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First published online 15 April 2009
doi: 10.1242/dev.028373
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1 UPMC Univ Paris 06, UMR 7622, Laboratoire de Biologie du Développement,
F-75005, Paris, France.
2 CNRS, UMR 7622, Laboratoire de Biologie du Développement, F-75005,
Paris, France.
3 Life and Health Sciences Research Institute, School of Health Sciences,
University of Minho, 4710-057 Braga, Portugal.
Author for correspondence (e-mail:
liliana.osorio-da-silva{at}snv.jussieu.fr)
Accepted 13 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Apoptosis, Chick, Delamination, Msx1, Noggin, Wnt1, Neural crest, Neuronal differentiation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). It has been suggested that NCC specification occurs very
early during development and involves the activity of Pax7, first detected at
Hamburger and Hamilton stage 4+ (HH4+)
(Hamburger and Hamilton, 1951)
in the epiblast layer (Basch et al.,
2006). As development proceeds, Pax7 expression gradually
shifts to the neural folds where the NCCs will arise. A specific
transcriptional program, resulting from a variety of signals secreted by the
surrounding tissues, is then activated
(Sauka-Spengler and Bronner-Fraser,
2008). Initially, a group of genes, often described as early
markers, are activated in the prospective NCCs. These genes include several
members of the Pax, Msx and Zic families. They collectively act to define a
broad region containing cells competent to form the neural crest (NC). The
subsequent generation of trunk NCCs requires the coordinated activity of
FoxD3, Snail2 and Sox9, considered as the trunk NCC
`specifying' genes. Their combined expression defines cells that manifest all
the principal transcriptional and morphological characteristics of NCCs
(Cheung et al., 2005). Once
specified, NCCs delaminate from the dorsal neuroepithelium, undergoing an
epithelial-to-mesenchymal transition
(Duband, 2006). In addition,
the onset of trunk NCC migration is thought to depend on Bmp-dependent Wnt
activity coordinated by somitogenesis
(Kalcheim and Burstyn-Cohen,
2005). All of these characteristics of NCC generation have been
described principally in regions of primary neurulation. We recently showed
that the same events occur more caudally in regions belonging to secondary
neurulation (Osorio et al.,
2009).
After detachment from the NT, NCCs migrate into the periphery along
stereotypical pathways and arrive at specific locations, where they
differentiate into a wide variety of cellular derivatives
(Le Douarin and Kalcheim,
1999). Mesectodermal derivatives, like the craniofacial skeleton,
derive only from the cephalic NC. Sensory neurons and glia derive from both
the cephalic and trunk levels, whereas sympathetic neurons are trunk-specific.
Melanocytes are produced along the entire rostrocaudal axis.
The most caudal region in both mammals and birds is characterized by the
absence of motor nerves in the spinal cord and a lack of peripheral ganglia.
In humans, this portion of the spinal cord constitutes the filum terminale,
which extends through the sacrum to the first coccygeal vertebrae, whereas in
birds it is located at the level of the pygostyle formed by the fusion of
three to six caudal vertebrae (Catala et
al., 2000). In the chick embryo, this part of the NT corresponds
to the region of somites 47-53, which are the last pairs of somites to be
formed during development. This `caudal-most' part of the NT is formed at HH24
during the fourth day of embryonic development (E4). The lack of motor nerves
arising from the spinal cord in this region has been described previously
(Afonso and Catala, 2005).
Concerning the absence of sensory ganglia and nerves, pioneer studies have
shown that this is not due to a local lack of NCCs but rather to a restriction
of their developmental potentials (Catala
et al., 2000). Both in vitro culture and in vivo transplantation
experiments have shown that these caudal-most NCCs give rise to Schwann cells
and melanocytes, but never to neurons.
