Division of Molecular Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
* Present address: GlaxoSmithKline Biologicals, Rue de l'Institut 89, B-1330
Rixensart, Belgium
Present addresses: Department of Clinical Genetics, Erasmus University, Dr
Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
Author for correspondence (e-mail:
vpachni{at}nimr.mrc.ac.uk)
Accepted 28 August 2002
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SUMMARY |
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Key words: Neural crest, Cell migration, Enteric nervous system (ENS), Receptor tyrosine kinase (RTK), RET, Glial cell line-derived neurotrophic factor (GDNF), Mouse
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INTRODUCTION |
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Several genes have been identified that play crucial roles in the
development of the mammalian ENS. Among them is Ret, which encodes
for the receptor tyrosine kinase (RTK) RET
(Takahashi et al., 1988;
Takahashi and Cooper, 1987
).
Ret is induced in PENCCs when they first reach the dorsal aorta and
continues to be expressed in ENCCs throughout their rostrocaudal migration
within the foetal gut (Durbec et al.,
1996
; Pachnis et al.,
1993
; Tsuzuki et al.,
1995
). Mutations of RET in humans lead to absence of
enteric ganglia from the distal colon (congenital megacolon, Hirschsprung's
disease-HSCR) (Parisi and Kapur,
2000
), whereas mice homozygous for a targeted mutation of
Ret (Ret.k) have complete intestinal aganglionosis
(Durbec et al., 1996
;
Schuchardt et al., 1994
). RET
is the signalling component of multisubunit receptors for GDNF and the other
members of its family, such as neurturin, artemin and persephin
(Baloh et al., 2000
;
Saarma, 2000
). The interaction
between these signalling molecules and RET is mediated by
glycosyl-phosphatidyl-inositol (GPI)-linked cell-surface glycoproteins, called
GFR
1-4 (Airaksinen et al.,
1999
). A series of in vitro and in vivo studies has established a
crucial role for GDNF, GFR
1 and RET for the survival and
differentiation of enteric NC cells (Baloh
et al., 2000
; Taraviras and
Pachnis, 1999
). In addition, it has recently been reported that
GDNF can function as a chemoattractant of ENS progenitors in vitro
(Young et al., 2001
). Despite
these studies, the role of RET and its functional ligands in the migration of
ENS progenitors and the patterning of enteric neurone processes in vivo
remains unclear. We show that GDNF/RET signalling is required for the invasion
of the foregut by PENCCs and the subsequent migration of ENCCs along the
length of the bowel. These migratory processes are ultimately controlled by
the spatial and temporal regulation of GDNF expression in the mesenchyme of
the foetal gut.
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MATERIALS AND METHODS |
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Explant cultures
Explant cultures in collagen gel matrices have been described previously
(Natarajan et al., 1999).
Briefly,
1-2 mm long segments from small intestine were dissected from
appropriate stage embryos, placed in three-dimensional collagen gel matrices
in four-well NUNC plates (Tessier-Lavigne
et al., 1988
) and cultured in a defined medium (optiMEM; Life
Technologies UK) supplemented with L-glutamine (1 mM; Life Technologies UK)
and antibiotic mixture (100 U/ml; Life Technologies UK) in an atmosphere of 5%
CO2. For the co-culture experiments, the distance between the
intestinal segments and either COS-7 cells or caecum, was
300 µm.
Foetal gut conditioned medium (FGCM), GDNF and inhibitors were added to the
culture medium. At the end of the culture period, explants were stained for
Tuj1, PGP9.5 or RET.
To prepare FGCM, eight to ten guts were dissected from wild-type E13.5 mouse embryos (Parkes), washed twice with Ca2+- and Mg2+- free PBS and incubated with 1 mg/ml dispase/collagenase mixture (Sigma) for 15 minutes at room temperature. The tissue was washed twice with PBS, dissociated by pipetting and plated onto fibronectin-coated dishes (20 µg/ml) in OptiMEM. After 3 days, the cell culture medium was collected, centrifuged (84 g for 5 minutes) and filtered (0.2 µ filter). FGCM was used at a 1:1 dilution.
rGDNF (Promega) was used at 10 ng/ml. Phosphatidylinsolitol-3 Kinase [PI(3)K] inhibitor (LY294002, New England Biolabs) and MEK1 inhibitor (PD98059, New England Biolabs) were added to the co-cultures of small intestine explants with COS/GDNF cells at the indicated concentrations (Fig. 5B). To quantitate the migratory response of ENCCs to GDNF, explants were counterstained with DAPI and the nuclei present in the space between small intestine explants and COS cells were counted. Although we noticed a qualitatively similar response of enteric axons to GDNF, no attempt was made to quantitate this response.
|
The anti-GDNF blocking antibody (from R&D Systems) was used at a
concentration of 10 µg/ml, as described previously
(Hashino et al., 2001;
Worley et al., 2000
).
