1 Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK
2 Institut dEmbryologie Cellulaire et Moleculaire, College de France et CNRS, Nogent-sur-Marne, 94736, France
*Author for correspondence (e-mail: a.burns{at}ich.ucl.ac.uk)
Accepted 2 April 2002
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SUMMARY |
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Key words: Enteric nervous system, Quail, Chick, Neural crest cells, Gut, Neurons, Glia
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INTRODUCTION |
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In common with the remainder of the autonomic nervous system, the neurons and glia that constitute the enteric nervous system (ENS) are entirely derived from the NC (Le Douarin and Teillet, 1973; Yntema and Hammond, 1954
). The rhombencephalic (vagal) region of the NC, adjacent to somites 1-7, contributes the vast majority of ENS precursors along the entire length of the gut (Epstein et al., 1994
; Le Douarin and Teillet, 1973
). Upon leaving the NC, these vagal-derived precursors enter the foregut then migrate in a rostrocaudal direction, reaching the terminal hindgut of the chick at embryonic day (E) 8-8.5 (Burns and Le Douarin, 1998
; Le Douarin and Teillet, 1973
) and of the mouse at E14 (Kapur et al., 1992
; Young et al., 1998
). A second source of enteric precursors, the sacral NC, which is situated caudal to somite 28 in the chick and somite 24 in the mouse, contributes cells to the postumbilical gut only (Burns et al., 2000
; Burns and Le Douarin, 1998
; Le Douarin and Teillet, 1973
; Pomeranz and Gershon, 1990
; Pomeranz et al., 1991
; Serbedzija et al., 1991
). Although initial studies (Pomeranz and Gershon, 1990
; Pomeranz et al., 1991
; Serbedzija et al., 1991
) suggested that sacral-derived precursors entered the hindgut early in development, before the arrival of vagal NCC, our studies in the chick embryo have demonstrated that sacral NC-derived precursors only begin to enter the gut after the colorectum has been colonised by vagal cells (Burns and Le Douarin, 1998
). This finding suggested that in order to colonise the hindgut, sacral NCC may require an interaction with vagal NCC, or with factors or signalling molecules produced by them. However, we have subsequently reported that sacral NCC colonise the hindgut independently of vagal NCC-derived enteric precursors (Burns et al., 2000
), a finding that has been confirmed in the chick using hindgut/NC co-culture experiments (Hearn and Newgreen, 2000
), and in the mouse using Wnt1-lacZ transgene expression as an early marker of murine NCC (Kapur, 2000
). Together, these data demonstrate that the hindgut ENS is formed by NCC that follow complex pathways to and within the gut. A first wave of vagal NCC migrate rostrocaudally (Burns and Le Douarin, 1998
; Le Douarin and Teillet, 1973
; Young et al., 1998
), and later arriving sacral NCC invade the hindgut migrating in an opposing caudorostral direction (Burns et al., 2000
; Hearn and Newgreen, 2000
; Kapur, 2000
).
In addition to the information concerning the spatiotemporal pathways that NCC follow, the recent understanding of the signalling mechanisms involved in the guidance of enteric precursors to and within the gut has rapidly progressed. Major defects in the ENS have been documented following the inactivation or overexpression of murine genes not previously known to affect the gut. Such genes include those encoding the components of the RET/GFR1/GDNF, ECE-1/endothelin-3/endothelin-B and RET/GFR
2/neurturin signalling pathways, the homeobox gene Hoxa4 and homeobox-related gene Hox11l1, the transcription factors Sox10 and Phox2b (Pmx2b Mouse Genome Informatics) and the proneural gene Mash1 (Ascl1 Mouse Genome Informatics) (Cacalano et al.,, 1998
; Enomoto et al., 1998
; Gershon, 1998
; Gershon, 1999
; Manie et al., 2001
; Pattyn et al., 1999
; Taraviras and Pachnis, 1999
; Tennyson et al., 1998
). In mice, where some of these genes have been knocked out, a wide spectrum of enteric phenotypes can arise depending on the specific genes involved. Such phenotypes range from a complete absence of ENS cells along the entire gastrointestinal tract (Sox10/), to varying degrees of regional deficiencies where sections of the gut are aganglionic (Mash1/ and knockout of genes within the RET/GFR
1/GDNF and ECE-1/EDN3/EDNRB signalling pathways). Less obvious phenotypes include those where only specific neuronal subpopulations are affected (neurturin-null and Gfra2/), and others where the numbers of neurons is actually increased within enteric ganglia (Hox11l1/) (reviewed by Young and Newgreen, 2001a
).