In the present study, our aim was to elucidate the mechanisms underlying the lack of neuronal derivatives that characterizes these caudal-most NCCs. We first determined the precise chronology of caudal-most NCC generation. Although the caudal-most NT is fully cavitated at E4/HH24 and adjacent somites are already differentiating, a very small number of NCCs are detected one day later, at E5/HH26. This is not due to a lack of specification of the NCCs. Instead, Bmp4-Wnt1 signaling, which is known to trigger trunk NCC delamination, is impaired in this region. In addition, an abnormal pattern of apoptosis is shown to take place at the same time (E4/HH24) in the dorsal half of the caudal-most NT. Both of these events undoubtedly contribute to the scarcity and delayed migration of caudal-most NCCs that in turn seem to culminate in their lack of neuronal potential. Results of heterotopic transplantation experiments of either the caudal-most somites or caudal-most NT into a more rostral region of younger embryos suggest that the features particular to caudal-most NCCs are the result of properties intrinsic to these cells. Furthermore, forced Nog expression in the trunk NT can reproduce the main characteristics observed for the caudal-most NCCs (scarcity and absence of neuronal derivatives), suggesting that impaired Bmp4 signaling is an event occurring upstream of the mechanisms operating in the caudal-most NT. Importantly, increased Bmp4-Wnt1 signaling, through inhibition of Nog in the caudal-most NT at E4/HH24, generates NCCs that express markers of specified neurogenic progenitors.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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In ovo embryonic manipulations
Quail-chick transplantation experiments
Tails of E4/HH24 quail embryos were excised posteriorly to somite pair 46.
After enzymatic dissociation, the rows of caudal-most somites and adjacent NT
were isolated and transplanted into different E2/HH11-12 chick embryos. The
caudal-most somites were transplanted bilaterally at the level of the
posterior presomitic mesoderm (PSM) and the caudal-most NT was transplanted at
the site of the endogenous NT, at the same level. Both types of grafts were
performed over a length corresponding to 
4-5 prospective somites,
keeping the correct anteroposterior (AP) and dorsoventral (DV) orientations.
Chimeras were incubated for an additional 16-48 hours and then fixed.
Electroporation experiments
For caudal-most electroporations, DNA was injected into the lumen of the
NT-facing somite 45 in E4/HH24 embryos. Electrodes were positioned in order to
target the dorsal region of the caudal-most NT and a Wave Stimulator (A-M
Systems, Model 2100) delivered seven pulses of 35 V, 50 milliseconds each. The
following expression plasmids were used: pcDNA3.1, containing mouse
full-length Bmp4 DNA in frame with a GFP coding sequence (0.8
µg/µl); pcDNA6.1 Gw/EmGFP-miR chick Nog (1 µg/µl),
constructed as described in the manufacturer's instructions using BLOCK-iT Pol
II miR RNAi Expression Vector Kit (Invitrogen, K4935-00; experimental details
are available upon request); and pMiwIII, containing chick full-length
Wnt1 DNA (1.0 µg/µl)
(Matsunaga et al., 2002),
coelectroporated with pEGFP-N1 (GenBank, U55762; Promega, 6085-1; 0.4
µg/µl).
For trunk electroporations, pCIG containing full-length mouse Nog DNA in frame with a GFP coding sequence (0.5 µg/µl) was injected into the lumen of the NT located at the level of the posterior PSM of E2/HH11-12 embryos. Five pulses of 25 V and 50 milliseconds each were delivered.
Embryos were incubated for an additional 8-48 hours and only those correctly electroporated, as verified by GFP expression, were used for posterior analyses.
In situ hybridization (ISH)
ISH was performed according to Henrique et al.
(Henrique et al., 1995). The
following chick-specific riboprobes were used: Bmp4
(Francis-West et al., 1994),
Cad6B and Cad7 (Nakagawa
and Takeichi, 1995), FoxD3
(Dottori et al., 2001;
Kos et al., 2001),
Msx1 and Msx2 (Coelho et
al., 1991; Coelho et al.,
1992), Ngn1 and Ngn2
(Perez et al., 1999),
Nog (Reshef et al.,
1998), Pax3 (Goulding
et al., 1993), Snail2
(Nieto et al., 1994),
Sox9 (Cheung and Briscoe,
2003), Sox10 (Cheng et
al., 2000), Uncx4.1
(Schrägle et al., 2004),
Wnt1 and Wnt3a (Megason
and McMahon, 2002). The following mouse-specific riboprobe was
used: mouse (m) Nog (McMahon et
al., 1998).
