Institut de Biologie du Développement de Marseille, Laboratoire de Génétique et Physiologie du Développement, CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 9, France
* Author for correspondence (e-mail: kodja{at}ibdm.univ-mrs.fr)
Accepted 11 November 2004
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SUMMARY |
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Key words: Xenopus, Vertebrates, Chordates, Neural induction, FGF, BMP, SMAD6, eFGF, Spemann's organiser, Mesoderm, Ectoderm, SU5402
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Introduction |
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In the ectoderm, the interplay between BMPs and their antagonists, such as
Chordin (Chd), is currently considered as being central to neural induction
(reviewed by Munoz-Sanjuan and Brivanlou,
2002). Xenopus ectodermal explants, which normally
express Bmp4, develop as epidermis when cultured in the absence of
organiser signals. When cell contacts and communication are disrupted in these
explants, neural tissue develops instead. Epidermis formation is, however,
restored when dissociated explants are cultured in the presence of BMP4
protein. Conversely, intact ectodermal explants form neural tissue upon
overexpression of BMP inhibitors, suggesting that these factors recapitulate
neural induction by the organiser. This series of ex vivo experiments served
as a basis for a model, known as the default model, which states that neural
induction is a direct consequence of BMP inhibition in the ectoderm
(Munoz-Sanjuan and Brivanlou,
2002
). In this model, neural identity constitutes the ground state
within the ectoderm and is revealed in the absence of instructive signals.
A major difficulty with the default model is that it does not appear to
account for neural induction in avian embryos. It was shown that organiser
graft, but not BMP inhibition, leads to neural development in chick lateral
epiblast (Streit et al.,
1998). Only the cells located at the border between neural and
non-neural territories can take on a neural identity upon BMP inhibition,
suggesting that these cells are exposed to additional neuralising cues
(Streit and Stern, 1999
).
Recent reports have indicated that FGF may constitute one of these signals
(Streit et al., 2000
;
Wilson et al., 2000
). Similar
to organiser grafts, soluble FGF8 can induce early neural marker gene
expression in competent epiblast. The organiser does not, however, elicit
neural induction if FGF receptor (FGFR) activity is blocked, in spite of the
normal expression of chd in the graft
(Streit et al., 2000
).
Interestingly, some authors suggested that perhaps FGF signalling in the chick
is primarily involved in repressing the expression of Bmp genes
(Munoz-Sanjuan and Brivanlou,
2002
; Wilson and Edlund,
2001
). A similar role has been proposed for early ß-catenin
activity in the Xenopus ectoderm
(Baker et al., 1999
). According
to this hypothesis, BMP inhibition in the chick, like in Xenopus,
remains central to neural fate acquisition, and is achieved both via
transcriptional repression and the activity of secreted antagonists. However,
recent experiments on chick epiblast explants have suggested that FGF
signalling also functions in a BMP-independent manner during neural induction
(Wilson et al., 2001
). Recent
work in ascidians, which represent basal chordates, suggested that BMP
inhibition is not involved in neural specification
(Darras and Nishida, 2001
),
but that, here also, FGF plays an essential role as a direct inducer of early
neural genes (Bertrand et al.,
2003
).
By contrast, FGFR signalling in Xenopus is currently considered to
be essential for posterior neural development but not for neural induction
(reviewed by Munoz-Sanjuan and Brivanlou,
2001; Munoz-Sanjuan and
Brivanlou, 2002
). This conclusion is primarily based on
overexpression of a dominant-negative form of the FGFR1 receptor, which does
not apparently suppress the formation of anterior neural features
(Amaya et al., 1991
;
Holowacz and Sokol, 1999
;
Kroll and Amaya, 1996
;
Ribisi et al., 2000
). However,
several reports using antimorphic FGFR1 or FGFR4 receptors have suggested
instead that FGF signalling plays a role during neural tissue development,
leaving this controversial issue still unresolved
(Hongo et al., 1999
;
Launay et al., 1996
;
Pera et al., 2003
;
Sasai et al., 1996
).
We therefore made use of a broad panel of molecular and pharmacological tools to readdress the roles of the BMP and FGF signalling pathways during neural induction in Xenopus embryos. The in vivo results presented here are consistent with a model whereby pre-gastrula FGF signalling functions in addition to BMP inhibition to induce the nervous system, thus providing an evolutionary conserved alternative to the default model.
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Materials and methods |
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SU5402, and Noggin protein treatments
SU5402 (Calbiochem) was dissolved in DMSO (120 mM), and diluted in
0.1xMBS for whole-embryo treatments, and in a low Ca2+, low
Mg2+ Ringer's solution for animal explants treated with Noggin
protein (LCMR, 43 mM NaCl, 0.85 mM KCl, 0.37 mM CaCl2, 5 mM HEPES,
pH 7.2, 50 µg/ml gentamycin). We did not observe any effect of SU5402 below
5 µM. The severity of the phenotypes exhibited by SU5402-treated embryos
increased in a dose-dependent manner. We arbitrarily defined five phenotypic
classes at tadpole stage (Fig.