COS-GDNF cells were generated by transiently transfecting COS-7 cells (using Lipofectamine-Gibco BRL) with a mammalian expression vector (pcDNA3) in which a cDNA fragment encoding human GDNF (kindly provided by Dr M Saarma; Institute of Biotechnology, Helsinki) had been subcloned. COS (control) cells were generated by transfecting COS-7 cells with the empty vector. Small clumps of COS or COS/GDNF cells were formed by hanging drop cultures. More specifically, 24 hours after transfection, cells were collected using a cell scraper in 500 µl of complete medium. 30 µl droplets of the cell suspension were placed on the inside surface of the lid of a tissue culture plate and cultured overnight over complete medium.
In situ hybridisation
Whole-mount in situ hybridisation histochemistry was performed as
previously described (Durbec et al.,
1996). The riboprobe used for detecting Ret mRNA was
generated from pmcRet7 (Durbec et al.,
1996
). To isolate cDNA clones for mouse Gdnf, we screened
an E13.5 mouse embryo brain cDNA library under low stringency conditions using
as a probe a 700 bp cDNA fragment that corresponds to the coding region of
human GDNF. Several clones were identified, and a subset of them were
mapped and sequenced. The cRNA probed used for the present experiments was
derived from a 1.7 kb EcoRI fragment that corresponds to the 3'
untranslated region of the Gdnf mRNA (C. M.-G. and V. P.,
unpublished). The Sox10 cRNA was generated from a cDNA clone that was kindly
provided by Dr M. Wegner (Institute of Biochemistry, Erlangen).
Immunostaining
For immunostaining, explants were fixed for 2 hours in 4% paraformaldehyde
(made in 1xPBS) at 4°C. After washing twice in PBT (1xPBS+0.1%
Triton X-100) they were incubated overnight (at 4°C) with blocking
solution (PBT+1% BSA +0.15% glycine). Primary antibodies were diluted in
blocking solution as follows: Tuj1 (mouse; BABCO) 1:1000, PGP9.5 (rabbit;
Biogenesis) 1:400 and RET (goat; R&D Systems) 1:50. After washing several
times with blocking solution, secondary antibodies [anti-mouse Alexa
FluorTM (Molecular Probes) 1:500; anti-rabbit FITC-conjugated (from
Jackson Labs) 1:500; anti-goat FITC-conjugated (Jackson Labs) 1:500] were
added in blocking solution for 6 hours at room temperature. After washing for
three times with blocking solution explants were counterstained with DAPI and
analysed in a Zeiss Axiophot microscope.
To detect apoptotic cells, explants were fixed in 4% paraformaldehyde, cryoprotected, embedded in OCT and sectioned at 12 µM. Sections were then processed for TUNEL staining according to the manufacturer's instructions.
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RESULTS |
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Given the crucial role of GDNF and RET in the development of the mammalian
ENS (Taraviras and Pachnis,
1999), we wished to examine whether purified GDNF could influence
the migration of ENCCs. To test this, small intestine explants from E11.0-11.5
mouse embryos were cultured for 12-16 hours in the presence of recombinant (r)
GDNF (10 ng/ml) and analysed as described before. As shown in
Fig. 1A, parts e-h, the
presence of rGDNF in the medium induces (n=37/40) profuse but
non-directed migration of PGP9.5+ cells and Tuj1-labelled axons
away from the explant and into the collagen gel matrix. No PGP9.5- or
Tuj1-positive cells or axons were detected in explant cultures (n=12)
derived from homozygous Ret.k embryos in which vagal NC fail
to colonise the intestine (Fig. 1A, parts
i,j), confirming that the responding cells were of NC origin. In
addition to GDNF, neurturin (10 ng/ml) had a similar (albeit weaker) effect on
cell and axonal migration from foetal small intestine explants (data not
shown). Finally, netrin or endothelin 3 (ET3) failed to have any detectable
effect on explants cultured under similar conditions (D. N. and V. P,
unpublished; A. Barlow and V. P., unpublished).
