGF2 (Kubota and Ito, 2000
) and an uncharacterised chemoattractant released from the otic vesicle (Sechrist et al., 1994
). Where studied in vivo, these factors appear to be involved in maintaining the segregation of NCC streams at the level of the branchial arches. However, the cues that enforce NCC segregation further dorsally, adjacent to the neuroepithelium, remain unknown.
In this study, we show that cues from r3 neuroepithelium and the overlying surface ectoderm are required to exclude subpopulations of NCCs from r3-adjacent mesenchyme.
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MATERIALS AND METHODS |
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Surgery
Eggs were windowed (Mason, 1999) and finely drawn glass needles were used for surgery. For neuroepithelial ablation, the left half of r3 was freed from surrounding tissue by incisions made just lateral to the rhombomere, along its mid-line, and along the r3/4 and r3/2 boundaries. For surface ectoderm ablation, a rectangle was cut superficially into the surface ectoderm, which was then carefully peeled off. The incisions extended from the dorsal mid-line laterally through 90 degrees, while along the AP axis they extended into the neighbouring r2 and r4 ectoderm by up to half a segment.
Cell tracing and cell grafting
For tracing migrating cells a 3 mg/ml solution of DiI or DiO (D-282, D-275; Molecular Probes) in dimethylformamide was microinjected into dorsal r2 or r4.
For cell grafting experiments, r2 or r4 were cleanly removed from donor embryos and labelled for 3 minutes with 250 µg/ml CM-DiI (C-7000, Molecular Probes) dissolved in Tyrodes solution containing 5% ethanol, 5% sucrose and 25% foetal calf serum. Labelled rhombomeres were washed with Tyrodes solution before either grafting into host embryos or dissecting the dorsal half into smaller fragments for injection into dorsal r2 or r4 of host embryos. After labelling, host r3 or r3 surface ectoderm were unilaterally removed. Embryos were allowed to develop for a further 5-45 hours.
Axon tracing
Forty-five hours after r3 removal, embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). DiI (3 mg/ml in dimethylformamide) was injected bilaterally into r2 or r4 basal plate, or into the ectopic cranial nerve. Embryos were viewed 3 days later.
In situ hybridisation
Whole-mount in situ hybridisation (Grove et al., 1998) was performed using 1 µg/ml digoxigenin-labelled riboprobe at 70°C. Chick Hoxb1, Hoxa2 and EphA4 plasmids were gifts from Robb Krumlauf. Chick Sox10 plasmid was a gift of Paul Scotting and Yi-Chuan Cheng.
Immunohistochemistry
Whole-mount immunohistochemistry (Lumsden and Keynes, 1989) was performed using anti-HNK1 (clone VC1.1; C0678, Sigma; 1:100), anti-neurofilament-160 kDa (clone RMO-270; Zymed Laboratories; 1:500) or anti-quail antibodies (Q¢PN; gift of Andrew Lumsden; 1:4). Anti-mouse Ig secondary antibodies were HRP or Cy3 conjugated (Amersham; 1:300).
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RESULTS |
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In subsequent r3 removal experiments, the full dorsoventral extent of r3 was excised unilaterally in 9ss-11ss embryos. This was found to be technically more reproducible and resulted in a higher proportion of embryos with the aberrant Sox10+ NCC migration phenotype (47/52 embryos after 20 hours). To investigate the origins of the misguided NCCs and to monitor the progression of the phenotype, we injected a fluorescent dye (DiI) into left dorsal r4 or r2, to label pre-migratory NCCs, just prior to r3 removal on the left side. The distribution of migrating DiI-labelled cells was examined at 5, 10, 20 and 30 hours post-surgery and compared with the distribution of Sox10-expressing NCCs in the same embryos (Fig. 2). In embryos where r4 was DiI labelled, large numbers of DiI-labelled NCCs migrated into ba2 within 5 hours, although in only 4/17 embryos did cells migrate aberrantly into r3* mesenchyme (arrow in Fig. 2B). In addition, several r4-derived cells entered r3*, the gap left in the neuroepithelium after removing r3 (arrowhead in Fig. 2B). However, Sox10 riboprobe labelled no cells within r3* or r3* mesenchyme at 5 hours postsurgery (Fig. 2C, although DiI/Sox10+ cells were sometimes found in association with the dorsal surface ectoderm). Only by 10 hours after surgery could a distinct subpopulation of r4-derived NCCs be detected within r3* mesenchyme (11/14 embryos), as revealed both by DiI labelling (Fig. 2D,E) and Sox10 in situ hybridisation (Fig. 2F; aberrant cells indicated by an arrow in Fig. 2E), although Sox10 was not detectable in all DiI-labelled ectopic cells (arrowhead in Fig. 2E). We do not know the identity of the Sox10/DiI+ cells. By 20 hours postsurgery a bridge of aberrantly migrating r4-derived cells connected the r4 and r2 NCC streams (23/29 embryos) and some r4-derived cells could be detected within the r2 NCC stream (Fig. 2G-I). By this stage, the distributions of DiI-labelled cells and Sox10-expressing cells coincided within r3* mesenchyme. Similarly, at 30 hours postsurgery, aberrantly migrating DiI+/Sox10+ r4-derived NCCs were detected within r3* mesenchyme and now also within the developing trigeminal ganglion (Fig. 2J-L) (11/12 embryos). Occasionally, DiI-labelled cells were also seen in ba1 (data not shown).
