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First published online 6 August 2008
doi: 10.1242/dev.025163
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Research Report |
1 Division of Molecular 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, MO
64110, USA.
* Author for correspondence (e-mail: vpachni{at}nimr.mrc.ac.uk)
Accepted 21 July 2008
SUMMARY
Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.
Key words: Enteric nervous system, Neural crest, Ret
INTRODUCTION
The enteric nervous system (ENS) of vertebrates is composed of a network of
interconnected ganglia that are embedded within the gut wall and control its
peristaltic activity, secretions and blood flow
(Furness, 2006
). ENS
progenitors are derived from a subpopulation of vagal neural crest cells that
emigrate from the post-otic hindbrain in mouse embryos at embryonic day (E)
8.5-8.75 and assemble transiently at the dorsal aorta. At E9.0-9.5, these
pre-enteric neural crest cells (NCCs) invade the foregut to become enteric
NCCs, which migrate in a rostrocaudal direction to colonise the entire
gastrointestinal tract (Burns,
2005; Durbec et al.,
1996; Le Douarin and Teillet,
1973). Many of the studies that defined the origin, migratory
pathways and fate of pre-enteric and enteric NCCs have been carried out in
avian embryos, owing to the ease of experimental manipulations such as
cell-lineage tracing and transplantation
(Le Douarin and Kalcheim,
1999; Yntema and Hammond,
1954). Insight into molecular pathways has come from genetic
studies in mice, which have identified several genes required for the
development of the ENS (Gershon,
1997; Heanue and Pachnis,
2007). One limitation of the mouse studies is that they rely
mostly on end-point phenotypic analysis because of the difficulty in
effectively monitoring or manipulating ENS progenitors in the dynamic stages
of their formation, migration and differentiation during embryogenesis.
One of the genes that is crucial for ENS development is Ret, which
encodes a receptor tyrosine kinase (RTK)
(Takahashi et al., 1988).
During mouse embryogenesis, Ret is first expressed in pre-enteric
NCCs assembling at the dorsal aorta and its expression is maintained
throughout the colonisation of the gut
(Durbec et al., 1996). Mice
homozygous for Ret deletion (Ret-/-) have
complete intestinal aganglionosis
(Schuchardt et al., 1994),
whereas partial loss-of-function mutations in this gene lead to colonic
aganglionosis (Asai et al.,
2006; de Graaff et al.,
2001; Uesaka et al.,
2008). Interestingly, hypomorphic mutations of RET are
responsible for 
50% of familial cases of Hirschsprung's disease (HSCR), a
congenital condition characterised by the absence of enteric ganglia from the
distal colon (Amiel et al.,
2008). By grafting wild-type ENS progenitors into aganglionic
Ret mutant intestine in culture, we have shown that the effect of
Ret deletion on enteric NCCs is cell-autonomous and that Ret controls
the response of individual cells to their microenvironment
(Natarajan et al., 1999).
To further explore the plasticity of enteric NCCs and the role of Ret in ENS development, we have followed the fate of genetically marked cells in cultured post-implantation mouse embryos and organotypic cultures of their gut. We demonstrate that like their endogenous counterparts, exogenous ENS progenitors grafted into the vagal NCC pathway of cultured embryos invade the foregut and colonise the entire intestine. Moreover, by transplanting wild-type ENS progenitors into the vagal NCC pathway of Ret-deficient embryos, we demonstrate that the earlier stages of gut colonisation by NCCs are controlled by Ret in a non-cell-autonomous manner. Our studies reveal novel complexities in the mechanisms of Ret function and provide an experimental paradigm for analysing the effects of mouse mutations on NCCs in general, and on the ENS in particular.
MATERIALS AND METHODS
Animals
Rosa26βgeo, Rosa26R-EYFP and
Ret-/- mice have been described previously
(Schuchardt et al., 1994;
Srinivas et al., 2001;
Zambrowicz et al., 1997).
Wild-type embryos were obtained from timed matings of CBA/ca females with
C57BL/10 males. E8.5 embryos were cultured as described previously
(Sturm and Tam, 1993).
Isolation of Ret+ cells from foetal mouse gut and grafting into cultured mouse embryos
Ret+ cells were isolated as described
(Lo and Anderson, 1995;
Natarajan et al., 1999).
