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
*Author for correspondence (e-mail: rek{at}stowers-institute.org)
Accepted 24 October 2001
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SUMMARY |
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Key words: Neural crest, Hindbrain, Segmentation, Cell migration, Cell death, Head patterning, Mouse, Chick, Transplantation
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INTRODUCTION |
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The function and the mechanism by which the r3 and r5 crest-free zones are established are contentious. On one hand, analyses in avian embryos reported elevated levels of cell death in the premigratory neural crest populations of r3 and r5 (Graham et al., 1993), mediated by Bmp4 signalling from the even rhombomeres. This induces Msx2 in r3 and r5, leading to the elimination of neural crest before migration (Ellies et al., 2000; Graham et al., 1994; Graham et al., 1993). Hence, inter-rhombomeric signalling would be the key factor in modulating the generation of crest cells from odd rhombomeres. In contrast, lineage and time-lapse analyses in avians clearly show that r3 and r5 do generate neural crest cells, but they move anteriorly and posteriorly, joining the even rhombomere streams (Kulesa, 1998; Kulesa et al., 2000; Kulesa and Fraser, 2000; Sechrist et al., 1993). Furthermore, in frog and fish embryos, r5 produces equivalent amounts of crest cells that migrate laterally, compared to the even rhombomeres (Smith et al., 1997). The variation between species in the amount and migratory pathways of neural crest cells generated by r5, implies that the crest-free zones are not an intrinsic property of odd-numbered rhombomeres themselves. This suggests that environmental influences adjacent to r3 and r5 may be important in controlling the pathways of hindbrain neural crest migration (Farlie et al., 1999), and distinct mechanisms may be utilised by different species to pattern these events. Therefore it is important to understand the mechanisms responsible for segregating the branchial arch streams in mouse embryos and determine if they are functional in other vertebrates.
In this study, we used cell grafting and lineage-tracing techniques in cultured mouse embryos to investigate the interactions between the hindbrain and the environment with respect to patterning the pathways of mouse cranial neural crest cells. We demonstrate that the patterns of cell death in the hindbrain do not correlate with the generation or migration of neural crest. Furthermore, odd-numbered rhombomeres have the same capacity to generate neural crest cells as the even segments. Reciprocal transpositions between even and odd rhombomeres show that specific regions are inhibitory to neural crest cell migration. Our findings demonstrate that the pathways of hindbrain neural crest migration and crest-free zones adjacent to r3 in the mouse are generated by combinatorial signalling events between the hindbrain and the adjacent environment, and this mechanism appears to be conserved in the chick.
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MATERIALS AND METHODS |
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Isolation and labelling of rhombomeric, mesoderm and ectoderm tissue
Consistent neuromeric landmarks, described by Trainor and Tam (Trainor and Tam, 1995), were used to identify the axial level of tissue to be grafted and the correct site of transplantation. Finely polished alloy and glass needles were used to separate the neuroectoderm from adjacent tissues. Tissue fragments that could not be cleanly separated from adjacent tissues were incubated in 0.5% trypsin, 0.25% pancreatin, 0.2% glucose and 0.1% polyvinylpyrolidone in phosphate-buffered saline (PBS) for 5 minutes at 37°C or in Dispase for 5 minutes at 37°C to ensure a pure population. Isolated tissue fragments were then washed in DMEM (Dulbeccos modified Eagles medium), before being labelled with DiI or DiO by soaking in a 1:1 mix of DiI:DR50 or DiO:DR50 (Manzanares et al., 1999), for 2 minutes. Tissue fragments were then washed in DMEM and dissected in DR50 (Sturm and Tam, 1993) using glass needles into smaller fragments consisting of approximately 15-30 cells.
Tissue transplantations
Odd rhombomere to even rhombomere grafts
Small groups of approximately 15-30 cells from r3 or r5 of 8.5 d.p.c. embryos were orthotopically transplanted back to the same sites and heterotopically transplanted into r2 or r4 of isochronic embryos.
Even rhombomere to odd rhombomere grafts
Small groups of approximately 15-30 cells from r4 of 8.5 d.p.c. embryos were orthotopically transplanted back to the same site and heterotopically transplanted into r2 and r3 of isochronic embryos.
Mesoderm and ectoderm grafts
Small fragments of mesoderm or ectoderm were isolated from adjacent to r3 and transplanted next to r4 in isochronic embryos.
Embryo analysis
Grafted embryos were analysed for DiI and green fluorescent protein (GFP) labelling by fluorescence microscopy and with a Leica TCS NT confocal microscope (567 nm excitation) before being assayed for cell death.