Many of the genes shown to affect ENS development in mice have also been implicated in humans with ENS disorders. For example, aganglionic megacolon or Hirschsprungs disease (HSCR), is characterised by the regional absence of enteric ganglion cells within the rectum and in a variable length of the colon (Kapur, 1999; Robertson et al., 1997
; Wartiovaara et al., 1998
). Susceptibility genes, including RET, GDNF, EDN3 and EDNRB have been documented in individuals with HSCR, but the condition is genetically complex, with multiple modes of inheritance, incomplete penetrance and variable expressivity known to exist (Kapur, 1999
; Wartiovaara et al., 1998
). As a result, the length of aganglionic bowel can vary, with short or long segment, or even total gut aganglionosis having been described (Nemeth et al., 2001
; Shimotake et al., 2001
).
We have recently studied an in ovo hypoganglionic hindgut model in the chick embryo by ablating part of the vagal NC so that insufficient NCC are available to colonise the entire length of the gut. After such ablations, the post-umbilical bowel is free from enteric plexuses although small, isolated ganglia derived from the sacral crest do occur (Burns et al., 2000). We decided to use this developmental model to investigate the ability of vagal NCC to colonise the hindgut when grafted into the sacral region of the neuraxis. The rationale for this study is based on previous findings that demonstrate the strongly invasive nature of vagal NCC for the gut. Thus, heterotopic vagal NC grafting and backtransplantation experiments have shown that vagal-derived ENS precursors were capable of colonising the gut either when transplanted to another region of the neuraxis (Le Douarin and Teillet, 1974
), or when segments of gut already colonised by vagal NCC were backtransplanted into younger embryos (Rothman et al., 1993
). By contrast, Erickson and Goins (Erickson and Goins, 2000
) recently proposed that sacral NCC have no special migratory properties that allow them to reach the gut, and it is the environment at the sacral level that is sufficient to allow NCC from any other axial levels to enter the gut mesenchyme. In this study we show that vagal NCC, transplanted into the sacral region, have the potential to invade the hindgut in sufficient numbers, and differentiate appropriately to form enteric plexuses and rescue an experimentally generated hypoganglionic hindgut phenotype in the chick embryo.
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MATERIALS AND METHODS |
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Microsurgical procedures: vagal neural crest ablation and heterotopic neural crest grafts
Access to the embryo was gained by cutting a window in the shell, then in order to facilitate visualisation of embryonic tissues, Indian ink, diluted 1:1 in phosphate-buffered saline (PBS), was injected into the sub-blastodermic cavity. The vitelline membrane was cut back to expose the embryo then the neural tube, including the NC, situated between somites 3-6 in 8- to 12-somite stage (ss) chick embryos, was excised using a fine microscalpel, as previously described (Burns et al., 2000) and as shown in Fig. 1.
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In a second series of grafts, following vagal NC ablation, the sacral crest was transplanted from 25 ss quail embryos into the vacant vagal region of the chick host embryos. In order to assess the ability of transplanted vagal NCC to migrate in a caudorostral direction, while normal vagal NCC were migrating in the opposing rostrocaudal direction, vagal-to-sacral transplantations were also performed without first ablating the vagal NC in the recipient embryo.
