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First published online 19 April 2006
doi: 10.1242/dev.02375
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Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
¶ Author for correspondence (e-mail: vpachni{at}nimr.mrc.ac.uk)
Accepted 20 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mouse, SOX10, Endothelin 3
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Swenson, 2002). The familial
form of HSCR has been associated mainly with mutations in RET
(Brooks et al., 2005;
Chakravarti, 2001), a locus
encoding a tyrosine kinase receptor for members of the GDNF family of ligands
(Baloh et al., 2000;
Saarma, 2000). However, other
genes, such as SOX10 and EDNRB, have also been implicated in
the pathogenesis of congenital megacolon
(Amiel and Lyonnet, 2001;
Brooks et al., 2005). Thus,
haploinsufficiency for SOX10, which encodes an SRY-related
HMG transcription factor (Wegner,
1999), is responsible for a significant proportion of cases of
HSCR, primarily those associated with Waardenburg syndrome
(Waardenburg-Hirschsprung's disease or Shah-Waardenburg syndrome), which, in
addition to colonic aganglionosis, is characterised by deafness and
hypopigmentation (Inoue et al.,
2004; Pingault et al.,
1998; Read and Newton,
1997). Consistent with the phenotype of SOX10 mutations
in humans, hemizygocity of this locus in mice is associated with aganglionosis
of the terminal colon and white spotting, while animals homozygous for null
mutations of Sox10 have severe deficits of the entire peripheral
nervous system (PNS) (Britsch et al.,
2001; Herbarth et al.,
1998; Kapur, 1999;
Southard-Smith et al., 1998).
The cellular and molecular mechanisms underlying such deficits are not clear,
but studies on mouse embryos heterozygous for a targeted deletion of
Sox10 (Sox10LacZ) have suggested that decreased
levels of SOX10 result in a smaller ENS progenitor pool
(Britsch et al., 2001;
Paratore et al., 2002). By
contrast, SOX10 overexpression in neural crest stem cells (NCSCs) allows them
to maintain their neurogenic and gliogenic potential
(Kim et al., 2003).
EDNRB, which encodes a G protein-coupled receptor for endothelin 3
(EDN3), is also necessary for normal development of the ENS and is responsible
for 
5% of cases of familial HSCR
(Amiel and Lyonnet, 2001;
Chakravarti, 2001;
Edery et al., 1996;
Hofstra et al., 1996;
McCallion and Chakravarti,
2001; Puffenberger et al.,
1994). The mechanisms by which the EDN3/EDNRB signalling pathway
regulates enteric neurogenesis are currently unclear. Studies with mixed ENS
cultures have shown that EDN3 inhibits neuronal differentiation
(Hearn et al., 1998;
Wu et al., 1999) and has a
permissive effect on the proliferation of ENS progenitors
(Barlow et al., 2003). However,
other experiments have suggested that EDN3 signalling impairs the self-renewal
of ENS progenitors by promoting their differentiation into myofibroblasts
(Kruger et al., 2003).
Cultures of multilineage neural crest cell progenitors isolated from avian
and mammalian embryos have served as valuable in vitro systems to study the
role of extracellular signals and intracellular transcription factors in cell
commitment and differentiation in the PNS
(Cohen and Konigsberg, 1975;
Dupin and Le Douarin, 1995;
Dupin et al., 2001;
Ito et al., 1993;
Morrison et al., 2000;
Sieber-Blum and Cohen, 1980;
Stemple and Anderson, 1992).
Multilineage progenitors of the ENS have also been isolated from the gut of
rat and mouse embryos. Thus, NCSCs have been identified in the gut of rat
embryos and postnatal animals using immunostaining for cell surface markers
and fluorescence-activated cell sorting (FACS)
(Bixby et al., 2002;
Kruger et al., 2002). Enteric
NCSCs generate colonies containing neurons, glia and myofibroblasts, while
they colonise various PNS ganglia upon grafting into the migratory path of
neural crest cells in avian embryos
(Kruger et al., 2002). More
recently, we have reported the isolation and characterisation of multilineage
ENS progenitors from cultures of dissociated embryonic and postnatal mouse
gut. Under appropriate conditions, such cultures generate neurosphere like
bodies (NLBs), which are composed of differentiated cells as well as
multipotential EPCs (ENS progenitor cells), which upon re-plating generate
colonies containing enteric neurons and glia
(Bondurand et al., 2003). EPCs
represent pre-enteric vagal neural crest cells expressing Sox10 and
Phox2b but lack early neurogenic markers, such as MASH1 and RET.
However, upon commitment to the neurogenic lineage, EPC progeny induce MASH1
and RET and differentiate into neurons which downregulate Sox10 and
Mash1 (Ascl1 - Mouse Genome Informatics) but further upregulate
Ret (Bondurand et al.,
2003). Glial cells can also be identified in EPC-derived colonies
subsequent to neuronal differentiation
(Bondurand et al., 2003).