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Heterotopic grafting reveals intrinsic differences in the responsiveness of r2 NCCs and r4 NCCs to r3* mesenchyme
The observation that predominantly r4 NCCs initially migrate aberrantly into r3* mesenchyme might relate to intrinsic differences between r2 NCCs and r4 NCCs, or to local differences in the mesenchymal environment. In order to differentiate between these possibilities, we performed unilateral heterotopic transplantations of either entire DiI-labelled rhombomeres or of small clusters of DiI-labelled dorsal neuroepithelium (containing pre-migratory NCCs) followed by unilateral removal of host r3. Ten hours after surgery, we found that when r2 was unilaterally grafted in place of r4, DiI-labelled r2 NCCs migrated along the appropriate (r4) pathway for their new location, but seldom migrated rostrally into r3* mesenchyme (in only 4/11 embryos; Fig. 3A-C). However, when r4 unilaterally replaced r2, DiI-labelled r4 NCCs migrated along the r2 pathway but many cells also deviated caudally into r3* mesenchyme (in 7/10 embryos; Fig. 3E-G). In control experiments, where rhombomeres were transplanted but r3 was left intact, no aberrant NCC migration was observed (four embryos each; not shown).
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In contrast to the results obtained by transplanting entire rhombomeres, when smaller clusters of r2 cells were grafted into r4, several of the ectopically placed cells migrated rostrally into r3* mesenchyme (in 6/10 embryos; Fig. 3I-K). Conversely, when r4 cells were grafted into r2, the r4-derived cells seldom migrated caudally into r3* mesenchyme (in only 2/12 embryos; Fig. 3M-O). In these embryos we detected no Hoxb1-negative r2 cells within r4 neuroepithelium (Fig. 3L) and found very few r4 cells maintaining Hoxb1 expression within r2 neuroepithelium (Fig. 3P), suggesting that grafted cells may change positional identity to match that of their new environment. In the chick, even-to-even-numbered rhombomere cell transplants become dispersed within the ectopic neuroepithelial environment (Guthrie et al., 1993), while in the mouse, as engrafted r4 cells disperse within r2 they lose Hoxb1 expression (Trainor and Krumlauf, 2000b
).
Our data reveal intrinsic differences between r2 NCCs and r4 NCCs, and suggest that multiple repulsive cues may exist within r3 mesenchyme. Thus, r2 NCCs are reluctant to enter r3 mesenchyme whether r3 is present or not, while several r4 NCCs enter r3 mesenchyme in the absence of the repulsive cues associated with r3.
After r3 removal, r3* mesenchyme gradually becomes permissive to NCCs
At least two explanations could account for the observed delay of 5 hours between r3 removal and the initial appearance of aberrantly migrating r4 NCCs within r3* mesenchyme. The r3-dependent repulsive activity might disappear rapidly from the mesenchyme after r3 removal, but only late-migrating NCCs might be competent to respond to this changed environment. Alternatively, the repulsive activity might require several hours to dissipate/degrade after r3 removal and the age of the NCCs could be relatively unimportant. To discriminate between these possibilities, we studied the migration of r4 cells in a number of cell tracing/grafting paradigms (summarised in Fig. 4A,B).