3A): class I embryos showed a slightly truncated tail; class II
embryos lacked a tail and showed a slightly shorter trunk; class III embryos
lacked all trunk and tail tissues; class IV embryos additionally showed a
reduced head; class V embryos lacked all axial tissues, including head
structures, but still formed a cement gland. As we noticed a variability
between experiments in the amount of inhibitor necessary to yield class V
embryos, we used in each experiment several concentrations ranging between 80
and 160 µM, and report data regarding the fifth class irrespective of the
concentration. There was no obvious defects in cell cleavage, or in cell
viability within the range of concentration used here. Gastrula stage
SU5402-treated embryos were classified according to the phenotype of sibling
embryos treated identically and grown up to tailbud or tadpole stage. Mouse
recombinant Noggin/Fc chimeric protein (R&D systems) was resuspended in
PBS, 0.1% BSA (50 µg/ml). Animal explants were treated with 2 µg/ml
Noggin/Fc in LCMR supplemented with 0.5% BSA (47% of the fusion protein
consists of the Noggin peptide), and stage 8 embryos were injected with 2 ng
Noggin/Fc in the blastocoel.
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Results |
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We first analysed the fate of presumptive neural cells subjected to ectopic
BMP activation, via RNA microinjection of a constitutively active BMP receptor
(CABR) in a single dorsal animal blastomere at the eight- or 16-cell
stage. At the late gastrula stage, injected cells did not express the early
neural markers Sox3 (9/10 negative) and Sox2 (45/47
negative), and instead expressed the epidermal marker cytokeratin 81
(K81) (35/35 positive; Fig.
1A, and not shown). Thus, consistent with ex vivo assays
(Munoz-Sanjuan and Brivanlou,
2002), BMP activation in vivo is incompatible with neural
induction, and elicits epidermis differentiation.
|
Another possibility to explain the lack of neural induction in vivo in our assays could be that ventral ectoderm cells are not able at all to respond to BMP inhibitors. We turned back to the animal cap assay to address this issue. We injected 1 ng Smad6 RNA in a single ventral ectoderm cell at the 16-cell stage, explanted animal caps at late blastula stage, and analysed Sox2 expression in caps and in intact injected siblings. We found that neuralisation occurred in response to Smad6 expression in injected cells in explanted animal caps (15/24 Sox2 positive; Fig. 1G), but not in intact embryos (11/11 Sox2 negative; Fig. 1E). These results indicate that our conditions of BMP inhibition are indeed sufficient to neuralise ventral ectoderm cells, which further supports the idea that the lack of neural induction by BMP inhibition in vivo is not merely quantitative. This also demonstrates that extirpation of the blastula ectoderm modifies the response to BMP inhibitors, which suggests that animal cap assays do not unambiguously reflect neural induction in the embryo. In summary, BMP/SMAD1 inhibition in vivo is sufficient to neuralise ectoderm cells in the vicinity but not away from the endogenous neural plate, suggesting that, as in the chick, additional inputs are required for neural induction.
Neural induction in vivo by conjugated FGF signalling and BMP inhibition
Because of works in amniotes (Streit et
al., 2000; Wilson et al.,
2000
; Ying et al.,
2003
) and ascidians (Bertrand
et al., 2003
), we asked whether FGF signalling could play such a
role by testing eFGF, a frog homolog of FGF4, in our ventral
ectoderm assay. Consistent with previous work
(Isaacs et al., 1994
),
injection of 3.2 pg eFGF RNA induced mesoderm as revealed by
Xbra expression (23/23 positive), within and around injected
cells (not shown). By contrast, injection of 0.16 pg eFGF RNA did not
activate Xbra expression (9/9 negative), nor did it significantly
suppress K81 expression (4/10 no repression; 6/10 light reduction,
example in Fig. 2A, centre), or
activate Slug (12/12 negative, not shown) or Sox2 expression
(11/12 negative; Fig. 2A
right). This suggests that at this dose eFGF does not efficiently
antagonise the BMP pathway, which we showed in the previous section is a
necessary condition for neural induction. We thus co-injected 0.16 pg
eFGF and 1 ng Smad6 RNAs, and found that this was now
sufficient to repress K81 (12/12 negative) and activate Sox2
(21/21 positive), in absence of Xbra expression (20/20 negative) at
the late gastrula stage (Fig.