Here, we have used clonogenic cultures of EPCs to examine the role of SOX10 and endothelin signalling in the commitment and differentiation of multilineage ENS progenitors. In addition, we have analysed the ENS progenitor pool size, proliferation and differentiation in wild-type and EDN3-deficient mouse embryos. Our experiments indicate that SOX10 and EDN3 control the commitment and differentiation of ENS progenitors into enteric neurons and glia.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
Ret51/51 (de Graaff et
al., 2001) and EdnrbLacZ/LacZ
(Lee et al., 2003) embryos.
Tissue from wild-type littermates was used as control. Mice carrying the
Edn3ls allele (Lyon, 1996) were obtained from the Medical
Research Council Mammalian Genetics Unit (Harwell, UK) and backcrossed to
C57Bl/6 mice for at least six generations. Heterozygous and homozygous
Edn3ls animals were identified by PCR
(Rice et al., 2000). The
generation of TgWnt1Cre and R26YFPStop
animals have been described previously
(Danielian et al., 1998;
Srinivas et al., 2001). The
day of vaginal plug detection was considered to be E0.5.
Dissociated gut cultures and generation of EPCs
Generation of NLBs from dissociated gut cultures, isolation of EPCs and
clonogenic cultures of EPCs have been described previously
(Bondurand et al., 2003).
Endothelin 3 (EDN3) (Calbiochem) was added to the culture medium at either 100
nM (dissociated gut cultures for NLB formation) or 10 nM (clonal cultures of
FACS isolated EPCs). BQ788 (inhibitor of EDNRB signalling; Calbiochem) was
used at a concentration of 100 nM. Cultures were re-fed every 2 days.
Acute cultures of dissociated intestine
Intestines were dissected from E11.5-E12.5 mouse embryos and incubated with
dispase/collagenase (0.5 mg/ml in Phosphate Buffered Saline-1xPBS;
Roche) for 3 minutes at room temperature. Tissue was washed three times in
1xPBS, dissociated into single cells by pipetting and plated onto
fibronectin-coated (20 µg/ml) SONIC-SEAL slide wells (VWR) in optiMEM (Life
Technologies) supplemented with L-glutamine (1 mM; Life Technologies) and
penicillin/streptomycin antibiotic mixture (Life Technologies). Cultures were
maintained for up to 3 hours prior to fixation in an atmosphere of 5%
CO2.
Retrovirus generation and infection
Details for the retrovirus generation and transduction have been reported
previously (Bondurand et al.,
2003). The SOX10-GFP retroviral transgene was generated by
subcloning the human SOX10 cDNA (Bondurand
et al., 2000) into the EcoRI and XhoI cloning
sites of the pMX-IRES-GFP vector
(Kitamura, 1998).
BrdU incorporation
BrdU (Sigma) stock solution (10 mg/ml) was made in 0.9% sodium chloride
(Sigma). This solution was injected intraperitoneally (10 µl/g of animal
weight) into pregnant mice and embryos were harvested 1 hour after the BrdU
injection. Whole guts were dissected, fixed for 2 hours in 4% paraformaldehyde
(PFA; in 1xPBS) at 4°C and immunostained as described below.
Alternatively, embryonic intestines from BrdU-injected pregnant animals were
dissociated into single-cell suspension, plated and maintained in optiMEM for
2-3 hours (acute cultures).
Immunostaining
Clonal cultures of EPCs were fixed in 4% PFA for 10 minutes at room
temperature. After washing twice in PBS + 0.1% Triton X-100 (PBT), cells were
incubated with blocking solution (PBT + 1%BSA + 0.15% glycine) at 4°C
(overnight) or at room temperature (for 2-3 hours). Primary antibodies were
diluted in blocking solution as follows: TuJ1 (mouse; BABCO, UK), 1:1000;
GFP/YFP (mouse or Rabbit; Molecular Probes), 1:1000; RET (rabbit;
Immuno-Biological Labs, Japan), 1:50; GFAP (rabbit; DAKO, USA), 1:400; B-FABP
(rabbit; kind gift from Thomas Muller), 1:1000; SOX10 (mouse; kindly provided
by Dr David Anderson), 1:10; SOX10 (rabbit; Chemicon), 1:200; MASH1 (mouse;
kindly provided by Dr D. Anderson), 1:1; SMA (mouse clone 1A4; Sigma), 1:500;
and MITF (mouse; Stratech), 1:200. Cultures were incubated with primary
antibodies at room temperature (5-6 hours) or at 4°C (overnight). After
several washes with PBT, secondary antibodies were added in blocking solution
for 2-4 hours at room temperature at the following dilutions: anti-mouse
FITC-conjugated (Jackson Labs), 1:500; anti-rabbit FITC-conjugated (Jackson
Labs), 1:500; anti-mouse AlexaFluor (Molecular Probes), 1:500; anti rabbit
AlexaFluor (Molecular Probes), 1:500.