Detection of cell death
8.5-9.5 d.p.c. embryos were assessed for rhombomeric cell death via Nile Blue, Acridine Orange or TUNEL staining. For Nile Blue and Acridine Orange staining, embryos were cultured in DR50 (containing a 1:400 dilution of 1.5% Nile Blue in water or 5 µg/ml of Acridine Orange) for 30-40 minutes. Apoptotic cells stain intensely blue when incubated in Nile Blue and are epifluorescent (rhodamine) after incubation with Acridine Orange. TUNEL staining labels apoptotic cells fluorescently (fluorescein) and was performed according to the manufacturers instructions (Boehringer Mannheim).
Whole-mount in situ hybridisation
Bmp4 and Msx2 digoxigenin-labelled riboprobes were synthesised (Boehringer Mannheim) and whole-mount in situ hybridisation was performed as previously described (Wilkinson, 1992).
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RESULTS |
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To score for general patterns of apoptosis, we used the vital dyes Nile Blue Sulphate and Acridine Orange, which stain the highly chromatin-rich apoptotic bodies. This enabled the detection of apoptotic cells in embryos cultured in vitro, so that a picture of cell death can be accumulated over a number of hours during development rather than at a single time point. Using mouse embryos from 8.5-9.5 d.p.c., vital staining demonstrated that, during the period of neural crest migration, cell death occurs in a temporally and spatially dynamic manner (Fig. 2A-D). Although we observed consistent and reproducible cell death in the hindbrain, there was no specific pattern that could be attributed to either odd or even rhombomeres. Even within a single embryo, the unfused neural folds often display completely different patterns of cell death, highlighting the dynamic nature of events (Fig. 2A, arrowheads). The elevated levels of cell death detected between 8.5-9.5 d.p.c. were associated with the normal caudal-to-rostral progression of neural tube closure and subsequent formation of the roof plate in the hindbrain (Fig. 2A-D). Similar results were obtained independently by TUNEL staining of fixed embryos (data not shown).
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The ability of r4 to generate neural crest cells also depends upon the environment
These experiments suggest that the environment adjacent to r3 plays an important role in restricting the delamination, migration and/or proliferation of neural crest cells in ba2. Hence, an important issue is whether even-numbered rhombomeres retain their ability to generate substantial amounts of neural crest cells in ectopic r3 environments. To test this, we used a Hoxb1-lacZ transgenic line expressing the reporter specifically in r4, to isolate, DiI-label and graft small groups of r4 cells into host embryos (Fig. 5). In control transplantations, r4 cells grafted back into r4 gave rise to numerous neural crest cells, which extensively populated ba2 (Fig. 5A). Similarly, when r4 cells were transposed into r2 they retained the ability to generate numerous neural crest cells, which migrate into ba1 (Fig. 5B). In contrast, when r4 cells were transplanted into r3, their capacity to produce migrating neural crest cells was repressed (Fig. 5C,D). We confirmed that this repression is not due to a change in their segmental identity, as indicated by continued lacZ expression in the grafted hindbrain tissue (data not shown). To exclude the possibility that the absence of migrating neural crest cells was due to cell death of the grafted tissue, we performed TUNEL staining (Fig. 5E-G). TUNEL and staining for ß-gal show that the grafted cells are retained in r3, express the appropriate marker and are hence not eliminated by cell death. These results imply that the local environment adjacent to r3 prohibits the formation and migration of neural crest cells equally well from odd- and even-numbered rhombomeres.
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Conservation of signalling interactions between mouse and chick
Our rhombomere transpositions in mouse showed that the relatively low production of r3-derived neural crest cells is not intrinsic to the rhombomere itself. Rather, combinatorial interactions between r3 and the surrounding arch environment as it develops, restrict the generation and migration of cranial neural crest cells. In contrast, in chick embryos it has been postulated that inter-rhombomeric signalling between even- and odd-numbered rhombomeres is responsible for limiting the production of neural crest (Ellies et al., 2000; Graham et al., 1994; Graham et al., 1993). In this model the ability of r3 to generate neural crest cells is repressed by signals received from its interaction with the adjacent r2 and r4 territories.