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In the second paradigm, we investigated the role of the environment. We unilaterally removed r3 at 10ss and incubated these embryos for 5 hours (to 13ss). Then host dorsal r4 cells were labelled by DiI injection (Fig. 4B part I; six embryos). After a further 3 hours, we found that labelled r4 cells had migrated in the r4 NCC stream towards ba2 and many labelled r4 cells had also migrated into r3* mesenchyme (Fig. 4F-H). To control for any injury-induced changes in host r4 NCCs, labelled r4 cells from unoperated 13ss donor embryos were grafted homotopically into 13ss host embryos in which r3 had been unilaterally removed at 10ss (Fig. 4B, part II; 10 embryos). The migration of labelled cells was examined 3 hours later. Donor r4 cells migrated in the r4 NCC stream towards ba2 and also into r3* mesenchyme (Fig. 4I-K).
Taken together, these data favour a model in which subsets of r4 cells from a wide age range are sensitive to a repulsive activity that is gradually lost from r3* mesenchyme.
Positional identity markers are unchanged after r3 removal
To determine whether aberrantly migrating r4 NCCs maintain expression of r4 segment identity markers within r3* mesenchyme, we used the r4 marker Hoxa2, which is expressed transiently by migrating r4 NCCs, but not by r2 NCCs (Prince and Lumsden, 1994). Pre-migratory r4 NCCs were labelled unilaterally with DiI at 10ss, before unilateral r3 removal, and embryos were processed for Hoxa2 in situ hybridisation 20 hours later. In cases where a dense stream of DiI-labelled r4 NCCs entered r3* mesenchyme (Fig. 5A,B), this aberrant NCC stream maintained Hoxa2 expression (Fig. 5C arrow), even within ba1 (Fig. 5C, arrowhead). However, when r4 cells entering r3* mesenchyme were few and dispersed (Fig. 5D,E), Hoxa2 expression could not be detected in this region (Fig. 5F arrow). This suggests that in the absence of cues from neighbouring r4 NCCs or the appropriate mesenchymal environment, r4 NCCs fail to maintain normal levels of a marker of their original AP identity. This finding is consistent with data from mice that demonstrate the importance of local environmental cues in reinforcing the positional identity of migrating NCCs (Golding et al., 2000
; Trainor and Krumlauf, 2000b
).
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Our initial studies demonstrated that cells from r2 and r4 entered r3* (the space previously occupied by r3; see Figs 1 and 2). However, in this ectopic environment, no incoming cells were found to express Hoxb1, an r4 neuroepithelial marker (Maden et al., 1991). Hoxb1 continued to show a sharp limit of expression at the r4/r3* boundary, although the position of r4 on the operated side of embryos often appeared shifted slightly rostrally (Fig. 5G). This suggests that r4 cells that enter r3* are either NCCs that have already downregulated Hoxb1, or r4 neuroepithelial cells that downregulate Hoxb1 in the ectopic r3* environment. Previous work indicates that the cells that infill ablated rhombomeres can readjust their Hox gene expression (Hunt et al., 1995
). To further investigate the identity of cells that entered r3* we used an EphA4 (Sek) riboprobe, which identifies r3 and r5 neuroepithelial cells (Nieto et al., 1992
). Embryos were processed for EphA4 in situ hybridisation either immediately after unilateral r3 removal (Fig. 5H), or 20 hours after surgery (Fig. 5I). At neither time point was EphA4 expression detected within r3*. These data confirm that our surgical procedures cleanly remove r3 and that although cells infiltrate r3* from neighbouring r2 and r4, this does not result in the regeneration of an r3 phenotype. In addition to providing information on cells that re-populate r3*, our results indicate that r4 positional identity markers are not altered by r3 removal.
Axon misprojections following r3 removal
Cranial NCCs give rise to several differentiated cell types, including components of the peripheral nervous system. Therefore, we investigated whether, in addition to aberrant NCC migration, there were any changes in cranial nerve anatomy, after r3 removals.