For immunostaining of acute cultures of intestines from embryos of BrdU-injected pregnant females, cells were fixed for 10 minutes in 4% PFA at 4°C, washed three times in PBT (5 minutes at room temperature) and incubated in PBS + 10% heat inactivated sheep serum (HISS) (overnight at 4°C). Antibodies for SOX10 and GFP were used as above. Following incubation with antibodies, cultures were washed three times (5 minutes each at room temperature) in PBT, post-fixed in 4% PFA for 10 minutes and incubated in 2 M HCl at 37°C for 30 minutes. HCl was removed using 0.1 M Borate Buffer (three washes, 10 minutes each). Incubation with BrdU antibody (rat; Oxford Biotechnology, 1:20) was for 5 hours at room temperature in PBT + 10% HISS and secondary antibody was anti-rat Alexa Fluor 568 (Molecular Probes, 1:500), which was added for 5 hours at room temperature followed by three 1-hour washes in PBT. The cultures were then mounted in Vectashield.
For whole-mount immunostaining, guts were fixed in 4% PFA at 4°C for 2 hours, washed three times (1 hour each) in PBT at room temperature and placed into PBT containing 10% heat-inactivated sheep serum (HISS) at room temperature for 5 hours. The explants were then incubated overnight at 4°C with antibodies for GFP (rabbit from Molecular Probes; diluted 1:1000 in PBT + 10% HISS) or TuJ1 (mouse from BABCO, UK; diluted 1:1000 in PBT + 10% HISS). Following antibody incubation, explants were rinsed three times in PBT (1 hour each) and incubated with the secondary antibody in PBT for 5 hours at room temperature (anti-rabbit Alexa Fluor 568; Molecular Probes, 1:500 or anti-mouse FITC-conjugated; Jackson Labs, 1:500). For BrdU immunostaining, preparations were then washed three times in PBT, post-fixed in 4% PFA and washed again with PBT (three times for 1 hour each). Following these washes, explants were incubated with 2 M HCl for 30 minutes at 37°C and then treated with 0.1 M borate buffer (three times for 10 minutes at room temperature). Incubation with anti-BrdU antibody (rat; Oxford Biotechnology, 1:20) was overnight at 4°C, while incubation with secondary antibody (anti-rat Alexa Fluor 568; Molecular Probes, 1:500) was for 5 hours at room temperature. Preparations were mounted using Vectashield (Vector Laboratories) or Vectashield containing DAPI.
Differentiation at the migratory wavefront was analysed using an Axiophot Zeiss epifluorescence microscope. The number of differentiated cells, as identified by TuJ1 expression, was determined at the migration wave-front using two different methods. First, the 30 more advanced YFP+ cells at the front of migration in each gut were identified and then scored for TuJ1 immunostaining. Second, 50 single YFP+ cells present within the migration wave-front of each gut were counted and their double immunoreactivlty for TuJ1 was determined. Both methods gave identical results presented in Fig. 7. Images were analysed using Metamorph software package (Universal Imaging). For the proliferation analysis at the front of migration, YFP and BrdU immunostained preparations were examined using a Bio-Rad confocal microscope. Optical sections of 0.5 µm were photographed using a 60x lens and individual YFP+ cells present within the migration wave-front were identified and scored for immunoreactivity for BrdU. A minimum of 50 cells were counted within each gut preparation. All figures were compiled using Adobe Photoshop 7 software. Statistical analysis was carried out using a t-test. Differences were considered to be significant if P value was less that 0.05.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
McKeown et al., 2005). To
examine the role of SOX10 in the differentiation of ENS progenitors, we used
retroviral vectors to introduce either GFP or SOX10-IRES-GFP (SOX10-GFP)
transgenes into clonogenic cultures of E11.5 EPCs
(Bondurand et al., 2003) and
determined the percentage of neurons and glial cells present in 5, 7 or 10
day-old colonies. We observed that in SOX10-GFP-expressing colonies, the
percentage of neurons was significantly reduced relative to GFP controls
(Fig. 1A-C). Despite this
reduction, MASH1 and RET immunostaining was detected in a similar number of
cells in GFP and SOX10-GFP colonies 5 days after plating, suggesting that
SOX10 function does not interfere with the commitment of EPCs to the neuronal
lineage (Fig. 1D-K). The effect
of SOX10 overexpression on neuronal differentiation was unlikely to be a
consequence of selective cell death, as the plating efficiency of the founder
cells and the size of colonies generated by control and SOX10-GFP-expressing
EPCs were similar (data not shown). In addition to neuronal differentiation,
glial differentiation, as assessed by immunostaining for glial fibrillary
acidic protein (GFAP), was also reduced in day-10 SOX10-GFP colonies relative
to those expressing GFP alone (Fig.
1L,M). These findings indicate that the effects of SOX10
overexpression on EPC differentiation are similar to those observed in NCSCs
(Kim et al., 2003) and suggest
that one of the functions of this transcriptional regulator is to maintain the
neurogenic and gliogenic potential of multilineage ENS progenitors.