Immunohistochemistry
Intact embryos or dissected gastrointestinal tracts were fixed in Carnoys fluid for 10-30 minutes, embedded in paraffin wax and sectioned at a thickness of 7.5 µm. After rehydration and rinsing in PBS, sections were placed in 10% serum in PBS for 45 minutes, then incubated overnight at 4°C with primary antibody. In this study, antibodies to QCPN (Developmental Studies Hybridoma Bank; mouse IgG1, culture supernatant), ANNA-1 (Altermatt et al., 1991) (human IgG, diluted 1:1000 in PBS), GFAP (Dako; rabbit IgG, diluted 1:200) and RET (IBL, Japan; rabbit IgG, diluted 1:50) were used for immunostaining. Tissues were extensively rinsed in PBS prior to appropriate secondary antibody labelling (all Southern Biotechnology), which was performed for 1 hour at room temperature. Double-immunostaining with QCPN and ANNA-1 was performed sequentially on wax sections as previously described (Burns and Le Douarin, 1998
). Double-labelling with RET and QCPN was sequentially performed on paraformaldehyde-fixed cryostat sections, prepared and treated as described (Burns and Le Douarin, 1998
), using alkaline phosphatase (Vector) and DAB (Sigma) to identify RET- and QCPN-labelled cells respectively.
NADPH-diaphorase whole-mount staining
At E16, dissected segments of gut were cut open adjacent to the mesentery, pinned flat and fixed in 4% paraformaldehyde in PBS for 12 hours. After rinsing in PBS, staining for NADPH-diaphorase activity was performed by incubation in PBS (pH 7.4) containing 1 mg/ml ß-NADPH, 0.5 mg/ml nitroblue tetrazolium and 0.05% Triton-X-100 (all Sigma) for 30-60 minutes at 37°C. The NADPH-diaphorase reaction product appeared as a dark blue granular deposit and the reaction was terminated by immersing the tissues in cold PBS.
Labelled tissues and sections were photographed using Fuji Provia 100 ASA or 400 ASA colour slide film with a Nikon microphot photomicroscope. The photographic slides were then scanned using a Nikon Coolscan III 35 mm digital scanner and figures prepared using Adobe Photoshop 6 software.
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RESULTS |
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Hindgut colonisation by vagal NCC transplanted to the sacral level
Embryos were first examined at E3, 12 hours after transplantation of the vagal neural tube into the sacral region of the neuraxis. At this stage, NCC were present lateral to the neural tube underneath and within the ectoderm (Fig. 2A). Dorsoventral migration also occurred between the ectoderm and paraxial mesoderm, with transplanted cells migrating to the primitive gut wall where association with splanchnopleural mesenchyme occurred (Fig. 2A,B). The presence of transplanted vagal NCC within the gut at this early stage of development suggested that these cells commenced migration away from the neural tube immediately, or very soon after grafting to the new level of the neuraxis.
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At E10, the stage at which sacral NCC normally begin to colonise the hindgut in large numbers, transplanted vagal-derived cells were grouped on either side of the circular muscle layers in the colorectum (Fig. 5A,B) and appeared to comprise the vast majority of cells within the myenteric and submucosal plexuses (Fig. 5B). Transplanted vagal cells were also evident within the post-umbilical intestine, where the majority occurred external to the circular muscle layers within myenteric ganglia (Fig. 5C). Occasional vagal cells were also present within the pre-umbilical intestine (not shown). In control animals at E10, few sacral NC-derived cells were evident within the myenteric and submucosal plexus regions of the colorectum (Fig. 5A',B'), and none were observed within the gut wall of the post-umbilical intestine (Fig. 5C'). Again, at this stage of development, in transverse sections the nerve of Remak appeared to be much finer in diameter in vagal NC transplanted animals than in non-operated or sacral NC grafted embryos.