Immunohistochemistry
Immunohistochemistry was performed as previously described
(Afonso and Catala, 2005),
using the following primary antibodies: anti-phospho Histone H3 (pH3; Upstate
Biotechnology, 06-570), anti-Isl1/2 (DSHB, 39.4D5), anti-N-cadherin (Sigma,
FA-5), anti-NC1/HNK1 (Vincent et al.,
1983; Tucker et al.,
1984), anti-Pax7 (DSHB), anti-QCPN (DSHB), anti-TuJ1 (Chemicon,
MAB1632), and anti-WRS (Reedy et al.,
1998; Harris et al.,
2008).
Nile Blue Sulfate (NBS) staining
NBS staining (Jeffs and Osmond,
1992), modified as described in Teillet et al.
(Teillet et al., 1998), was
used to detect cell death in whole embryos. The embryos were photographed and
then fixed for TUNEL assay.
TUNEL
Detection of apoptotic cells was performed using the TUNEL assay, following
the manufacturer's instructions (Roche, 12156792910).
Acquisition and analysis of images and art graphics
Whole-mount embryos were photographed using a Nikon DXM1200 camera coupled
to a Leica MZFLIII microscope. Sections were photographed with an Evolution VF
camera coupled to a Nikon Eclipse E800 microscope, using Image-Pro Plus
software (Media Cybernetics) and OptiGrid System (Optem). Images were analyzed
in Adobe Photoshop CS3.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Thiery et al., 1982;
Teillet et al., 1987;
Osório et al.,
2009).
NCC specification in the caudal-most region
Several hypotheses might account for the scarcity and delayed migration of
the caudal-most NCCs, one of which is a possible defect in their
specification. We therefore analyzed the expression of a number of genes with
known involvement in NCC specification
(Sauka-Spengler and Bronner-Fraser,
2008). Pax3 and Pax7 were detected in the dorsal aspect
of the caudal-most NT as early as E4/HH24
(Fig. 2A-B''). By
contrast, Msx1 was not observed
(Fig. 2C-C''), even at
E5/HH26 (see Fig. S2A-A'' in the supplementary material). However,
Msx2 was already expressed at E4/HH24
(Fig. 2D-D''). In
addition, FoxD3, Snail2 and Sox9 were also present in the
dorsal aspect of the caudal-most NT at E4/HH24
(Fig. 2E-G''). We thus
conclude that NCC specification has indeed occurred in the caudal-most NT at
E4/HH24.
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). Signals emanating from epithelial somites inhibit
Nog transcription in the dorsal NT and trigger a Bmp4-Wnt1 cascade,
leading to NCC delamination
(Sela-Donenfeld and Kalcheim,
1999; Sela-Donenfeld and
Kalcheim, 2000; Burstyn-Cohen
et al., 2004). Since NCC specification in the caudal-most region of the chick embryo (at E4/HH24) is followed by a temporal lag and delayed migration of these cells (at E5/HH26), we analyzed the `status' of both Bmp and Wnt pathways in this region. We observed that Bmp4 is homogeneously distributed along the entire length of the caudal-most NT at E4/HH24 (Fig. 3A,A'). However, contrary to more anterior levels, Nog is not downregulated in the dorsal NT at the level of somites 47-53 as they form at E4/HH24 (Fig. 3B,B') and continues to be expressed even after their complete dissociation at E5/HH26 (see Fig. S2B-B'' in the supplementary material). Interestingly, we did not detect Wnt1 in the dorsal caudal-most NT (Fig. 3C,C'), even at E5/HH26 (see Fig. S2C-C'' in the supplementary material), whereas Wnt3a was expressed as soon as E4/HH24 (Fig. 3D,D'). These results show a lack of both Bmp4 and Wnt1 signaling in the dorsal caudal-most NT, which might contribute to the delayed caudal-most NCC migration.