Endothelin 3 signalling regulates neuronal and glial differentiation of EPCs
A potential mechanism by which SOX10 inhibits neuronal and glial
differentiation of EPCs is the regulation of expression of a receptor(s) that,
upon activation by its cognate ligand, maintains the undifferentiated state of
these cells. In light of this, it is interesting that SOX10 regulates the
expression of EDNRB (Zhu et al.,
2004), which, upon activation by EDN3, inhibits neuronal
differentiation in mixed ENS cultures
(Hearn et al., 1998;
Wu et al., 1999). Here, we
have examined the potential role of endothelin signalling in the maintenance
of multilineage ENS progenitors by analysing the effects of EDN3 on the
differentiation of clonogenic EPC cultures. For this, EPCs derived from
embryonic (E11.5) mouse gut were plated at clonal densities and then allowed
to form colonies in standard culture medium or in medium supplemented with
EDN3 (10 nM). As expected, an increasing number of TuJ1+ cells were
observed in EPC colonies maintained in standard (control) medium (CM) for 5 to
10 days (Fig. 2A,C,G). By
contrast, colonies forming in the presence of EDN3 had either no or very few
neurons (Fig. 2B,D,G). The
differentiation inhibiting effect of EDN3 was also evident in clonogenic
cultures of EPCs derived from postnatal (PN) day 10 mouse gut
(Fig. 2E,F). Unlike the
constitutive expression of SOX10, endothelin signalling prevented induction of
Mash1 and Ret in EPC progeny
(Fig. 2H-K). Thus, control day
5 colonies contained 24±7.4% and 26.7±9.4% of MASH1+
and RET+ cells respectively, but in the presence of EDN3 (10 nM)
very few cells expressed these markers (0.8±0.3% and 2.5±0.7%,
respectively) for at least 10 days. Mash1 and Ret were not
induced even after 10 days in the presence of BMP2, a factor known to have a
neurogenic effect on NCSCs and EPCs [(Kim
et al., 2003; Shah et al.,
1996) and data not shown]. EDN3 also blocked glial differentiation
in clonogenic cultures of EPCs. This is indicated by the dramatic reduction
among the progeny of embryonic and postnatal EPCs of cells expressing the
glial-specific markers brain-specific fatty acid binding protein (B-FABP)
(Kurtz et al., 1994;
Young et al., 2003) on day 5
or GFAP on day 10 (Fig. 2L-Q).
In EPC colonies maintained in control medium, expression of Sox10 is
normally downregulated in enteric neurons but maintained in glial cells
(Bondurand et al., 2003).
Despite the inhibition of glial differentiation, the vast majority of cells
(at least 95%) in EPC colonies maintained in EDN3-supplemented medium
maintained high levels of SOX10, indicating their undifferentiated progenitor
status (Fig. 2R-U,V). Addition
of EDN3 to our standard culture medium generally did not alter colony size.
Moreover, immunostaining for activated caspase 3 revealed no difference in the
extent of apoptosis detected in the presence (2.2±2.0%) or absence
(2.3±2.2%) of EDN3. Thus, EDN3 inhibits neuronal and glial
differentiation of EPCs but this differentiation-inhibiting effect is unlikely
to result from selective elimination of differentiated cells.
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). To
explore the possibility that endothelin signalling drives differentiation of
mouse ENS progenitors towards cell types that lack neurogenic and gliogenic
potential, EPC colonies forming in the presence of EDN3 were immunostained for
SMA and MITF [a marker of neural crest-derived melanocytes
(Widlund and Fisher, 2003)].
Neither of these markers was detected in colonies maintained for up to 15 days
in the presence or absence of EDN3 (Fig.
3A-F), suggesting that irreversible differentiation of EPCs into
myofibroblasts or melanocytes may not account for the lack of neuronal and
glial differentiation. To further address this issue, we also examined whether the differentiation-inhibiting effect of EDN3 on EPC colonies was reversible. For this, freshly isolated EPCs were cultured in the presence of EDN3 for up to 7 days and then transferred to EDN3-free medium supplemented with BQ788 (a specific inhibitor of EDNRB used here to block residual EDN3 activity), for an additional 5 days. As expected, colonies maintained in standard medium throughout the culture period (up to 12 days) contained many neurons and glia (Fig. 3G-I), while in the presence of EDN3, the percentage of both cell types was drastically reduced (Fig. 3G,J,K). Interestingly, removal of endothelin signalling 5-7 days after plating resulted in significant recovery of neuronal and glial differentiation (Fig. 3G,L-O). Taken together, our studies suggest that EDN3 inhibits reversibly the commitment and differentiation of multipotent ENS progenitors along the neuronal and glial lineages, and that this effect is unlikely to result from the early commitment of EPCs to an irreversible non-neurogenic or non-gliogenic differentiation pathway.
Normal Sox10 activity and endothelin signalling are required for the formation of NLBs and EPCs in dissociated gut cultures
We have previously suggested that formation of NLBs and recovery of EPCs
from dissociated gut cultures depends on the self-renewing capacity of
undifferentiated ENS progenitors (Bondurand
et al., 2003). This idea is further supported by our present
studies, demonstrating a significant reduction in the size and number of NLBs
and EPCs generated from cultures of gut isolated from E11.5 embryos hemizygous
for a deletion of Sox10 (Sox10+/LacZ)
(Britsch et al., 2001),
relative to similar cultures from wild-type littermates
(Fig. 4A,B). This deficit does
not appear to reflect the reduced number of neural crest cells in the gut of
Sox10+/LacZ embryos
(Paratore et al., 2002), as
parallel experiments with similar stage embryos homozygous for
Ret51, which have a severe reduction in the number of
enteric neural crest cells [(de Graaff et
al., 2001); D.N. and V.P., unpublished], yield normal sized and
numbers of NLBs and EPCs [Fig.