Neuronal fate of transplanted vagal NCC
The anatomical locations of vagal-derived cells within the hindgut, i.e. on either side of the circular muscle layer in plexus-like groupings (Fig. 6A), strongly suggested a neuronal fate for these NCC. Using the pan-neuronal marker ANNA-1 (Altermatt et al., 1991), enteric neurons were identified within the hindgut of vagal NC transplanted embryos as immunopositive groups of cells situated on either side of the circular muscle layer (Fig. 6B). When QCPN/ANNA-1 double antibody-labelled ganglia were examined in the same animals, all neurons were found to carry the QCPN-positive quail nucleus (Fig. 6C-C'') indicating that neurons within the colorectum were derived from vagal NCC transplanted into the sacral neuraxis.
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DISCUSSION |
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Transplanted vagal NCC colonise the hindgut more rapidly than sacral NCC
Following heterotopic grafting of vagal NC into the sacral region of the neuraxis, we found that transplanted vagal NCC immediately began to migrate away from the neural tube along characteristic pathways. Transplanted cells were evident in a superficial lateral pathway, underneath and within the ectoderm, and within a dorsoventral pathway leading to the developing gut. These pathways are similar to those previously reported for migrating vagal NCC in the more rostral region of the embryo (Burns and Le Douarin, 1998). Although the majority of vagal-derived cells appeared within characteristic pathways, occasional transplanted cells were also observed in ectopic locations, such as the mesonephros. Although the fate of these cells was not determined in this investigation, in experiments where quail vagal NC was grafted to the adrenomedullary region of the chick embryo (Le Douarin and Teillet, 1974
), these authors subsequently documented connective cells and cartilage within the mesonephritic tissue as heterotopic vagal NC derivatives. A similar fate is likely for the transplanted vagal NCC observed within the mesonephros in our current study.
In contrast to the highly invasive transplanted vagal NCC that enter the developing gut mesenchyme in large numbers almost immediately, sacral NCC do not initially enter the gut, but accumulate in the region adjacent to the gut wall where they form the nerve of Remak from E4. We have previously shown that sacral NCC do not begin to enter the gut wall until at least 4 days later at E8, when the hindgut is already colonised by vagal NCC (Burns and Le Douarin, 1998). The mode of entry of sacral cells into the gut is via axons derived from the nerve of Remak that project to the outer layers of the gut wall, in the region corresponding to the presumptive myenteric plexus (Burns and Le Douarin, 1998
). The study of Shepherd and Raper (Shepherd and Raper, 1999
) demonstrated that prior to this stage, at E6, the secreted glycoprotein collapsin-1, which belongs to the semaphorin family of molecules (Luo et al., 1993
) and acts as an axon repellent (Behar et al., 1996
; Messersmith et al., 1995
; Puschel et al., 1995
), is expressed throughout the rectal wall. However, at E8, collapsin-1 expression retreats from the outer muscle layers to the inner submucosal and mucosal regions allowing axons to project from the nerve of Remak into the gut, which in turn facilitates entry of sacral NCC into the hindgut. Although it is not clear whether collapsin-1 affects the migration of sacral NCC directly, as it affects the axons along which they migrate to gain entry to the gut, collapsin-1 has been reported to be a repulsive signal for NCC migrating in both the hindbrain and trunk regions (Eickholt et al., 1999
). Furthermore, these authors also reported that a collapsin-1 receptor, neuropilin-1, is expressed by migrating NCC in these regions, further supporting the functional role of this molecule for NCC patterning. Although this evidence is convincing for these cell populations, it appears that collapsin-1 does not play a role in patterning vagal NCC within the gut. Our previous study has shown that the rectum is colonised by vagal NCC during E7.5-8.5, and that the primary migration pathway for these cells is within the submucosal region of the hindgut (Burns and Le Douarin, 1998
). According to the findings of Shepherd and Raper (Shepherd and Raper, 1999
), this is the precise period of development when collapsin-1 expression becomes restricted to the sub-mucosal region of the hindgut. Therefore it seems highly unlikely that collapsin-1, in its function role as a repulsive signal for NCC (Eickholt et al., 1999
), affects the migration of vagal NCC in this region of the embryo, as these cells are undergoing extensive migration in the specific gut region where collapsin-1 is highly expressed.