Extensive cell apoptosis in the caudal-most NT at E4/HH24
In addition to the impairment in the signals normally triggering NCC
delamination, other mechanisms, such as cell death, might account for the
scarcity and delayed migration of the caudal-most NCCs. When compared with a
more rostral region at an equivalent developmental stage
(Fig. 4A,B), more pronounced
cell death occurs along the caudal-most NT at E4/HH24, as shown by NBS
staining (Fig. 4C) and TUNEL
assay (Fig. 4D), which revealed
massive apoptosis throughout the entire dorsal moiety of the caudal-most NT
and the overlaying ectoderm. This is an exceptional and transient phenomenon,
as no further apoptosis was observed in the caudal-most NT at E5/HH26
(Fig. 4E,F). To our knowledge,
such apoptosis affecting the entire dorsal half of the NT has never been
described at any level of the AP axis. Indeed, apoptotic cells within the
trunk secondary NT showed no particular localization along the DV axis, like
those in the primary NT (Hirata and Hall,
2000). Together with the lack of Bmp4-Wnt1 signaling, the massive
apoptosis, which affects the dorsal moiety of the caudal-most NT at E4/HH24,
must contribute to the observed drastic reduction in the number of NCCs formed
in this region of the embryo.
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). Quail caudal-most somites were grafted bilaterally at the
level of the posterior PSM of E2/HH11-12 chick embryos
(Fig. 5A'). Since NCC
specification has already occurred at this level of the embryo
(Sela-Donenfeld and Kalcheim,
1999), this allows us to evaluate the influence of caudal-most
somites on NCC delamination and further development.
Most of the chimeras collected at 16-48 hours post-transplantation (hpt)
were morphologically normal (21/25), and the grafts were always found between
somites 22 and 28, validating their initial location. No change in
Sox10 expression was found at 16 hpt at the level of the graft, as
indicated by anti-QCPN immunolabeling (Fig.
5B), and NCCs were found dorsally to the NT and in the MSA
(Fig. 5B'; n=3).
At 24 hpt, a defect in the segmented pattern of migration of NCCs facing the
grafted caudal-most somites was apparent
(Fig. 5C,C';
n=4). We observed that Wnt1 expression in the dorsal NT was
not modified (Fig. 5D,D';
n=3), indicating that Bmp4 activity continued in the presence of the
grafted caudal-most somites. At 48 hpt, a continuous, non-segmented expression
of Sox10 (Fig.
5E,E') was observed (n=3). In spite of this, DRG,
formed by Isl1/2+ postmitotic neurons
(Avivi and Goldstein, 1999),
developed at the graft level (Fig.
5F,G; n=4). Interestingly, the DRG facing the grafted
caudal-most somites were irregularly segmented, smaller and more dorsally
located than the normal DRG, as was found following the transplantation of a
series of posterior half-somites at the place of the entire somites
(Kalcheim and Teillet, 1989).
However, normal striped Uncx4.1 expression
(Schrägle et al., 2004)
indicated that the caudal-most somites presented anterior and posterior
compartments (Fig.
5H,H'), as did the more rostral somites.
In conclusion, caudal-most somites provide the required signals for trunk NCC delamination and do not seem to be responsible for the lack of Bmp4-Wnt1 signaling occurring in the NT of the caudal-most region.
Rostral transplantation of the caudal-most NT does not restore DRG formation
To further investigate the role of the somitic environment in the defective
Bmp4-Wnt1 signaling occurring in the caudal-most NT, we grafted the quail
caudal-most NT at the posterior PSM level of E2/HH11-12 chick embryos
(Fig. 5A''), thereby
confronting it with all of the steps of trunk somitogenesis.