4C; see also Bondurand et al.
(Bondurand et al., 2003)].
Therefore, the effect of Sox10 hemizygocity on NLB and EPC formation
is specific and reflects the requirement of this transcriptional regulator in
the maintenance and propagation of multilineage ENS progenitors.
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)
and NLBs themselves (Fig. 4D,E;
data not shown). Consistent with a role of endothelin signalling in NLB
formation, NLBs and EPC formation was drastically reduced in gut cultures from
embryos homozygous for a targeted deletion of Ednrb
(Lee et al., 2003) (data not
shown). These findings indicate that formation of NLBs and generation of EPCs
in dissociated gut cultures requires EDN3 signalling. As no purified
endothelin is normally added to the culture medium, the putative source for
this (or a related) factor is the chicken embryo extract (CEE) supplement
(Bondurand et al., 2003;
Morrison et al., 1999;
Stemple and Anderson, 1992).
Consistent with this idea, NLBs and EPCs fail to form in cultures maintained
in CEE-free medium (Fig. 4F).
However, addition of EDN3 (100 nM) to CEE-free medium was sufficient to rescue
the formation of both NLBs and EPCs (Fig.
4G). Taken together, these experiments suggest that normal
activities of SOX10 and EDN3/EDNRB, are required for the propagation and
maintenance of multilineage ENS progenitors in dissociated gut cultures.
Reduced size of the pool of ENS progenitors in EDN3-deficient animals
In a recent study, Paratore and colleagues have shown that the colonic
aganglionosis of Sox10 haploinsufficient animals
(Sox10+/LacZ) is associated with depletion of ENS
progenitors (identified by expression of Sox10) in the midgut of
mutant embryos (Britsch et al.,
2001; Paratore et al.,
2002). To examine directly whether absence of EDN3 signalling
results in a similar reduction in the size of the ENS progenitor pool, we
determined the fraction of Sox10-expressing cells within the total
neural crest cell population in the gut of wild-type and EDN3-deficient
E11.5-E12.0 mouse embryos. At this stage, overt glial differentiation is not
apparent, and expression of Sox10 marks multilineage ENS progenitors
(Paratore et al., 2002;
Young et al., 2003) (N.B.,
D.N., A.B., N.T. and V.P., unpublished). To perform this analysis, we first
established an in vivo lineage marking system, which allowed us to identify
unambiguously the relatively small population of neural crest-derived cells
within the gut of mouse embryos. This system is based on the generation of
animals carrying a Wnt1-Cre transgene
(TgWnt1Cre), which encodes the bacterial Cre recombinase
under the control of Wnt1 regulatory DNA sequences
(Danielian et al., 1998), and
the R26YFPStop reporter allele, generated by inserting
into the wild-type Rosa26 (R26) locus, the coding sequence
of yellow fluorescent protein preceded by loxP-flanked stop sequence (YFPStop)
(Srinivas et al., 2001). As
Wnt1-Cre is specifically expressed along the length of the dorsal
neural tube and thus by all neural crest cells
(Danielian et al., 1998) (S.
Bogni and V.P., unpublished),
TgWnt1Cre;R26YFPStop animals express YFP in
neural crest cells and their derivatives, including those colonising the
embryonic gut (Fig. 5A). By
carrying out the appropriate crosses, we next introduced the loss of function
Edn3ls allele into the
TgWnt1Cre;R26YFPStop background. Fluorescent
E11.5-E12.0 embryos derived by intercrossing mice heterozygous at each of the
three loci (i.e. Edn3ls, TgWnt1Cre and
R26YFPStop) were identified and their intestines were
dissociated into single cell suspension and plated. Shortly after plating (at
most 2-3 hours) the cultures were fixed and double immunostained for YFP and
SOX10. The proportion of YFP+ cells in such acute cultures
established from Edn3ls homozygous embryos was decreased
by 65% relative to the equivalent percentage in wild-type embryos.
Furthermore, in cultures established from Edn3+/+ and
Edn3+/ls embryos, SOX10+ cells represented,
respectively, 71.6±3.9% and 68.5±3.9% of the total neural
crest-derived (YFP +) cell population within the gut
(Fig. 5B). By contrast, in the
gut of Edn3ls/ls embryos, the
SOX10+YFP+/YFP+ fraction of cells was
significantly reduced (56.2±5.8%; P<0.05, n=6;
Fig. 5B). Using a similar
approach, we have also observed that the proportion of SOX10+ cells
within the enteric neural crest cell lineage was reduced by 20% in embryos
homozygous for a targeted allele of Ednrb
(Lee et al., 2003) relative to
heterozygous or wild-type controls (data not shown). Therefore, severe deficit
in EDN3 signalling leads to a relative reduction in the pool of
Sox10-expressing ENS progenitors within the gut of mouse embryos.