To test whether GDNF can also induce a migratory response from pre-enteric ENS progenitors, the immediate postbranchial region containing RET-expressing PENCCs (boxed in Fig. 1B, part k), was dissected from E9.0-9.5 mouse embryos and cultured within collagen gel matrices in the absence or presence of rGDNF. In control medium, no RET+ cells left the postbranchial explant (n=4/4; Fig. 1B, part l). However, in the presence of 10 ng/ml of rGDNF, a large number of cells emigrated from the explant and invaded the collagen matrix (n=5/6; Fig. 1B, part m). Expression of Ret by the majority of responding cells indicates that they are of NC origin (inset in Fig. 1B, part m). Furthermore, no cell emigration was observed from explants derived from Ret.k homozygous embryos (data not shown). These findings suggest that GDNF promotes the migration of ENS progenitors prior to their entry into the foregut.
To distinguish between a non-directional motogenic and a chemoattractive effect of GDNF, we examined the response of ENCCs to a localised source of GDNF. For this, E11.0-11.5 small intestine explants were co-cultured (for 12-16 hours) with a clump of COS-7 cells transfected with either a mammalian expression vector encoding GDNF (COS/GDNF cells) or the empty vector (COS cells). COS cells failed to induce cellular or axonal migration from such explants (n=40/40; Fig. 1C, parts n,o). By contrast, COS/GDNF cells promoted extensive ENCC and axonal emigration from the side of the explants facing the source of GDNF (n=55/55; Fig. 1C, parts p,q). The majority of cells and axons converged towards the GDNF-expressing COS cells irrespective of their orientation at the exit point (Fig. 1C, parts p,q).
We have previously described the generation (by targeted mutagenesis) of
mice carrying a hypomorphic allele of Ret, which encodes only one of
the RET isoforms (miRet51)
(de Graaff et al., 2001).
Although newborn miRet51/miRet51 animals have
an apparently normal complement of enteric ganglia in the small intestine,
they fail to develop enteric neurones in the colon
(de Graaff et al., 2001
). To
examine whether the migratory response of ENCCs and enteric axons to GDNF
depends on the signalling properties of RET, co-culture experiments were
performed with small intestine explants derived from
miRet51 homozygous embryos and either COS or COS/GDNF
cells. Although the response of miRet51 explants was
qualitatively similar to that of their wild-type counterparts
(n=9/9), the number of cells and axons invading the collagen matrix
and their distance from the explant over the same culture period were reduced
(Fig. 1C, parts r,s; data not
shown). Our studies so far extend those of Young et al.
(Young et al., 2001
) and
suggest that GDNF can function as a chemoattractant that promotes the invasion
of the foregut by PENCCs, the rostrocaudal migration of ENCCs and the
outgrowth of enteric axons. Furthermore, our experiments indicate that these
processes are dependent on the signalling properties of the RET RTK.
Stage and region specific expression of Gdnf in mouse foetal
gut
To obtain further evidence for the role of GDNF as a chemoattractant of ENS
progenitors in vivo, we used in situ hybridisation to analyse the expression
of Gdnf in the gut of E8.5-13.5 mouse embryos. These stages encompass
the entire migratory phase of PENCCs and ENCCs, and the early stages of
enteric neurogenesis (Durbec et al.,
1996; Kapur et al.,
1992
; Pham et al.,
1991
). In E8.5-9.5 embryos, Gdnf mRNA was expressed at
high levels in the splachnic mesenchyme of the foregut (and particularly the
stomach) and in the pharyngeal pouches
(Fig. 2A,B,E). Twenty-four
hours later (E10.5), the expression of Gdnf in the stomach appeared
to be reduced, while a new well-defined domain of high expression was clearly
evident more posteriorly, in the prospective caecum
(Fig. 2; compare 2E with 2F).