Most of the chimeras that survived up to E4/HH24 were morphologically
normal (21/26), with some embryos presenting fused somites at the midline just
prior to and/or after the level of the graft, revealing a local interruption
of the NT due to the lack of growth of the grafted caudal-most NT. The
transplant was generally found in the trunk region between somites 20 and 25,
justifying its initial location. We first analyzed NCC generation from the
graft. At 24 hpt, no quail Sox10+ cells were observed
(Fig. 5I,I';
n=5), even when the adjacent somites were well formed. At 48 hpt,
quail HNK1+ cells were detected dorsally to the NT
(Fig. 5J,J'). They seemed
more numerous than NCCs of the caudal-most region in situ at an equivalent
stage. However, no DRG were ever detected
(Fig. 5J; n=5). It
should be noted that quail DRG were still absent in similar chimeras at E12,
where only quail melanocytes and glial cells had differentiated
(Catala et al., 2000).
Moreover, we found that some quail NCCs at 48 hpt were WRS+
(Fig. 5K), corresponding to
early melanocyte precursors (Reedy et al.,
1998). In addition, at 24 hpt, Wnt1 expression in the
dorsal region of the ectopically grafted caudal-most NT was not restored
(Fig. 5L,L';
n=3), indicating continued impairment of Bmp4 signaling.
Taken together, the results of the rostral transplantation of either the caudal-most somites or the caudal-most NT indicate that Bmp4-Wnt1 signaling defects and a lack of neuronal potential of the caudal-most NCCs are properties intrinsic to these cells.
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), our aim was to evaluate the effect of maintaining
Nog expression in the pre-migratory NCCs themselves. At 8 hours
post-electroporation (hpe), we found Sox10+ NCCs along the
entire length of the electroporated NT
(Fig. 6A,B; n=6).
Interestingly, these cells remained dorsal to the NT at the level of both
epithelial and dissociating somites and did not migrate ventrally
(Fig. 6B',B''), as
NCCs do in control GFP-electroporated embryos (see Fig. S3A-B'' in the
supplementary material). Moreover, delaminated GFP+ NCCs did not
express Nog, suggesting that they were influenced by Nog emanating
from the adjacent NT (Fig.
6C,C'; n=4). At 24 and 48 hpe, NCCs remained dorsal
to the NT and in the MSA, with no formation of DRG
(Fig. 6D-G; n=5). In
addition, Cad6B expression was decreased at 12 hpe
(Fig. 6H,I; n=3),
whereas at 24 hpe N-Cad was upregulated in both the dorsal NT and the NCCs
overlaying the NT, but absent from NCCs located in the MSA
(Fig. 6J,K; n=3).
Nevertheless, some Cad7+ NCCs were present dorsally at 48
hpe (Fig. 6L-M; n=3).
In addition, Msx1 (Fig.
6N; n=4), Wnt1
(Fig. 6O; n=4) and
FoxD3 (Fig. 6P;
n=3) were absent from the electroporated region at 24 hpe.
We went further in our analysis by examining the fate of the NCCs generated
under these conditions. We found that some of the cells were in fact neuronal
precursors Ngn1+ (Fig.
6Q-R; n=3) (Perez et
al., 1999). However, anti-TuJ1 and anti-Isl1/2 immunolabeling
showed differentiating neurons only inside the NT
(Fig. 6S,T; n=5). More
importantly, a great number of the migratory NCCs were TUNEL+ and
massive apoptosis also occurred in the electroporated NT in a non
cell-autonomous manner (Fig.
6U; n=5).