|
). However,
other reports have indicated that EDNRB signalling is not required for the
proliferation of enteric NCSCs of rat embryos
(Kruger et al., 2003). Here,
we have re-examined the role of EDN3 in the proliferation of endogenous ENS
progenitors by analysing the incorporation of the nucleotide analogue
bromodeoxyuridine (BrdU) into YFP+ or SOX10+ cells in
the intestine of wild-type, Edn3+/ls and
Edn3ls/ls embryos. For this, animals heterozygous for
Edn3ls, TgWnt1Cre and
R26YFPStop were intercrossed and pregnant females (at
E11.5) were injected with BrdU. One hour later, the intestines of individual
embryos were dissociated, plated as acute cultures, and immunostained for BrdU
and YFP or SOX10. We found that the fraction of
BrdU+YFP+/YFP+cells was similar in the
intestine of wild-type, Edn3+/ls or
Edn3ls/ls embryos (31.0±4.5%, 33.4±3.3% and
31.0±4.4%, respectively, n=5;
Fig. 6A,C). In addition, a
similar fraction of BrdU+SOX10+/SOX10+ cells
was found in the intestine of wild-type and Edn3+/ls E11.5
embryos (48.7±5.0% and 46.2±3.6%, respectively;
Fig. 6D). By contrast, the
fraction of BrdU+SOX10+/SOX10+ cells was
reduced in the gut of Edn3ls/ls embryos (39.9±4.8%)
relative to wild-type or Edn3+/ls littermates
(P<0.007, n=10; Fig.
6D). Therefore, severe reduction in EDN3 signalling results in a
smaller fraction of proliferating SOX10+ cells within the intestine
of Edn3ls homozygous embryos.
The above experiments were designed to examine the global effect of the
Edn3ls mutation on the proliferation of enteric neural
crest-derived cells irrespective of their position along the length of the
intestine. However, previous studies have demonstrated that expression of
Edn3 is spatially and temporally regulated during gut organogenesis,
with highest levels detected in intestinal segments harbouring the front of
rostrocaudally migrating neural crest cells. For example, in E11.5 embryos,
the front of migrating neural crest cells is crossing the ileocecal valve to
enter the cecum, while the highest levels of Edn3 mRNA are detected in the
cecum and the proximal colon (Barlow et
al., 2003; Leibl et al.,
1999). These data raise the possibility that enteric neural crest
cells at the migratory front are exposed to higher levels of EDN3 and thus may
be more dependent on this signalling molecule for their proliferation. To
examine this possibility, pregnant mice from Edn3ls,
TgWnt1Cre and R26YFPStop intercrosses were
pulsed for 1 hour with BrdU, and the guts of individual fluorescent embryos
(at E11.5) were double immunostained as whole-mount preparations with
antibodies specific for BrdU and YFP and analysed by confocal microscopy
(Fig. 6A,B). Contrary to the
acute culture experiments, we observed a significant reduction in the fraction
of BrdU+YFP+/YFP+ cells at the migratory
front of Edn3ls/ls embryos (48.9±6.7%) compared
with wild-type littermates (61.8±5.0; P<0.007,
n=6) (Fig. 6E). The
equivalent value for Edn3+/ls animals was
51.8±8.9%, which indicated a trend towards reduced proliferation even
in heterozygous embryos (Fig.
6E). The very high density of neural crest-derived cells behind
the front of migration precluded a similar analysis to be carried out in more
proximal gut regions. In addition, for technical reasons, we were unable to
perform a similar analysis using a combination of SOX10- and YFP-specific
antibodies. In summary, our experiments indicate that decreased endothelin
signalling in vivo leads to a reduction in the proliferation of
Sox10-expressing multilineage ENS progenitors. This requirement for
endothelin signalling appears to be spatially regulated along the intestine
and is highest in neural crest cells at the front of migration.
|
To examine whether the increase in neuronal differentiation was restricted to the front of migration, we also determined the percentage of TuJ1-expressing cells in the entire intestinal population of YFP+ cells by establishing acute cultures of dissociated intestine dissected from E11.5-12.0 embryos. We found that the overall proportion of TuJ1+YFP+/YFP+ cells within the gut of Edn3ls/ls embryos was decreased relative to wild-type or heterozygous mutants (data not shown), indicating that severe deficit in EDN3 signalling reduces the overall differentiation of enteric neural crest cells.
|
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), indicate that SOX10 functions during embryogenesis to
maintain and propagate the neurogenic and gliogenic progenitors of the ENS.
Independent experiments have shown that overexpression of SOX10 in the dorsal
neural tube can induce neural crest cells in avian embryos
(McKeown et al., 2005), while
studies on NCSCs have demonstrated that high levels of this factor are
sufficient to maintain their neurogenic and gliogenic potential
(Kim et al., 2003). Therefore,
SOX10 appears to have overlapping roles within the neural crest cell lineage,
contributing to its early induction in the neural tube and the maintenance of
its multipotential state.