2B). Consistent with the activation of a stable neural programme,
the injected territory was subsequently found to express the differentiation
marker NCAM at tailbud stage (10/10 positive;
Fig. 2C). Co-injection of 0.16
pg eFGF and 200 pg tBR RNAs gave similar results (11/11
K81 negative; 9/13 Sox2 positive; 12/12 Xbra
negative; Fig. 2D). FGF signals
can activate multiple transduction pathways, including the ras/MAPK pathway
(Powers et al., 2000
). As this
pathway is active in the early frog embryo
(Schohl and Fagotto, 2002
), we
asked whether activated ras was able to induce ectopic neural tissue in
BMP-deficient epidermis. We found that co-injection of 1 pg v-ras and
1 ng Smad6 RNAs led to Sox2 activation (12/12 positive),
without Xbra expression (11/11 negative;
Fig. 2E and not shown). We
conclude that in vivo and in the absence of detectable mesoderm induction,
weak FGF or ras signalling combined with BMP inhibition is sufficient to
induce neural tissue in ventral ectoderm.
|
As ras functions downstream of FGF receptors, we asked whether it could bypass the action of SU5402, and directly rescue neural plate tissue. We found that animal injection of 5 to 20 pg v-ras RNA could restore Sox2 expression in late gastrula class V embryos (14/14 Sox2 negative in class V; 12/23 Sox2 positive upon v-ras injection; Fig. 4B,C). Consistently, v-ras also rescued NCAM expression in most SU5402-treated embryos at tailbud stage (not shown).
|
Neural induction by FGF signalling is initiated prior to gastrulation
To assay the temporal requirements for FGFR activity upon neural
development, we made use of the reversibility of the SU5402 action following
washing, and varied the time of initiation and duration of treatment
(Fig. 5). First, we found that
treatment initiated at the two-cell stage and interrupted at the mid-blastula
transition (MBT) yielded normal embryos. This confirms that SU5402 can be
washed away, and indicates that FGFR activity is dispensable prior to MBT.
Treatments initiated at the MBT induced phenotypes progressively more severe
as their duration increased. Treatment starting at MBT and lasting 5.5 hours
yielded class V embryos, whereas embryos developed anterior neural tissue
(class IV) when the same 5.5 hours treatment started 2 or 3 hours past MBT.
This early requirement for FGFR activity is consistent with the loss of
Sox2 and opl expression in class V early gastrulae
(Fig. 3J,L). Thus, as in the
chick (Streit et al., 2000;
Wilson et al., 2000
),
Xenopus neural induction in vivo requires pre-gastrula FGFR
activity.
|
First, we analysed the effect of restricting R4 expression
to ectoderm cells by injection at the eight-cell stage in the two dorso-animal
blastomeres (200 pg/blastomere). We found that at the early gastrula stage
injected ectoderm cells did not express Sox2 (24/24 negative), thus
supporting a direct requirement for FGF signalling at the time of neural
induction in presumptive neural cells (Fig.
4J,K). We note that this is consistent with previous studies
showing that animal expression of
R4 suppressed NCAM or
N-tubulin expression, although these data could have been interpreted
in terms of a requirement of FGF signalling for the maintenance, rather than
induction of neural fates (Hongo et al.,
1999
; Pera et al.,
2003
).
In a second series of experiments, we addressed whether ectoderm submitted to FGF inhibition could respond to the natural neural inducers produced by the organiser. Thus, organiser mesoderm was recombined with ectoderm from embryos treated or not with 80 µM SU5402 until the time of explantation, and washed before recombination (Fig. 6A). When compared with controls, pre-treatment with SU5402 caused a 50% reduction in the number of NCAM-positive conjugates made at the early gastrula stage (Fig. 6C). This reduction rose to 80% when recombination was performed at the mid-gastrula stage (Fig. 6C). Moreover, not only the number of NCAM-positive conjugates dropped dramatically upon pre-treatment with SU5402, but the amount of NCAM-positive cells within these conjugates, and the intensity of the staining in those cells were severely reduced (asterisks in Fig. 6B). We further noticed that in most cases, NCAM-negative conjugates still contained a cement gland (arrows in Fig. 6B), similar to what is observed in class V whole embryos (Fig. 3A). We conclude that FGFR activity is required to confer to the ectoderm its capacity to become neural in response to organiser signals.
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Discussion |
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The default model of neural induction is based on assays where BMP
signalling is manipulated in frog ectoderm explanted at blastula stages. In
the most recent version of this model, ectoderm cells become epidermis when
submitted to strong BMP signals, they adopt a border fate (including neural
crest and cement gland) when submitted to weaker BMP signals, and they become
neural in absence of BMP signalling
(Munoz-Sanjuan and Brivanlou,
2002). Using assays in whole embryos, we first confirmed that BMP
signalling promoted epidermis formation, and repressed neural development.