Approximately 50 cells were grafted into cultured embryos using a
stereomicroscope and a micromanipulator. Cells were loaded individually into a
5 µm diameter needle (borosilicate glass tube, LASER) and delivered at both
sides of the embryo to the region between the neural tube and somites 2-4.
Analysis of the ENS of grafted embryos
Gut organ culture was performed as described
(Natarajan et al., 1999).
Immunostaining, X-Gal staining and in situ hybridisation on whole-mount
preparations and cryosections were performed as described
(Natarajan et al., 1999;
Wong et al., 2005).
|
To explore the plasticity of ENS progenitors in vivo, we adopted an ex
utero mouse embryo culture system that allows access to, and experimental
manipulation of, early NCCs. In this experimental paradigm, post-implantation
mouse embryos can be cultured for a short period with growth rate and
morphogenesis comparable to those of embryos developing in utero
(Sturm and Tam, 1993). For the
present experiments, E8.5 mouse embryos
(Fig. 1A) were cultured in
rolling vials for 3 days, in increasing concentrations of rat serum and oxygen
(Trainor et al., 1994). At the
end of the culture period (designated hereafter E8.5+3), the majority of
embryos (
90%) reached a stage comparable to E10.5
(Fig. 1B). We have previously
demonstrated that cranial NCCs delaminate normally from the dorsal neural tube
of cultured embryos and follow appropriate migratory routes to reach their
destination (Trainor and Tam,
1995). However, the migration of vagal NCCs, Ret
expression and the early stages of enteric neurogenesis have not been examined
in this experimental paradigm. Therefore, we carried out whole-mount in situ
hybridisation analysis of E8.5+3 embryos using a Ret-specific
riboprobe. At E10.5, Ret is expressed in defined subpopulations of
NCCs and their derivatives, including the migrating enteric NCCs and the
condensing autonomic and sensory ganglia
(Enomoto et al., 2001;
Pachnis et al., 1993).
Ret-expressing cells were observed in cranial sensory ganglia VII, IX
and X of E8.5+3 embryos in a pattern and distribution equivalent to those of
freshly dissected E10.5 embryos (Fig.
1C). Moreover, a large number of Ret+ cells were
observed in the foregut and midgut of cultured embryos. Consistent with the
requirement of Ret for ENS formation
(Schuchardt et al., 1994), ENS
progenitors were absent from the gut of E8.5+3 Ret-/-
embryos (data not shown). These experiments suggest that the processes
controlling gut colonisation by ENS progenitors in cultured mouse embryos are
similar to those for embryos developing in utero. Hence, mouse embryo culture
can be used to monitor and experimentally manipulate vagal NCCs at the early
stages of enteric neurogenesis ex utero.
Previous studies have shown that ENS progenitors from quail bowel
transplanted into the trunk NCC pathway of younger chick embryos can
contribute to the formation of peripheral nerves, sympathetic ganglia and
adrenals (Rothman et al.,
1990). Moreover, we have previously shown that enteric NCCs
isolated from E11.5 mouse embryos and transplanted isochronically into
embryonic gut in organotypic culture, colonised all segments of the organ and
differentiated into enteric neurons and glia
(Natarajan et al., 1999). We
aimed to extend these studies to earlier stages of development and explore the
capacity of intestinal ENS progenitors to re-enact the migratory behaviour and
developmental programme of pre-enteric vagal NCCs upon in vivo
transplantation. For this, Ret+ cells were isolated from the gut of
E11.5 embryos (using Ret-specific antibodies and FACS analysis)
(Natarajan et al., 1999) and
grafted heterotopically and heterochronically into the migratory pathway of
pre-enteric vagal NCCs. Pilot DiI-labelling experiments indicated that NCCs
emerging from the neural tube at the level of somites 2-4 efficiently colonise
the gut of cultured embryos (Fig.