The inhibitory role of endothelin signalling on neuronal differentiation
has been reported previously by two groups, who have shown that upon addition
of EDN3 (or the EDNRB agonist IRL1620), primary cultures of enteric neural
crest-derived cells yield fewer postmitotic enteric neurons
(Hearn et al., 1998;
Wu et al., 1999). However, in
these studies EDN3 and IRL1620 were added to a mixed population of neural
crest-derived gut cells representing various stages of commitment and
differentiation, thus precluding the identification of the precise cell target
for endothelin. Here, we have employed the experimental paradigm of clonogenic
EPC cultures to explore specifically the role of EDN3 in the commitment and
differentiation of multilineage ENS progenitors. Our studies show that,
similar to SOX10 overexpression, EDN3 inhibits overt neuronal and glial
differentiation. However, unlike SOX10, EDN3 prevents commitment of EPCs to
the neurogenic and gliogenic pathways. Taken together, these studies suggest
that one of the roles of endothelin signalling during gut organogenesis is to
maintain ENS progenitors in an uncommitted, multipotential and self-renewing
state. This suggestion is further supported by our in vivo analysis of the
intestine of E11.5-E12.0 mouse embryos homozygous for the
Edn3ls mutation, which demonstrated a relative reduction
of the pool of multipotential ENS progenitors.
|
). However,
expression of EDN1 or its receptor EDNRA has not been reported within the gut
of mouse embryos and the potential effects of Edn1 and Ednra
mutations (Pla and Larue,
2003) on the development of the mammalian ENS are unclear. Other
signalling pathways could also function in series or in parallel to EDN3 to
regulate the number and differentiation of ENS progenitors in vivo. For
example, the receptor tyrosine kinase RET is known to interact with the EDN3
signalling pathway to regulate the severity of the phenotype in individuals
with HSCR and the extent of colonisation by neural crest cells of the gut of
mouse embryos (Barlow et al.,
2003; Carrasquillo et al.,
2002; Kruger et al.,
2003; McCallion et al.,
2003). Marker analysis of vagal neural crest-derived cells in the
gut of E9.0-E10.5 embryos and clonogenic cultures of EPCs, have indicated that
the SOX10+RET- population of pre-enteric neural crest
cells that invades the foregut gradually induces expression of Ret
and (in the case of neurogenic sublineage) eventually downregulates
Sox10 (Anderson et al.,
2006; Bondurand et al.,
2003; Young et al.,
2003). Although absence of EDN3 during embryogenesis is expected
to reduce the number of early multilineage ENS progenitors and thus the supply
of RET+ progeny, activation of RET by GDNF could partly compensate
for this deficit by stimulating the proliferation of Ret-expressing
descendants (Gianino et al.,
2003; Heuckeroth et al.,
1998). This idea is consistent with the severe ENS phenotype
observed in embryos bearing mutations in both Ret and Edn3
genes (Barlow et al., 2003),
and suggests that EDN3 and GDNF control the size of partially overlapping cell
populations within the gut.
Our experiments, together with other genetic studies
(Cantrell et al., 2004),
indicate that Sox10 and Edn3/Ednrb are components of a
signalling cascade that controls commitment and differentiation in the
mammalian ENS. This idea is further supported by analysis of transgenic mice,
demonstrating that Sox10 regulates the spatial and temporal
expression of Ednrb in neural crest cells
(Zhu et al., 2004).
Interestingly, other studies have implicated SOX10 in the regulation of
expression of Ret in the ENS of mouse embryos
(Lang et al., 2000;
Lang et al., 2003). Therefore,
Sox10 activity appears to have antagonistic effects on enteric neural
crest cells by promoting the expression of receptors associated with
maintenance of cells in an uncommitted and undifferentiated state (EDNRB) but
also commitment and differentiation towards mature phenotypes (RET). This
apparent paradox may be explained by the specific challenges faced by neural
crest cells upon invasion of the foregut. Thus, as these cells migrate to
colonise the gut, the number of multilineage progenitors must remain
sufficiently high for a relatively long period in order for the appropriate
number of progeny to colonise an expanding gastrointestinal tube. Therefore,
exposure of pre-enteric neural crest cells to differentiation-promoting
signals from the splachnic mesenchyme of the gut must be initially neutralised
by the employment of differentiation-inhibiting mechanisms that preserve a
relatively high proportion of ENS progenitors in an uncommitted and
undifferentiated state. Consistent with this hypothesis, arrival of
pre-enteric neural crest cells to the vicinity of the aorta and the foregut is
associated with induction of Ret [presumably under the influence of
BMPs (Lo et al., 1997)],
which, upon activation by GDNF, is capable of promoting neuronal
differentiation (Taraviras et al.,
1999). Under these conditions, EDN3/EDNRB signalling would be
specifically required within the gut as an `antidote' to the neurogenic and
differentiation-inducing signals from the splachnic mesenchyme. In support of
this idea, we have observed that the differentiation-promoting effect of BMPs
and GDNF on EPC colonies can be blocked efficiently by EDN3 (N.B., D.N., A.B.,
N.T. and V.P., unpublished). However, the commitment and
differentiation-inhibiting effects of EDN3 on ENS progenitors must be
transient and reversible, as neural crest progenitors must eventually escape
the differentiation block to generate mature neuronal and glial phenotypes.
The molecular mechanisms controlling the release from such an `EDN3 hold' are
not known but may well include the downregulation of Ednrb and the
spatial and temporal regulation of Edn3 expression within the gut.