In conclusion, forced Nog expression in the dorsal trunk NT does not completely block NCC migration. However, the possibility that the migration process had begun before effective ectopic Nog expression occurred must be considered. In addition, forced Nog expression abolishes NCC ventral migration and prevents ganglia formation. Thus, it mimics the main features of the caudal-most NT (absence of Msx1 and Wnt1, and apoptosis) and the specific caudal-most NCC `phenotype' (scarcity and lack of neuronal derivatives). However, some differences concerning Cad6B, N-Cad and FoxD3 expressions were observed.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The first cells that delaminate at the trunk
level travel into the ventral pathway between the NT and the anterior half of
each somite in a segmented manner. These mainly give rise to neurons and glia
of both the sensory and sympathetic nervous systems. By contrast, later
migrating NCCs travel in a uniform and unsegmented fashion into the dorsal
pathway between the somites and the overlying ectoderm and generate skin
melanocytes. Migration into this last pathway is delayed by 24 hours in
the chick embryo (Erickson et al.,
1992). In light of such correlations, the `temporal lag' observed
in the caudal-most region between NCC specification at E4/HH24 and their
migration at E5/HH26, associated with the lack of neuronal derivatives, led us
to propose that early migrating NCCs, which normally give rise to neurons, are
lacking at this level. NCCs that delaminate at E5/HH26 most probably
correspond to the late migrating population that principally gives rise to
melanocytes.
Mechanisms of elimination of early migrating NCCs
Absence of the early migrating NCC population might be the result of the
massive cell apoptosis which occurs in the dorsal caudal-most NT. This
cellular event shows high temporal precision and occurs at E4/HH24 (see
Fig. 4), with no observable
apoptotic cells by E5/HH26 (see Fig. S2 in the supplementary material). This
argues in favor of a specific elimination of the early migrating NCC
population, with those NCCs arriving later being unaffected. In our study, we
were unable to identify the cause of this massive apoptosis. The relation
between defective NCC migration and neuroepithelial cell death remains
unclear. One possibility is that defective Bmp4-Wnt1 signaling prevents the
delamination of NCCs following their specification, thereby rendering the
cells incapable of survival, as has been reported in other studies
(Vogel and Weston, 1988;
Cheung et al., 2005).
Neurogenic cell death could also be a direct consequence of the absence of
Wnt1, described as a cell survival factor in distinct cell types
(He et al., 2004;
You et al., 2004;
Almeida et al., 2005). Other
roles for Wnt1 must also be considered, as it promotes the expansion of NCCs
(Ikeya et al., 1997;
Megason and McMahon, 2002;
Dunn et al., 2005). The
increase in NCC number obtained in our electroporation experiments in the
caudal-most NT is consistent with both putative Wnt1 activities. According to
our results, these might involve the activity of Msx1 and/or
FoxD3, both induced in response to ectopic Wnt1 expression.
In fact, both of these genes have been reported to play an important role in
the maintenance of NCC progenitors, by protecting pre-migratory or early
migrating NCCs from apoptosis (Ishii et
al., 2005; Lister et al.,
2006; Teng et al.,
2008). It is important to mention that FoxD3 is already
present in the dorsal caudal-most NT at E4/HH24, in contrast to Msx1,
which is lacking (see Fig. 2
and Fig. S1 in the supplementary material). This indicates that the lack of
Bmp4-Wnt1 signaling has no consequence on the initial expression of
FoxD3, in contrast to that of Msx1.
Pre-specification or pluripotency of NCCs in the caudal-most region?
Several studies in both chick and zebrafish embryos have shown that late
migrating NCCs are able to counterbalance the loss of the early migrating NCCs
(Raible and Eisen, 1996;
Baker et al., 1997),
highlighting the high level of plasticity of these cells, as well as the role
of the environment in determining their fate
(Le Douarin et al., 2004).