2A). Therefore, Ret+ cells were generally
grafted at this level between the somitic mesenchyme and the neural tube
(Fig. 1A). In order to trace
the fate of grafted cells, Ret+ cells were isolated from
embryos carrying the Rosa26βgeo or
Rosa26R-EYFP alleles, which ubiquitously express the β-geo
reporter or the fluorescent protein YFP, respectively. Similar results were
obtained with the two reporter lines. Following the graft, 72% (31/43) of
transplanted embryos contained β-geo+ cells at the end of the
culture period. Interestingly, in the majority of positive embryos, donor
cells were restricted to the gastrointestinal tract
(Fig. 2B). Occasionally,
however, a small number of β-geo+ cells were also detected
near the pharyngeal pouch of the third branchial arch (data not shown). Two
groups of embryos could be distinguished based on the extent of colonisation
of the gastrointestinal tract by the grafted cells. In 66% (20/31) of positive
embryos, clusters of β-geo+ cells were found exclusively in
the foregut (oesophagus and stomach) (Fig.
2C). However, in 33% (11/31) of embryos, grafted cells were found
in both the foregut and midgut (Fig.
2D). In most embryos of this latter group, β-geo+
cells formed a relatively dense network, similar to that formed by intrinsic
NCCs at E10.5. In addition, the number of β-geo+ cells in this
group clearly exceeded that of the grafted population, suggesting that they
had undergone extensive proliferation. Together, these data show that
intestinal ENS progenitors retain the capacity to re-enter the foregut and
migrate rostrocaudally to colonise more-distal gut segments.
In mouse embryos, Ret is not expressed in the early vagal NCCs,
but is specifically induced in the subpopulation that reaches the dorsal aorta
and invades the foregut (Durbec et al.,
1996; Lo et al.,
1997). Induction of Ret expression appears to control the
response of prospective enteric NCCs to the chemoattractive activity of Gdnf,
which at this stage is expressed by the stomach anlage. Consistent with this
view, Ret is necessary for the chemoattraction of enteric NCCs by Gdnf in
vitro (Natarajan et al., 2002;
Young et al., 2001).
Furthermore, in mouse embryos homozygous for a hypomorphic mutation of
Ret, vagal NCCs delay their invasion of the foregut and accumulate
transiently at the dorsal aorta (D.N. and V.P., unpublished). Our data suggest
a model in which Ret plays a key role in `sorting' vagal NCC populations, as
expression of this RTK is sufficient to guide them into the gut.
Interestingly, exogenous Ret+ cells have occasionally been detected
in the third pharyngeal pouch (our unpublished observations), a region that
expresses relatively high levels of Gdnf. Although at present we cannot
exclude the possibility that apoptotic cell death eliminates Ret+
cells that happen to migrate into Gdnf-free embryonic regions, our findings
support the hypothesis that growth factors and their cognate tyrosine kinase
receptors serve as guidance systems for the colonisation of specific embryonic
sites by distinct subpopulations of NCCs
(Wehrle-Haller and Weston,
1997).
|
|
). We
reasoned, therefore, that a combination of embryo culture followed by
organotypic culture of the gut would allow us to explore the further migration
of grafted enteric Ret+ cells and assess their differentiation
capacity by extending the period of analysis. For this, the gut of E8.5+3
embryos grafted with genetically marked Ret+ cells was dissected
and placed in organ culture for a further 7 days. At the end of this period
(designated E8.5+3+7), the grafted cells and their progeny had colonised the
entire intestine (Fig. 3A,B)
and were arranged radially along with the intrinsic NCC derivatives
(Fig. 3C). Therefore, a
relatively small number of Ret+ enteric NCCs isolated from the gut
of E11.5 mouse embryos and heterochronically transplanted into the vagal
region of E8.5 embryos, are able to invade the foregut and migrate in a
rostrocaudal direction to colonise its entire length.
To examine the developmental potential of genetically marked
Ret+ cells (isolated from the intestine of E11.5
Rosa26R-EYFP embryos) in our experimental paradigm, cryosections of
E8.5+3+7 gut were immunostained for YFP (to identify the grafted cells) and
either TuJ1 (Tubb3 - Mouse Genome Informatics) (to identify neurons) or B-FABP
(Fabp7) (to identify glia) (Kurtz et al.,
1994). We observed significant co-localisation of YFP with these
markers, suggesting that grafted Ret+ cells were capable of
differentiating into both neurons and glial cells
(Fig. 3D,E). The close
association of YFP+ cells with the endogenous Tuj1+ and
B-FABP+ cells suggests that the transplanted cells and their
descendants are capable of homing to the appropriate gut layer, raising the
possibility that they can integrate into the intrinsic network of enteric
ganglia and establish functional contacts.