Therefore, by regulating the expression of receptors for multiple signalling
pathways, Sox10 is uniquely capable of coordinating the transition
from a multilineage progenitor to a differentiated cell type in the specific
microenvironment of the developing mammalian ENS.
The smaller pool of Sox10-expressing ENS progenitors observed in
Edn3ls homozygote embryos results, at least partly, from
reduced proliferation of these cells. Although EDN3 is a mitogenic factor for
avian neural crest (Lahav,
2005; Lahav et al.,
1998; Lahav et al.,
1996), no direct proliferative effect has so far been described
for mammalian enteric neural crest cells or EPCs. Therefore, EDN3 might
influence the proliferation of endogenous ENS progenitors indirectly
(Barlow et al., 2003), perhaps
by maintaining their undifferentiated state. Contrary to our findings, other
investigators have reported that Ednrb mutations in rats have no
effect on the proliferation of enteric neural crest cells during embryogenesis
(Kruger et al., 2003).
Moreover, these authors presented evidence that EDN3 promotes the
differentiation of cultured NCSCs to myofibroblasts and that absence of EDNRB
signalling in vivo has no effect on neuronal differentiation. The reasons for
the discrepancy between this report and our studies are currently unclear. It
is possible that species specific differences could determine the extent to
which endothelin signalling can influence the proliferative and
differentiation responses of neural crest cells. Alternatively, these
discrepancies could reflect potential differences in the cell populations
analysed by the two laboratories. Further experimentation will be necessary to
explore these issues.
One of the most striking findings of our work is that the effect of the Edn3ls mutation on endogenous enteric neural crest cells varies along the length of the embryonic gut. Thus, cells located at the migratory front are more dependent on endothelin signalling for their proliferation relative to other more posterior cell groups. Moreover, Edn3ls/ls embryos show increased differentiation at the front of migration, although overall the differentiation of enteric neural crest cells is reduced. The increased differentiation and reduced proliferation of neural crest cells at the migratory front are likely to reflect primary effects of EDN3 on ENS progenitors, consistent with the distribution of Edn3 mRNA in vivo and the observed effects of EDN3 on clonogenic EPC cultures. By contrast, the effects of the Edn3ls mutation on more posterior groups of enteric neural crest cells might reflect secondary effects of the EDN3 reduction. For example, the overall reduction in the fraction of TuJ1+ cells in the gut of mutant embryos could be a consequence of a reduced total number of neuroectodermal cells within the mutant gut (itself the result of reduced proliferation and increased differentiation of the early neural crest invading the gut), which could prevent the necessary cell-cell interactions to drive cell differentiation. Irrespective of the specific mechanisms underlying this effect, our findings reveal that the effect of mutations in Edn3 are spatially and temporally regulated, consistent with the implication of this signalling pathway in the integration, in time and in space, of cellular activities necessary for normal ENS neurogenesis.
CEE is a key ingredient of the culture medium used for the propagation of
multipotential self-renewing progenitors of the PNS, including NCSCs and EPCs
(Bondurand et al., 2003;
Morrison et al., 1999;
Stemple and Anderson, 1992).
As CEE is an unfractionated homogenate of chick embryos, its crucial
ingredient(s) are currently unknown. Here, we demonstrate that EDN3 signalling
is necessary and sufficient to support the formation of NLBs and the recovery
of EPCs in dissociated embryonic gut cultures in the absence of CEE,
suggesting that EDN3 is an active ingredient of CEE and mediates its effects
on PNS progenitors. Although the mechanisms by which endothelin signalling
preserves the undifferentiated state of EPCs are currently unclear, our
findings could have profound effects on stem cell biology as they suggest that
EDN3 is uniquely capable of converting an asymmetric pattern of cell divisions
of ENS progenitors (EPCs), which in standard media generate colonies
containing a mixture of multilineage progenitors and differentiated progeny,
to a symmetrical one that gives rise to colonies composed almost exclusively
of multilineage progenitors. A similar potential has been assigned recently to
FGF2 and EGF, which, in combination, suppress the differentiation of neural
stem (NS) cell lines and support their continuous symmetrical self-renewal
divisions (Conti et al.,
2005). It is currently unknown whether EDN3, alone or in
combination with other factors, can maintain the long-term self-renewal of
undifferentiated neural crest cells in culture. Nevertheless, our findings
suggest that EDN3 could be a key ingredient of fully defined culture media
that would allow the expansion of a homogeneous population of ENS stem cells
that, upon removal of this factor, would be able to differentiate into enteric
neurons and glia. Such a cell population could be used for cell
transplantation experiments aimed at restoring the peristaltic activity of
aganglionic gut segments.
Note added in proof
Just before our paper was accepted another publication appeared that, in
general, is in agreement with the conclusions of our work
(Nagy and Goldstein,
2006).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: INSERM U654, Bases moléculaires et cellulaires des
maladies génétiques, Hôpital Henri Mondor, 94010
Créteil Cedex, France
Present address: Neural Development Unit, UCL Institute of Child Health, 30
Guilford Street, London WC1N 1EH, UK
Present address: Gastroenterology and Neural Development Units, UCL
Institute of Child Health and Great Ormond Street Hospital, 30 Guilford
Street, London WC1N 1EH, UK
|
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