However, in the caudal-most region, the NCCs generated later do not compensate
for the loss of the early migrating population, as they do not contribute to
the formation of neuronal derivatives. A similar observation has been reported
in other experimental situations (Maynard
et al., 2000). Cumulating evidence suggests that NCCs constitute a
heterogeneous population of cells containing both pluripotent and
fate-restricted progenitors (Harris and
Erickson, 2007). The existence of `pre-specified' lineages, an
early migrating lineage endowed with neurogenic potential and a later
migrating lineage with melanogenic ability, has already been described
(Erickson and Goins, 1995;
Henion and Weston, 1997;
Reedy et al., 1998). In light
of such observations, the early migrating population of NCCs that is absent in
the caudal-most region most probably corresponds to the population of cells
with neurogenic potential. In addition, the fact that no neuronal derivatives
are formed even when the caudal-most NT is transplanted into a more rostral
somitic environment indicates that this is an intrinsic property of all
caudal-most NCCs. It also suggests that the signals required for the
establishment of this neurogenic potential are not effective in the dorsal
caudal-most NT. Although formal in vivo evidence of such a pre-specified
population of neurogenic progenitors is still lacking in the chick embryo, its
existence has been demonstrated in zebrafish and mouse
(Raible and Eisen, 1994;
Wilson et al., 2004).
Variations along the AP axis in the mechanisms triggering NCC delamination
Despite maintained levels of Nog expression and lack of Bmp4-Wnt1
signaling in the caudal-most NT, NCCs are still able to delaminate at this
level in the chick embryo (see Fig.
1). This suggests that mechanisms other than those based on
Bmp-dependent Wnt signaling, currently proposed for the trunk
(Shoval et al., 2007), are
operating in this region. It is tempting to consider the existence of a
correlation between the type of mechanisms involved in NCC delamination and
the nature of their derivatives. In such a way, early emigrating NCCs
contributing to neuronal derivatives would be dependent on Bmp4-Wnt1
activities, whereas those migrating later and giving rise to melanocytes would
not. In addition, whilst we did demonstrate that Nog overexpression
in the trunk NT was able to mimic the main features of the caudal-most NT and
NCCs, we also ascertained some important differences. In this experimental
situation, we found decreased Cad6B expression in prospective NCCs,
whereas N-Cad was upregulated both in the dorsal NT and in the NCCs located
dorsally to it (see Fig. 6). In
the caudal-most region however, although Nog expression is
maintained, Cad6B is found in the prospective NCCs, whereas N-Cad is
absent from these cells (data not shown). This supports our statement that
mechanisms governing NCC delamination in the caudal-most region of the embryo
are specific to this region. It should be noted that specific Bmp
signaling-independent and Ets1 activity-dependent mechanisms have already been
reported in the control of cranial NCC delamination
(Théveneau et al.,
2007).
In spite of the obvious changes in the adhesion properties of the
prospective NCCs induced by ectopic Nog expression in the trunk NT,
we still found some delaminating NCCs. This is puzzling in the light of recent
data showing that Nog overexpression maintains N-Cad, causing an
almost complete failure of NCC delamination
(Shoval et al., 2007).
However, one possibility to explain this discrepancy might be of technical
order: in our experiments, we have used a concentration of 0.5 µg/µl of
DNA for Nog electroporation, whereas Shoval and colleagues used a DNA
concentration of 3-5 µg/µl. In fact, it seems that the effect of
Nog is dose-dependent, as we were able to completely block trunk NCC
delamination by using a 2.0 µg/µl concentration of Nog
expression plasmid (our unpublished data).
The Bmp-dependent Wnt signaling model, first described in relation to the
onset of trunk NCC delamination (Kalcheim
and Burstyn-Cohen, 2005), could be more complex than previously
believed. In fact, the results of our heterotopic transplantation experiments
raise important questions concerning the specific role of the epithelial
somites in the regulation of Wnt1 signaling by Nog downregulation.
Firstly, we found that Wnt1 was not restored in rostrally
transplanted caudal-most NT (see Fig.
5L,L') and secondly, caudal-most somites did not prevent
Wnt1 expression in the trunk NT (see
Fig. 5D,D'). This
suggests that the dorsal NT itself, in particular the caudal-most NT, plays an
important role in NCC delamination by its own ability to respond to the
somitic signal(s).