At this stage, relatively low level of Gdnf expression was detected
throughout the small intestine. The appearance of the two domains of high
Gdnf expression (i.e. stomach and caecum) preceded the arrival of
RET-expressing NC cells at the corresponding regions. Thus, invasion of the
foregut by PENCCs (white arrow in Fig.
2C) is taking place at E9.0-9.5 (at a stage when Gdnf is
already expressed at high levels in the stomach anlage)
(Fig. 2; compare 2B with 2C),
while ENCCs arrive at the GDNF-expressing caecum at E11.0
(Fig. 2; compare 2F with 2G).
These findings are consistent with a role of GDNF as an attractant of ENS
progenitors. Furthermore, these data suggest that expression of Gdnf
in the developing gastrointestinal tract is controlled independently of the
invading ENS progenitors, an idea that is further supported by the normal
pattern of Gdnf expression in the foregut of RET-deficient mouse
embryos (Fig. 2D). During later
stages of embryogenesis (E11.5-13.5), the posterior boundary of the caecal
domain of Gdnf expression (arrow in
Fig. 2H) extended progressively
more caudally, until it spread throughout the entire length of the hindgut.
This caudal extension of Gdnf expression coincided with the posterior
migration of the front of ENCCs (arrow in
Fig. 2I). At these stages,
lower but easily detectable levels of GDNF mRNA were also present throughout
the precaecal gastrointestinal tract.
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Endogenous GDNF is likely to control the migration of ENS
progenitors
To test whether GDNF produced by foetal gut can influence the migration of
ENCCs, we co-cultured segments of proximal small intestine from E10.5 embryos
(expressing relatively low levels of Gdnf), with caeca dissected from
E11.0-11.5 mouse embryos (containing relatively high levels of GDNF). ENCCs
emigrating from the small intestine explants were identified at the end of the
culture period by immunostaining for RET and counterstaining for DAPI. In
small intestine explants cultured alone (n=26), a small but similar
number of NC-derived cells emigrated from either end of the explant
(Fig. 3A-D). However, in the
case of co-culture experiments (n=38), an increased number of
RET-expressing cells emigrated from the small intestine explant.
Interestingly, the majority of these cells emerged from the end of the small
intestine closest to the caecum and filled the space between the two explants
(Fig. 3E-H). To determine
whether endogenous GDNF is responsible for the effect of caecum on ENCCs,
similar co-culture experiments were performed with caeca isolated from mouse
embryos homozygous for a null allele of Gdnf (Gdnf-)
(Moore et al., 1996). As shown
in Fig. 3I-L, GDNF-deficient
caeca fail to induce cell emigration from wild-type small intestine explants
(n=6/6). Finally, significantly reduced cell emigration was also
observed from wild-type small intestine/caecum co-cultures in the presence of
blocking anti-GDNF antibodies (n=13/13;
Fig. 3M-P)
(Hashino et al., 2001
;
Worley et al., 2000
).
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Mutations of Ret affect the rate of migration of ENS
progenitors
Although reduced cell survival of PENCCs and ENCCs plays an important role
in the generation of intestinal aganglionosis in RET-deficient mice
(Taraviras et al., 1999), the
contribution of a migratory defect to this phenotype has not been examined
directly. To determine whether RET signalling is required in vivo for the
invasion of the foregut by PENCCs, we compared the distribution of early ENS
progenitors in the foregut of wild-type and RET-deficient
(Ret.k) E.9.5 mouse embryos using Sox10 as an
independent marker of early NC cells
(Britsch et al., 2001
;
Southard-Smith et al., 1998
).
In addition to Sox10-expressing cells present in sensory and autonomic ganglia
(arrows in Fig. 4A,B), a large
number of positive ENS progenitors were detected in the postbranchial region
(black arrowhead in Fig. 4A)
and the foregut mesenchyme (white arrowhead in
Fig. 4A) of wild-type embryos.