The ability of Ret+ intestinal cells grafted into the wall of
E11.5 Ret-deficient gut to colonise the entire organ
(Natarajan et al., 1999)
argues that the effect of the Ret-null mutation on ENS progenitors is
cell-autonomous and suggests that the intestine of mutant embryos can support
the formation of enteric ganglia by wild-type NCCs. However, these experiments
did not address the interaction of wild-type Ret+ cells with the
environment of Ret mutant embryos at earlier stages of ENS
development, particularly during the invasion of the foregut by nascent
Ret+ vagal NCCs emerging at the dorsal aorta. To address this,
Ret+ cells from the intestine of E11.5
Rosa26βgeo embryos were grafted into the vagal pathway
of E8.5 embryos resulting from intercrosses of Ret+/-
mice. At the end of the E8.5+3+7 period, cultured guts were analysed for the
presence of β-geo+ cells. In 85% (17/20) of
Ret+/- guts, we observed a significant number of
β-geo+ cells that had colonised the stomach and the entire
intestine (Fig. 4A). Although
exogenous β-geo+ cells were found in the foregut of both
Ret+/- and Ret-/- embryos at the end
of the 3-day culture period (Fig.
4C,D), in all cases of Ret-deficient E8.5+3+7 cultures (5/5),
grafted cells were restricted to the proximal foregut while the distal stomach
and the entire intestine were devoid of both intrinsic and extrinsic NCC
derivatives (Fig. 4B). These
findings suggest that although Ret+ cells can invade the foregut of
wild-type and Ret-deficient host embryos, in the case of the mutant embryos
they are unable to progress further to colonise the remainder of the
gastrointestinal tract.
|
), whereas transplantation into the vagal pathway
restricts the extrinsic cells to the proximal foregut (this study). Our
findings have revealed a previously unrecognised non-cell-autonomous effect of
the Ret- mutation on the invasion of the foregut by NCCs.
The mechanistic basis for the differential fates in the two experimental
paradigms has not been determined. Ret might, directly or indirectly, mediate
specific intercellular interactions among vagal NCCs that are necessary for
foregut invasion. Such interactions might require a minimal number of
Ret+ cells and therefore would not materialise in Ret
mutant embryos in which apoptotic cell death eliminates the majority of early
ENS progenitors (Taraviras et al.,
1999). The requirement of a minimal number of NCCs for the
efficient colonisation of the gut has also been suggested by recent studies on
avian embryos, which showed that when pre-migratory NCCs are reduced below a
critical threshold, they are restricted to the foregut
(Barlow et al., 2008;
Simpson et al., 2007). Our
present findings could also be explained by a model whereby Ret activity
within vagal NCCs induces specific changes to the foregut mesenchyme, which in
turn facilitates the invasion and rostrocaudal migration of enteric NCCs.
Consistent with this idea, it has been demonstrated previously that the
absence of enteric neurons and glia from the gut of Ret mutant
embryos induces characteristic changes in gene expression in the
non-NCC-derived mesenchyme and epithelial cells of the gut
(Heanue and Pachnis, 2006;
Vohra et al., 2006).
Irrespective of the mechanisms implicated, our studies reveal previously
unappreciated complexities in the means by which Ret signalling controls the
colonisation of mammalian gut by NCCs, and argue that the site of
transplantation and the number of wild-type ENS progenitors are critical
factors in our efforts to rescue the aganglionic phenotype of animal models of
congenital megacolon (HSCR). In summary, the experimental system described here allows us to monitor the ENS of wild-type embryos ex utero, from the earliest stages of vagal NCC formation to the complete colonisation of the gut. Furthermore, this system allows the generation of chimaeric embryos in which host and grafted cells are of the desired genotype. Such chimaeric embryos will enable us to explore further the interaction between NCCs and the gut microenvironment.
ACKNOWLEDGMENTS
This work was supported by the Medical Research Council (MRC, UK) and by a Telethon pre-doctoral fellowship to S.B. (438/b).
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