Nature of the signals involved in the generation of distinct NCC lineages
One main conclusion from our study concerns the identity of the Wnt signal
involved in melanocyte specification. Although the implication of Wnt
signaling in the generation of melanocytes has been clearly demonstrated,
previous studies have been unable to discriminate the exact nature of the
signal involved (Jin et al.,
2001; Ikeya et al.,
1997; Dorsky et al.,
1998; Hari et al.,
2002). Our results in the chick embryo suggest that the generation
of the later migrating NCCs, and thus melanocyte progenitors, specifically
depends on a Wnt3a, and not Wnt1, signal. This is supported by other data
showing that Wnt3a but not Wnt1 promotes melanocyte differentiation at the
expense of other derivatives (Dunn et al.,
2005). Nevertheless, we observed a large number of NCCs migrating
through the dorsal pathway after Wnt1 overexpression in the
caudal-most NT (see Fig. 7).
Ectopic EphB2 or EDNRB2 expression was recently shown to be
sufficient to induce dorso-lateral migration of non-melanogenic NCCs
(Harris et al., 2008). Ectopic
Wnt1 expression might produce a similar effect. In fact, NCCs
migrating along the dorsal pathway under these conditions are not
WRS+ (data not shown), indicating that they are not melanoblasts.
Wakamatsu et al. (Wakamatsu et al.,
1998) previously reported the brief presence of some NC-derived
neuronal cells on the dorsal pathway, before their removal by an episode of
apoptosis.
The nature of the signals controlling neuronal differentiation has been
more difficult to identify. Our results in the caudal-most region suggest that
both Bmp4 and Wnt1 are required for the generation of early migrating NCCs and
thus neuronal derivatives. In the chick and mouse embryos, Bmp and Wnt
signaling has been implicated in the generation of sympathetic and sensory
neuronal derivatives (Huber,
2006; Sommer,
2006). In addition, Wnt1 seems to have an instructive influence on
the sensory fate in multipotent NCCs
(Kleber et al., 2005;
Lee et al., 2004).
Importantly, we have shown that increased Bmp4-Wnt1 signaling in the
caudal-most NT induces NCCs to be specified as neurogenic progenitors (see
Fig. 7). At 48 hpe, the longest
survival time of the electroporated embryos that we were able to obtain, we
observed no definitive neuronal differentiation. It should be noted here that
in order to target the caudal-most NT, our electroporation experiments needed
to be performed at E4/HH24. This is precisely the stage at which a massive
apoptosis is occurring and thus a selective elimination of early migrating
NCCs might already have been underway. In addition, in the zebrafish embryo,
early migrating NCCs endowed with the ability to give rise to DRG neurons
require appropriate environmental factors to express this intrinsic ability
(Raible and Eisen, 1996).
Thus, we cannot exclude the possibility that an asynchrony between NT and
adjacent somite maturation contributes to the absence of peripheral ganglia in
the caudal-most region.
|
|
Footnotes |
|---|
Supplementary material available online at http://dev.biologists.org/cgi/content/full/136/10/1717/DC1
We thank Prof. Isabel Palmeirim for her friendship, scientific comments and unfailing support; and Prof. Carol A. Erickson, Dr Roberto Mayor and Dr Elizabeth Dupin for insightful discussions. We are grateful to Dr Jean-Loup Duband for use of the technical facilities, his scientific advice and his gift of the NC1 antibody; to the reviewers for their relevant comments; and to Claire Fournier-Thibault for valuable help in setting up the electroporation experiments. We thank Dr Catherine Jessus for her support; Dr Eric Théveneau for his remarks; and Dr Gillian Butler-Browne for manuscript edition. We are grateful to Dr James Briscoe for the Sox9 plasmid; Dr Jane Johnson for Ngn1 and Ngn2 plasmids; Dr Eric Agius for Bmp4 and noggin; Dr Sylvie Schneider-Maunoury for the mNog plasmid; and Dr Marion Wassef for Wnt1 expression plasmids. This work has been supported by the CNRS, UPMC, FCT and AFM. L.O. is a recipient of a grant from FCT (SFRH/BD/11858/2003) and from ARC.
* Present address: Department of Biochemistry, University of Hong-Kong,
Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Hong-Kong 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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