By contrast, no Sox10+ cells were detected within the foregut of
Ret.k embryos, despite the presence of PENCCs in the
postbranchial region (black arrowhead in
Fig. 4B) and the apparently
normal expression of Sox10 in all other regions of the PNS (compare
Fig. 4A with Fig. 4B).
|
We next compared the position of ENCCs within the gut of E10.5 wild-type
and miRet51 homozygous embryos
(de Graaff et al., 2001) using
in situ hybridisation with Sox10-specific riboprobes. In wild-type embryos,
the front of migrating ENS progenitors had reached the midgut loop (halway
between the caudal end of the pancreas and the caecum; black arrow in
Fig. 4C). However, in similar
stage miRet51 embryos, the front of migrating ENCCs was
located more anteriorly, just beyond the pancreatic anlage (black arrow in
Fig. 4D). In addition, we
observed that the total number of Sox10-expressing cells was reduced in
miRet51 homozygous embryos. Thus, normal RET signalling is
likely to be necessary for the initial invasion and subsequent rostrocaudal
migration of ENS progenitors within the gut of mouse embryos.
Mitogen activated protein (MAP) kinase and PI(3) kinase activation
are required for the GDNF-induced migratory response of ENCCs
Previous studies have identified a number of intracellular signalling
pathways that are activated upon binding of the GDNF/GFR-1 complex to
the extracellular domain of RET. Thus, activation of RET expressed by
established cells lines and primary cell cultures, promotes cell survival and
differentiation via activation of the MAP kinase and the PI(3) kinase pathways
(Besset et al., 2000
;
Chen et al., 2001
;
De Vita et al., 2000
;
Hayashi et al., 2000
;
van Weering and Bos, 1998
). To
determine the relative roles of these intracellular signalling systems in ENCC
and axonal migration in the gut during embryogenesis, small intestine explants
and COS/GDNF cells were co-cultured (within collagen gel matrices) in the
presence of PD98059 or LY294002, specific inhibitors of the MEK1 and PI(3)
kinase, respectively (Atwal et al.,
2000
). Both inhibitors reduced the number of ENCCs and axons
migrating towards the GDNF-producing COS cells
(Fig. 5A, parts a-f) and their
effect was additive (Fig. 5C).
The effect of the inhibitors is unlikely to result from elimination of ENCCs
as TUNEL staining of similar explants cultured in the presence of LY29002 (30
µM) or PD98059 (60 µM) failed to induce a significant change in the
number of apoptotic nuclei (Fig.
5B and data not shown). Interestingly, increasing concentrations
of the PI(3)K inhibitor LY294002 resulted in complete abrogation of the ENCC
response to GDNF (Fig. 5C). By
contrast, the response of ENCCs observed at low concentration (1 µM) of the
MEK1 inhibitor PD98059 (approx. 30% relative to control), remained essentially
unchanged even at 60-fold higher concentration
(Fig. 5C). These findings
suggest that activation of both the MAPK and PI(3)K signalling pathways in
response to GDNF signalling is required for the normal migration of ENCCs and
the correct patterning of enteric neurone projections. Furthermore, PI(3)K
appears to have a crucial function in ENCC migration that cannot be
compensated for by MAPK.
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DISCUSSION |
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Our analysis of Gdnf expression is consistent with (and indeed
extends) the studies of Young et al., who have previously reported high levels
of GDNF mRNA in the caecum of E11.5-13.5 mouse embryos
(Young et al., 2001). However,
these authors did not report expression of Gdnf in the foregut,
possibly because the earliest stage they analysed was E10.5. In addition,
these authors suggested that the intense GDNF signal observed in the caecum,
is likely to result from the increased thickness of this region of the
gastrointestinal tract. Although after a certain point of embryogenesis, the
mesenchymal walls of the caecum and (in particular) the stomach are thicker
relative to other parts of the gut, we believe that the stronger GDNF signal
observed in these regions is likely to reflect increased levels of
Gdnf expression. This is based on the observation that the stomach-
and caecum-specific upregulation of Gdnf is observed prior to these
regions acquiring distinct anatomical characteristics that distinguish them
from the rest of the gut (Fig.
2; D. N., C. M.-G., V. P. and E. d. G., unpublished). Indeed, high
levels of GDNF mRNA are detected in the precursors of the splachnic mesenchyme
of the stomach at a stage (E8.0-8.5) when they are still part of the lateral
plate mesoderm (data not shown). In addition, analysis of sections of the
foetal gut preparations shown in Fig.
2, indicates that mesenchymal cells in the stomach and caecum
regions express higher levels of GDNF mRNA (data not shown).