Neurogenic NCCs from r2 and r4 contribute to the trigeminal ganglion and the facial/acoustic ganglia, respectively. By 20 hours after r3 removal, whole-mount anti-neurofilament antibody staining revealed a small number of axons extending through r3* mesenchyme, between the developing trigeminal ganglion and the facial/acoustic ganglia (Fig. 6A,B). No neuronal cell bodies were detected within r3* mesenchyme and we did not detect any axons entering r3* mesenchyme directly from the lesioned neuroepithelium (Fig. 6A,B). By 30 hours after r3 removal, the number of mis-projecting axons was greater and they had fasciculated into a thin bridge between the trigeminal and facial/acoustic ganglia (Fig. 6D,E). By 45 hours after r3 removal, a substantial ectopic nerve was present between these ganglia (Fig. 6G), which persisted at 72 hours (Fig. 6H).
The origin of axons contributing to the ectopic cranial nerve was investigated by injecting DiI into the nerve 45 hours after r3 removal. Peripherally, axons and neuronal cell bodies within the trigeminal and facial ganglia were labelled (Fig. 6I). Centrally, longitudinally running sensory axons were labelled. Motoneurone cell bodies within r1, r2, r4 and r5 were sometimes labelled (Fig. 6J,K). However, when DiI was applied to medial r2 (Fig. 6L,M) or medial r4 (Fig. 6N,O) (in the vicinity of motoneurones), aberrant axon projections were rarely detected peripherally within r3* mesenchyme after r2 labelling and never after r4 labelling.
Removal of r3 surface ectoderm also alters NCC migration pathways
Two distinct cell types abut the cranial mesenchyme. Medially, it contacts neuroepithelium, while laterally it contacts surface ectoderm. In order to investigate the contribution that surface ectoderm-derived cues might make to cranial NCC pathfinding, we unilaterally removed r3 surface ectoderm and examined the distribution of Sox10-expressing cells 20 hours later. Similar to the r3 removal phenotype, we detected a cohort of Sox10-expressing cells extending through r3 mesenchyme (Fig. 7A-D; 15/39 embryos).
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Although our data suggest that surface ectoderm and neuroepithelium independently provide patterning cues, there remained the possibility that only one of these tissues was involved. This is because at the most dorsal region of r3 the neuroepithelium and surface ectoderm are closely apposed, and both tissues are likely to be removed in either type of ablation experiment. To address this issue, we performed control experiments in which the most dorsal part of r3 neuroepithelium plus surface ectoderm was unilaterally removed and the embryos processed with Sox10 riboprobe 20 hours later. No aberrant NCC migration was detected in any of these (6) embryos (not shown), indicating that dorsalmost r3+ectoderm is not sufficient to pattern NCC migration. Moreover, this experiment reveals that, in more ventral locations, where r3 neuroepithelium and r3 surface ectoderm can be separately removed, both of these tissues are required to pattern NCC migration correctly.
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DISCUSSION |
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NCCs respond heterogeneously to mesenchymal pathfinding cues
Two features of NCC pathfinding are demonstrated by our results. First, the migration of only subpopulations of r2 NCCs and r4 NCCs are affected by our surgical interventions. Second, it is initially mainly r4 NCC migration that is affected by surface ectoderm removal or r3 removal, although the onset of aberrant migration is sooner following surface ectoderm removal. These observations suggest first the existence of multiple NCC repulsive activities in r3 mesenchyme, some of which persist in the absence of neighbouring neuroepithelium and ectoderm, and second, intrinsic differences in responsiveness of r4 NCCs and r2 NCCs to these repulsive activities.
Our results do not allow us to determine which tissues actually synthesise the repulsive activity but as the ectoderm-dependent activity dissipates faster than the neuroepithelial-dependent activity, it is possible that a sequence of signals needs to be relayed from neuroepithelium to ectoderm to mesenchyme.
An alternative interpretation of the first point is that removal of r3 or r3 ectoderm might make r3 mesenchyme permissive to all NCCs, but adhesive interactions with appropriate NCC pathways (Bronner-Fraser, 1984; Bronner-Fraser, 1985
; Bronner-Fraser, 1986
; Bronner-Fraser, 1987
; Kil et al., 1996
) or chemoattraction from target tissues (Kubota and Ito, 2000
; Sechrist et al., 1994
) exert stronger influences on the migration of most NCCs than the lure of the new territory opened up by the loss of the mesenchymal repulsive activity.