In terms of ENS development in the chick, it therefore appears that sacral NCC initially form the nerve of Remak beginning at E4, and projections from this nerve extend into the gut when collapsin-1 expression retreats from the outer muscle layers at E8. Sacral NCC then migrate along these penetrating nerve fibres into the gut and subsequently contribute to the enteric neuronal and glial populations. By contrast, vagal NCC appear to be inherently much more invasive. When transplanted to the sacral region of the neuraxis, these cells, which are unaffected by collapsin-1, immediately enter the developing gut mesenchyme in large numbers, and while an instructive cue to form the nerve of Remak appears to exist, fewer cells are available to contribute to this nerve with the result that the Remak is much reduced in size.
Prespecification of vagal NCC as ENS precursors may permit transplanted vagal NCC to invade the gut from different locations
Vagal NCC appear to be inherently more invasive of the gut than sacral NCC, as these cells were capable of migrating to the gut when (1) transplanted to the sacral level of the neuraxis (results of this study), (2) transplanted to the thoracic level of the neuraxis (Le Douarin and Teillet, 1974) and (3) sections of embryonic gut containing vagal NCC were backtransplanted into younger embryos (Rothman et al., 1993
). It was initially suggested by Le Douarin and Teillet (Le Douarin and Teillet, 1974
) that some vagal NCC may be pre-specified as ENS precursors, thus enabling them to follow a chemotactic guidance cue to the gut. This theory of pre-specification for vagal NCC is supported by more recent findings which showed that premigratory vagal NCC express RET (Robertson and Mason, 1995
) and CCK-lacZ (Lay et al., 1999
), respectively. As both RET and CCK are subsequently expressed in enteric ganglia (in addition to other ganglia), it is possible that the NCC positive for RET and CCK prior to migration are those pre-specified to restricted lineages. The experiments carried out by Natarajan et al. (Natarajan et al., 1999
) have confirmed that RET-positive cells isolated from mouse bowel are multipotential ENS progenitors. When small numbers, or even individual RET-positive cells were microinjected into the stomach of aganglionic gastrointestinal tracts that were grown in organ culture for 7 days, enteric neurons and glia were subsequently found in the oesophagus, and the small and large intestine, thus highlighting the extensive migratory and proliferative capacities of RET-positive vagal NCC. In this current investigation we have demonstrated that a subpopulation of sacral NCC within the myenteric and submucosal ganglia of the chick hindgut is RET positive. As these cells are less invasive of the gut than are vagal NCC, as discussed above, it appears that other factors, not including RET, account for the difference in invasive capacity of vagal and sacral NCC. To date, it is unclear whether sacral NCC express CCK-lacZ.
In contrast to the evidence concerning ENS prespecification for vagal NCC, Erickson and Goins (Erickson and Goins, 2000) have demonstrated that sacral NCC have no special prespecification or migratory properties that allow them to migrate to the gut, as these cells failed to colonise the gut when heterotopically grafted to the thoracic level of the neuraxis in the chick. However, these authors suggested that the environment at the sacral level is sufficient to allow NCC from other axial levels to enter the mesentery and gut mesenchyme, since thoracic NCC transplanted to the sacral level do colonise the gut. We also performed experiments to test the ability of sacral NC to invade the gut when grafted to the vagal region of the neuraxis. After such grafts, sacral NCC colonised the gut and were evident within the presumptive myenteric and to a lesser extent, submucosal regions. However, colonisation was less extensive than for normal vagal NCC at similar stages of development, because at E8, a stage at which the entire length of the gut is colonised by vagal ENS precursors, transplanted sacral cells were not found within the hindgut. It is possible that transplanted sacral NCC may be able to colonise the gut as they are exposed to stronger chemotactic signals when grafted to the vagal level of the neuraxis, or that preferential pathways leading to the gut from the vagal region are more permissive for migration, as originally suggested by Le Douarin and Teillet (Le Douarin and Teillet, 1974
) following grafting of truncal crest to the vagal level. Therefore, although the migration pathways to the gut from the sacral level of the neuraxis appear to be sufficient to allow sacral (Burns and Le Douarin, 1998
), and even transplanted thoracic NCC to reach the gut (Erickson and Goins, 2000
), they do not appear to be as permissive as those at the vagal level where sacral NCC can colonise the gut in significant numbers.