The temporal and spatial distribution of GDNF in the developing gut and its capacity to function as a chemoattractant could provide a mechanism for the ordered colonisation of the oesophagus, stomach and small intestine. However, it is unclear whether a similar mechanism is responsible for the colonisation of the postcaecal region of the gut; our studies so far have failed to identify additional regions of GDNF expression that could function as chemoattraction centres for the colonisation of the hindgut. Instead, it appears that during hindgut colonisation by ENS progenitors, the caecal domain of GDNF expression extends posteriorly alongside the front of migrating cells. This suggests a permissive, rather than an instructive role for GDNF in the postcaecal region of the gastrointestinal tract. It is therefore likely that the mechanisms underlying the colonisation of the hindgut by ENS progenitors appear to be distinct from those operating in the fore- and midgut.
Activation of RTKs by their cognate ligands is likely to represent a
general mechanism adopted by vertebrate embryos for the guidance of NC cells
to their final destination sites. In addition to the evidence presented here,
analysis of mice homozygous for a targeted mutation in neurofibromin
(Nf1) has suggested that activation of the KIT RTK by stem-cell
factor (SCF or mast cell growth factor-MGF) is required for the directed
migration of melanocyte precursors in the lateral migratory pathway
(Wehrle-Haller et al., 2001).
It is likely therefore, that expression of distinct RTKs by specific groups of
migrating NC cells and the complementary expression of their ligand(s) at the
target site, creates a `chemoattraction code' that is used to allocate
subpopulations of NC cells to various locations within vertebrate embryos.
Phenotypic analysis of mice homozygous for miRet51 has
shown that these animals have an apparently normal complement of enteric
ganglia in the foregut and small intestine but lack enteric neurones and glia
from the majority of the colon (de Graaff
et al., 2001). This situation mimics the phenotype of congenital
megacolon (HSCR) encountered in individuals with loss-of-function mutations of
RET (Parisi and Kapur,
2000
). The restriction of aganglionosis in the terminal part of
the gut could suggest that full activity of RET signalling is required only
during colonisation of the hindgut, and that hypomorphic alleles of
Ret do not affect the pattern of migration of ENCCs in the foregut
and midgut. However, our analysis of miRet51 homozygous
embryos (Fig. 1C, parts r,s;
Fig. 4C,D) has suggested that
both the rate of migration and the number of ENCCs is already significantly
reduced at E10.5 (when the midgut has only been partially colonised),
suggesting that normal levels of RET signalling are required throughout the
colonisation of the mammalian gut. In light of this, it is remarkable that
newborn miRet51 animals appear to have normal enteric
plexi in the foregut and small intestine, indicating a certain degree of
recovery at later stages of embryogenesis. However, such compensatory
mechanisms fail to restore a normal ENS in the colon of
miRet51 animals, providing further support for the idea
that the successful colonisation of the gut by ENS progenitors is determined
by the interaction between NC-intrinsic mechanisms and region-specific
mesenchymal signalling factors (de Graaff
et al., 2001
).
The normal patterning of enteric axonal projections is crucial for the
coordinated peristaltic activity of the gut and the rostrocaudal movement of
its contents (Gershon et al.,
1994). Thus, rostrally projecting excitatory cholinergic neurones
are responsible for contracting the musculature orally to a bolus, while
inhibitory NOS-containing neurones project caudally and relax the musculature
ahead of it (Brookes, 2001
). So
far, very little information is available on the mechanisms that control the
growth and polarity of enteric axons and the extent to which such mechanisms
are related to the directional migration of ENS progenitors within the foetal
gut. However, the ability of GDNF to chemoattract neuronal processes present
in the gut of E11.5 mouse embryos, suggests that at least a subpopulation of
early enteric neurones is likely to respond to signals that are similar to
those controlling the rostrocaudal migration of ENC cells. Supporting this
suggestion is the finding that the majority of axonal processes of the
early-born catecholaminergic neurones present in the small intestine of E11.5
mouse embryos project caudally - towards the high GDNF expressing caecum. To
what extent the polarity of enteric axons is controlled directly by GDNF or
via the effect of this molecule on the migration of ENC cells is currently
unclear.
Binding of GDNF to GFRalpha1 and RET results in the concerted activation of
the MAP kinase and the PI(3) kinase intracellular signalling pathways which
are key biological effectors of the receptor
(Besset et al., 2000;
Chen et al., 2001
;
De Vita et al., 2000
;
Hayashi et al., 2000
;
van Weering and Bos, 1998
).