To distinguish between these mechanisms we performed transpositions from mouse to chick. Mouse r3 tissue grafted into r4 in the chick, becomes incorporated and proliferates, generating neural crest cells, which populate ba2 (Fig. 7A). This is further evidence that r3 has the intrinsic capacity to generate neural crest cells. Conversely the ability of r4 to generate neural crest is lost when mouse r4 tissue is transplanted into r3 in the chick (Fig. 7B). This is not due to cell death or changes in identity as the grafted cells continue to express the lacZ reporter (Fig. 7C). This illustrates that the environment adjacent to r3 in the chick also restricts the ability of r4 to generate migratory neural crest and that mouse rhombomeric cells have conserved the ability to respond to these signalling interactions. Since in both of these types of transpositions, r3 and r4 cells are juxtaposed and capable of interacting with each other, our experiments show that interactions between the arch environment and rhombomeres are a critical determinant in controlling the production of neural crest. Together with the neural crest-free zones adjacent to r3 in other vertebrates, these results suggest that there is a common mechanism involving interactions between r3 and arch tissues used to govern the patterning of neural crest derived from r3.
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DISCUSSION |
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The transposition of cranial mesoderm and surface ectoderm suggest that the signals influencing the generation and migration of neural crest are established by interactions between the hindbrain and these tissues. This suggests several possibilities. Positive, emigration-provoking signals, potentially in concert with extracellular matrix differences, may be present in some mesodermal populations either intrinsically or through interactions with the endoderm. Furthermore, neural crest survival, emigration and migration into the arches may result from the sequential interactions initiated in the hindbrain during neural plate stages. Hence, in a manner similar to what we have found in studying cranial neural crest plasticity in A-P patterning, the proper program of events governing the migration of crest may need first to be established in the hindbrain, to allow migratory crest cells to interpret and respond to environmental signals set up through a series of tissue interactions. This contrasts with the trunk, where the segmental migratory pattern of neural crest through the rostral sclerotome is directly imposed by the rostrocaudal character of the mesoderm (Bronner-Fraser and Stern, 1991; Keynes and Stern, 1984). The exclusion of neural crest adjacent to r3, and the rostral and caudal patterns of migration of r3-derived neural crest observed in the mouse, create a neural crest-free zone. This is a common feature in vertebrates (Farlie et al., 1999) and suggests that the signalling interactions we have observed in the mouse reflect a conserved mechanism for patterning the distribution of neural crest cells from r3. This raises several interesting issues with respect to neural crest and craniofacial patterning.
Model for signals influencing neural crest migration
An important question is why is it necessary to establish exclusion zones and restrict r3 neural crest migration? One major reason may be a need to prevent mixing between non-Hox and Hox expressing neural crest cells, which populate ba1 and ba2, respectively (see Fig. 8). A variety of gain- and loss-of-function studies have shown that Hoxa2 is primarily responsible for specifying second branchial arch fate and can also inhibit the lower jaw skeleton (Couly et al., 1998; Gendron-Maguire et al., 1993; Grammatopoulos et al., 2000; Kanzler et al., 1998; Pasqualetti et al., 2000; Rijli et al., 1998; Rijli et al., 1993). Therefore Hoxa2-expressing populations must be excluded from the first branchial arch neural crest. This is achieved in part by restricting the generation of neural crest from r3 and also by changes in identity of the small number of anteriorly migrating cells, due to plasticity and cell community effects (Schilling, 2001; Trainor and Krumlauf, 2000b). Furthermore, the tight contacts and communication between migrating neural crest cells derived from a similar location may help to feed back the influences of inhibitory signals in the arch environment (Kulesa et al., 2000; Kulesa and Fraser, 2000).
While this mechanism excludes the lateral migration of r3-derived neural crest, other mechanisms must operate to prevent in-filling and mixing of neural crest from adjacent territories to maintain an exclusion zone. For example, in ErbB4 mutants a population of r4-derived neural crest cells acquires the ability to migrate through the dorsal mesenchyme adjacent to r3 (Golding et al., 2000). This phenotype arises due to changes in the mesenchyme and is not autonomous to the neural crest (Golding et al., 2000). Since ErbB4 is expressed only in r3 and r5, this phenotype reflects defects in signalling between r3 and its adjacent environment (Golding et al., 2000). There is also evidence that Eph/ephrin signalling is another mechanism that contributes to restrictions in the mixing of branchial arch neural crest populations (Fig. 8). In Xenopus embryos, the neural crest delaminates as a contiguous A-P band and separation is achieved only during the later phases of migration through the differential expression of the Eph receptor and ephrin ligand families of genes (Sadaghiani and Theibaud, 1987; Smith et al., 1997). Hence these mechanisms keep neural crest streams segregated and prevent infilling from adjacent territories, working in concert with restrictions in the lateral migration of r3 neural crest to establish crest-free zones.