Vagal NCC simultaneously migrate in opposing directions within the gut
We have demonstrated that vagal NCC transplanted to the sacral region of the neuraxis colonise the gut in a caudorostral direction, with the migration front of cells reaching the post-umbilical intestine by E4, the level of the umbilicus by E5.5 and the pre-umbilical intestine by E7. The most rostral region of gut populated by transplanted vagal NCC was found to be within the pre-umbilical intestine, midway between the umbilicus and stomach. This level was reached by E8.5, and even though later stages were examined, transplanted NCC were not observed in more rostral regions. This colonisation pattern is intriguing because in the course of their migration, transplanted vagal NCC encountered (in experiments where the vagal NC was not ablated) normal vagal NCC migrating in the opposing rostrocaudal direction, with the migration fronts of the two NCC populations crossing over and intermingling from E5.5 to E8. During this time period transplanted vagal NCC moved from the post-umbilical intestine to pre-umbilical intestine, while the converse was true of normal vagal NCC. However, as the cecal region and all of the colorectum was extensively colonised by transplanted vagal NCC as early as E4, migration of normal vagal NCC into these hindgut regions did not occur and careful examination of the enteric plexus revealed that virtually all cells were of graft origin (i.e. derived from transplanted vagal NCC).
The mechanisms that guide two populations of vagal NCC simultaneously in opposing directions in the gut is unclear, although a possible explanation may involve cell population pressure as a source of migratory drive, as proposed by Hearn et al. (Hearn et al., 1998) for vagal NCC grown in vitro. This theory suggests that population pressure would cause NCC to move away from their point of entry to the gut. Therefore normal vagal NCC that enter the foregut migrate rostrocaudally to the unpopulated regions, and transplanted vagal cells that enter the hindgut in large numbers are forced to move to less populated rostral regions. Some intermingling of these cells was found to occur in the intestine, but normal vagal NCC did not migrate to the hindgut, which was already colonised by transplanted NCC, while the rostral migration of transplanted vagal NCC was impeded when these cells encountered the normal vagal NCC that had extensively colonised the proximal intestine. However, the study by Natarajan et al. (Natarajan et al., 1999
) outlined above seemingly provides evidence countering the population pressure theory for gut colonisation. When these authors microinjected single RET-positive vagal-derived NCC into aneural mouse gut that was then grown in culture, individual cells were found to migrate relatively long distances from the point of entry. In these studies, cells were injected into the stomach region and the resulting migration was either rostrocaudally within the gut or caudorostrally into the oesophagus. It would be interesting, however, to inject isolated cells into the distal hindgut and map their subsequent migration from this alternative point of entry. In this current study, to test whether reducing the numbers of vagal NCC transplanted to the sacral region of the neuraxis affected colonisation of the gut, progressively smaller segments of vagal neural tube were used as donor grafts (not shown). Interestingly, even when the length of the grafted vagal neural tube was reduced by approximately 75%, similar spatiotemporal patterns of vagal NCC were observed within the hindgut, cecal region, and pre- and post-umbilical intestine. As the source of donor NCC was greatly reduced in these experiments, yet an apparently similar number of vagal cells were observed in the gut, at time points mirroring cells derived from full-length grafts, it appears that vagal NCC are not only highly invasive of the gut, but are capable of altering proliferation in order to form enteric ganglia. This is in contrast to sacral NCC, which in the absence of vagal NCC, colonise the hindgut but do not compensate for lack of vagal-derived ENS precursors (Burns et al., 2000
).