Crucial for the activation of these signalling pathways is the recruitment of
adaptor and signalling proteins at a multidocking site forming at tyrosine
1062 (Y1062). In vitro and in vivo mutagenesis of Y1062
results in abrogation of the transforming activity of RET and failure of renal
and ENS development during embryogenesis
(Besset et al., 2000
;
Ishiguro et al., 1999
;
Segouffin-Cariou and Billaud,
2000
) (S. Bogni, E. d. G. and V. P., unpublished). Using specific
inhibitors of MAPK and PI(3)K, we observed that the normal response of
RET-expressing ENS progenitors and enteric axons to a localised source of GDNF
requires the activation of both biochemical pathways. The effect of these
inhibitors is unlikely to be related to their potential role on cell survival.
This suggestion is based on the relatively short duration of the assay (less
than 12 hours) and the failure to observe during this period apoptotic cell
death in explants cultured in the presence of inhibitors (D. N., C. M.-G., V.
P. and E. d. G., unpublished). Despite the requirement of both signalling
pathways for optimal response to GDNF, we find that activation of PI(3)K is
the most crucial signalling event for the migration of ENS progenitors, and
its role could not be compensated for by activation of other signalling
pathways. This is based on the observation that efficient inhibition of the
PI(3)K signalling pathway completely abolishes the response of ENCCs to GDNF,
while a significant degree of chemotactic response was maintained even under
conditions of efficient inhibition of the MAPK pathway. The mechanism by which
PI(3)K activation results in asymmetric cytoskeletal rearrangements and
migration are currently unknown. However, our findings are consistent with
previous reports demonstrating that activation of the PI(3)K signalling
pathway is necessary for lamellipodia formation by cultured cells as a
response to GDNF (van Weering and Bos,
1997
). Furthermore, our data are consistent with the essential
role of PI(3)K signalling in the directional response of growth cones to
chemotactic stimuli in vitro (Song and
Poo, 2001
).
Asymmetric expression of key guidance cues or the uneven activation of
receptors on the cell surface, are necessary for the generation of directional
cell migration. For example, in Drosophila melanogaster, homogeneous
activation of RTKs by ligand overexpression or by mutations that render these
receptors ligand independent and constitutively active, result in cell
migration defects (Duchek and Rorth,
2001; Duchek et al.,
2001
). In that respect, it is interesting that germline mutations
that lead to the constitutive activation of RET and the cancer syndrome
multiple endocrine neoplasia type 2A (MEN2A), sometimes (15% of the cases)
result in congenital megacolon (HSCR)
(Decker et al., 1998
;
Mulligan et al., 1994
).
Although in some cases, HSCR could be due to the reduced expression or
inefficient translocation of the mutant receptor to the cell membrane
(Takahashi, 2001
), our data
provide an alternative explanation for the aganglionic phenotype encountered
in a subset of MEN2A mutations. According to this view, the constitutive
activation of RET on the cell surface of ENCCs prevents them from detecting or
correctly interpreting the surrounding GDNF gradients, resulting occasionally
in inefficient cell migration. Indeed, given our findings and those of Young
et al. (Young et al., 2001
),
it is unclear why aganglionosis is not encountered more frequently in
individuals with MEN2A patients. A potential explanation for this discrepancy
is based on the capacity of GDNF to `activate' MEN2A RET expressed on the cell
surface (Mograbi et al., 2001
)
and thus generate asymmetric responses that are presumably sufficient for
directional cell migration.
Several studies have established that members of the GDNF family of
signalling molecules can promote survival and differentiation of various
neuroectodermal derivatives, including the progenitors of the ENS
(Baloh et al., 2000;
Taraviras and Pachnis, 1999
).
The effect of GDNF and RET signalling in cell and axonal migration in vitro
(Young et al., 2001
) (this
study) and in vivo (this study), adds to the spectrum of activities of
Ret during development of the mammalian nervous system. Understanding
the molecular mechanisms by which RET can regulate the activation of distinct
but overlapping developmental processes that are crucial for mammalian ENS
histogenesis, constitutes one of the main challenges in this field.
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ACKNOWLEDGMENTS |
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