Differences between the r3 and r5 environments
There are important differences in the mechanisms governing neural crest migration from r3 and r5. Firstly, in mouse, fish and frog embryos r5 generates considerably more neural crest than r3. Secondly, the migration patterns of r5 neural crest are not as well conserved in vertebrates as they are for r3. In frog and fish embryos r5 generates equivalent amounts of neural crest compared to the even rhombomeres, which also migrate laterally (Smith et al., 1997). In contrast, in mouse (Fig. 1) and chick embryos (Kulesa, 1998; Kulesa et al., 2000; Kulesa and Fraser, 2000; Sechrist et al., 1993), crest from r5 migrates rostrally and caudally to join adjacent even-rhombomere streams (Fig. 8). Whilst the general migration pattern of r5-derived cells is similar to r3, it does not appear to arise by the same mechanism. The otic vesicle is positioned immediately adjacent to r5, which provides a physical barrier rather than an exclusion zone that inhibits lateral migration from r5 (Fig. 8). This is consistent with the fact that in fish and frog embryos, the otic vesicle is positioned more laterally, thus allowing the unimpeded lateral migration of neural crest cells from r5 that is seen in these species (Sadaghiani and Theibaud, 1987; Schilling and Kimmel, 1994; Smith et al., 1997; Snape et al., 1991). Furthermore, in mouse and chick transplantations, moving r5 from the proximity of the otic vesicle results in the lateral migration of r5-derived neural crest cells (Fig. 4D) (Saldivar et al., 1996). Thus there are differences in the ability of the environments adjacent to the odd-numbered rhombomeres to influence neural crest migration. Hence the interactions between r3 and the environment, which generate signals that establish a neural crest-free zone, appear to be unique to that rhombomere.
Similarities and differences between the mouse and chick patterning of neural crest
Our interspecies grafting experiments, in which mouse r3 was transposed into chick r4, confirms that r3 has the capacity to generate neural crest in a different species (Fig. 7). Conversely transplants of mouse r4 into chick r3 display a repressed ability to generate neural crest cells. This suggests that the environmental signals adjacent to r3, which restrict the initial generation and migration of neural crest cells, are conserved between the species. In contrast, other mechanisms contributing to neural crest patterning appear to have diverged. In the chick, interactions between odd and even rhombomeres generate segmental patterns of apoptosis mediated by a Bmp4-Msx2 signalling loop that contributes to the reduction of neural crest from r3 (Ellies et al., 2000; Graham et al., 1994; Graham et al., 1993). However, in the mouse hindbrain the patterns of Bmp4 and Msx2 expression are not segmentally restricted in comparison to the chick. Bmp4 is not expressed in the mouse hindbrain and Msx2 is expressed uniformly along the dorsal edge of the neural tube. While it is possible that other Bmps may be involved, neither Bmp7 or Bmp2 are segmentally expressed in odd rhombomeres. Furthermore, this is consistent with our findings in the mouse that while cell death occurs in a temporally and spatially dynamic manner, there is no evidence for cell death specifically in odd rhombomeres during the period of neural crest migration (Fig. 2).
Since cell death in odd-numbered rhombomeres does not contribute to the patterning of mouse cranial neural crest, this raises the issue of why there are differences between the species. One explanation may be related to fundamental differences in the timing and manner in which neural crest cells are generated in each species. Neural crest migration in the mouse occurs during neural fold elevation prior to neural tube closure, whereas in the chick, neural crest migration occurs well after neural tube closure (Le Douarin, 1983; Le Douarin and Kalcheim, 1999). As a consequence the conserved interactions adjacent to r3 that inhibit crest migration may cause crest cells to build up between the neural tube and surface ectoderm in the chick and not the mouse. Hence, in chick, cell death may be required to clear cells in r3 and r5 that are unable to migrate.
In conclusion, our grafting experiments, together with existing studies in the chick (Farlie et al., 1999; Saldivar et al., 1996), suggest that signalling interactions between r3 and the surrounding environment restrict the generation and migration of cranial neural crest. To date few molecules that influence the pathfinding of cranial neural crest cells have been identified, but examples include the ephrins and their Eph receptors (Helbling et al., 1998; Smith et al., 1997) and evidence is emerging to indicate that collapsin-1/semaphorin-III might also be involved (Eickholt et al., 1999). Future analyses in mouse embryos will be aimed at identifying the molecular networks that interact to generate and pattern the migration pathways of cranial neural crest cells.
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ACKNOWLEDGMENTS |
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