In addition to the population pressure theory for gut colonisation, there are also undoubtedly cell-cell signalling interactions that affect the migration of vagal NCC to and within the gut. Interactions may be between NCC and extracellular matrix components within the migration pathways, and/or diffusable chemoattractive molecules that originate in the gut and attract vagal (or sacral) NCC. This latter idea is supported by studies such as those of Rothman et al. (Rothman et al., 1993) mentioned above. In these investigations, when sections of gut already colonised by vagal NCC were backtransplanted into younger embryos, the vagal-derived ENS precursors left the bowel and entered into new host migration pathways. Similarly, in the study of Fontaine-Perus et al. (Fontaine-Perus et al., 1988
) when sensory ganglia containing cells of NC origin were either grafted into younger embryos, or cultured adjacent to segments of aneural gut, NC-derived cells migrated into the gut and became positioned within enteric ganglia. As the cells within the sensory ganglia had already reached their final migration sites prior to manipulation, the fact that these cells migrated out of the ganglia and colonised the gut provides strong evidence for the influence of chemotactic signals acting on vagal NCC. Although the precise nature of these signals is still unclear, recent work using organ-cultured gut and explants grown on collagen gel has demonstrated that GDNF, the ligand for the RET/GFR
1 receptor complex, is a chemoattractant for enteric neural cells that promotes directed axon outgrowth and migration of vagal NCC throughout the gut (Young et al., 2001b
). Although the mechanism by which GDNF could promote the rostrocaudal migration of vagal NCC is not clear, Young et al. (Young et al., 2001b
) surmised that GDNF protein levels could be higher in areas unoccupied by NCC which would act as GDNF sinks. NCC migration could then be influenced by chemoattraction towards these areas with higher levels of GDNF. Although weak GDNF expression has been described in the chick hindgut early in development (Homma et al., 2000
), it is unclear if such GDNF sinks exist in embryonic chick gut that could account for the caudorostral migration of transplanted vagal NCC described in this study. In addition to its novel role as a chemoattractant for ENS precursors, GDNF has previously been shown to be necessary for the proliferation and survival of ENS precursors (Hearn et al., 1998
; Heuckeroth et al., 1998
; Chalazonitis et al., 1998
), while EDN3 appears to modulate the effect of GDNF by inhibiting the differentiation of migrating ENS precursors (Hearn et al., 1998
; Wu et al., 1999
), thus ensuring that sufficient cells are available to colonise the entire length of the gut. Interestingly, in mutations affecting EDN3, either spontaneous (Bolande, 1975
; Jacobs-Cohen et al., 1987
; Kapur et al., 1993
) or targeted (Baynash et al., 1994
), only the distal hindgut is aganglionic, suggesting that ENS precursors differentiate, and thus stop migration, before they colonise the terminal bowel. Humans with Hirschsprungs disease also have been identified with EDN3 mutations and have a similar phenotype of aganglionic hindgut (Bidaud et al., 1997b
; Kusafuka and Puri, 1997
; Oue and Puri, 1999
; Kenny et al., 2000
).
In conclusion, we have shown that vagal and sacral NCC possess different invasive capacities of the gut. Vagal NCC, when transplanted to the sacral region of the neuraxis, immediately colonised the gut mesenchyme in large numbers, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses. We have also shown that RET, which is essential for ENS development, is expressed in vagal crest-derived ENS cells, sacral crest-derived cells that comprise the nerve of Remak and within a subpopulation of sacral NCC within hindgut enteric ganglia.
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ACKNOWLEDGMENTS |
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