The second point is less contentious, as the intrinsic behavioural differences between r4 NCCs and r2 NCCs revealed by our heterotopic rhombomere transplantation experiments could be related to well-established molecular differences between these populations of migrating NCCs for example in their expression of Hox genes (Hunt et al., 1991; Prince and Lumsden, 1994
; Trainor and Krumlauf, 2000a
) and Noggin (Smith and Graham, 2001
).
The observation that aberrantly migrating Sox10+ NCCs migrate in a tightly defined dorsolateral pathway raises the possibility that cryptic pathfinding cues might be specified within r3 mesenchyme, but that these are normally masked by the overriding influence of repulsive activities. Intriguingly, the aberrant NCC pathway lies in the same dorsoventral plane as the nerve exit points and cranial ganglia, suggesting that cues for NCC migration or differentiation may be produced by all rhombomeres at this DV level. However, these putative common cues are unlikely to specify exit points, as neither breaks in the neural tube basal lamina nor ectopic Sox10+ NCC boundary caps (Niederlander and Lumsden, 1996) were found alongside r3 after surface ectoderm removal (Fig. 7C).
Molecular mechanisms that pattern NCC migration
Several studies have shown alterations in cranial NCC migration or cranial nerve projection in response to respecifying rhombomere identity using retinoic acid or targeted changes in Hox gene expression (Alexandre et al., 1996; Bell et al., 1999
; Lee et al., 1995
). However, our analysis of Hoxa2 and EphA4 expression patterns provides no evidence for respecification of r2 or r4 identity after r3 removal.
Within the hindbrain, Eph/ephrin interactions are important for restricting cell intermixing between rhombomeres (Mellitzer et al., 1999; Xu et al., 1995
; Xu et al., 1999
). In Xenopus embryos Eph/ephrin interactions are also important for regulating cranial NCC migration pathways, as inactivation of these signalling mechanisms (specifically, EphA2, or EphA4, EphB1 and their cognate ligand ephrinB2) leads to ectopic migration of ba3 NCCs (from r5, r6 and r7) into ba2 and ba4 (Helbling et al., 1998
; Robinson et al., 1997
; Smith et al., 1997
). Similarly, in the mouse, loss of ephrinB2 leads to ectopic scattered migration of non-gliogenic r4 NCCs at the level of ba2 (Adams et al., 2001
). However, no regional differences in Eph or ephrin expression have been reported in more dorsal regions of cranial mesenchyme adjacent to r2-r4, and inactivation of Eph/ephrin signalling does not result in altered NCC migration patterns similar to those we report here, indicating that other molecules pattern these earlier stages of NCC migration.
One defect in cranial NCC migration that does resemble the phenotype we report here is seen in mice lacking the receptor tyrosine kinase erbB4 (Gassmann et al., 1995; Golding et al., 2000
). In these mice, a subpopulation of late-migrating r4 NCCs enter r3 mesenchyme and also contribute to the trigeminal ganglion. Subsequently, an ectopic cranial nerve is produced between the trigeminal and facial/acoustic ganglia. During the period of NCC migration, r3 expresses Erbb4 in mouse (Gassmann et al., 1995
) and chick (Dixon and Lumsden, 1999
), suggesting that the similar r3-dependent NCC phenotype we report here could be related to a loss of erbB4-mediated signalling.
In summary, our data reveal that tripartite signalling interactions between neuroepithelium, surface ectoderm and mesenchyme help sculpt the initial pathways taken by migrating cranial NCCs. Future work will attempt to identify these patterning cues and determine whether the repulsive activities within r3 mesenchyme are synthesised locally or are supplied by the neighbouring tissues. Several different signalling systems are probably required to pattern NCC migration correctly within the developing head, and a crucial goal of developmental biology is therefore to determine how these various cues co-operate or integrate with each other to direct head morphogenesis.
Note added in proof
A recent study by Trainor et al. (Trainor et al., 2002) demonstrates that in mouse embryos, the crest-free zone adjacent to r3 is maintained by combinatorial interactions between r3 neuroepithelium and the adjacent mesenchyme/surface ectoderm.
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ACKNOWLEDGMENTS |
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