Hence, epidermis development appears to be dominant over neural development.
However, reducing BMP/SMAD1 signalling in the ventral ectoderm distant from
the endogenous neural plate, to a level incompatible with epidermis, cement
gland and neural crest development, did not elicit neural specification,
strongly arguing against the default model. We demonstrate, however, that
under these conditions, BMP inhibition is sufficient to neuralise ventral
ectoderm cells upon explantation (Fig.
1G), making it unlikely that the lack of neural induction in vivo
is only due to partial BMP inhibition. These results argue that neural
induction in vivo does not consist of a simple epidermis-to-neural, or neural
border-to-neural switch, which would be regulated by distinct levels of BMP
signalling. Although it is necessary that BMP be downregulated for epidermis
and neural border to be suppressed, an additional signal(s) is required to
initiate neural induction in the BMP-negative territory.
Several arguments allow us to propose that FGF signalling plays a positive
role in this process. First, eFGF could transform BMP-deficient cells into
neural progenitors in vivo, without inducing mesoderm. Second, using all
available antimorphic FGF receptors and a pharmacological FGFR inhibitor, we
found that the earliest steps of neural tissue development critically and
specifically depends on FGF activity. These results confirm and extend
previous studies. We note that in our assays, R4 misexpression
consistently led to more penetrant neural deficiencies than XFD, when equal
amounts of RNAs were injected. This could suggest that FGFR4 is the main
receptor involved in neural induction at blastula stages, in agreement with
its higher level of expression (Hongo et
al., 1999
). Support for this idea also comes from the observation
that tR4, a form of FGFR4 that is active in the absence of FGF ligands, can
rescue neural phenotypes generated by
R4. In addition, our data
indicate that the transduction pathway at work downstream of FGF receptors may
involve ras, as it is sufficient to induce ectopic neural tissue in
BMP-deficient ectoderm, and it can rescue neural plate tissue in
SU5402-treated embryos. Our work thus provides initial hints as to the
transduction cascade responsible for neural induction in frogs.
Interestingly, we have shown that neural induction in intact embryos
requires FGF activity at pre-gastrula stages
(Fig. 4J,K,
Fig. 5). This early requirement
for FGF signalling appears to be conserved between Xenopus (this
work), zebrafish (Furthauer et al.,
2004), chick (Streit et al.,
2000
; Wilson et al.,
2000
) and even ascidians
(Bertrand et al., 2003
). As
this period of development is anterior to the formation of Spemann's
organiser, a possible interpretation of our results would be that neural
induction is initiated by FGF signalling prior to gastrulation, and that this
is maintained by organiser signals. However, it is equally possible that
signals produced by the organiser, and in particular BMP antagonists, also act
in the blastula at the time of FGF requirement
(Kuroda et al., 2004
;
Wessely et al., 2001
). Thus,
further work is needed to compare the periods of requirement of FGF signalling
and BMP inhibition for the normal activation of the neural programme.
We provide here multiple evidence that FGF signalling plays a direct role
in the ectoderm during the process of neural induction. First, eFGF can
cooperate with BMP inhibitors to neuralise ventral ectoderm cells in vivo,
without mesoderm induction. Second, targeted R4 injection in
presumptive neural plate cells is sufficient to suppress early neural marker
gene expression. Third, ectoderm explants submitted to the FGFR inhibitor
SU5402 showed a severely reduced ability to respond to neural-inducing signals
produced by the organiser, or to BMP inhibitors. In summary, both gain- and
loss-of-function experiments suggest an essential role of FGF signalling
directly in the ectoderm, consistent with ERK activation in prospective neural
tissue at blastula/gastrula stages (Schohl
and Fagotto, 2002
; Uzgare et
al., 1998
).
In chick, the neural-inducing activity of FGF seems to be accounted for by
both BMP-dependent and -independent functions. Our results point to a similar
situation in Xenopus. In support of a BMP-dependent action of FGF,
Bmp4 expression is upregulated in the dorsal ectoderm of
FGFR-deficient embryos, which appears to be a conserved feature in vertebrates
(Furthauer et al., 2004;
Wilson et al., 2000
).
Moreover, noggin expression is lost in class V embryos, which is
further expected to upregulate BMP signalling. Finally, as it has been
reported that activated ERK could inhibit SMAD1
(Pera et al., 2003
), this
mechanism could also attenuate BMP signalling in response to FGF. However, FGF
does not appear to act solely via inhibition of BMP/SMAD1 signalling. First,
the amount of eFGF that was found sufficient for neural specification in vivo,
when combined to BMP inhibitors (tBR or SMAD6), does not efficiently repress
epidermis formation on its own, nor does it activate a border fate
(Fig. 2A). As both SMAD6 and
FGF/ras are expected to block SMAD1 activity, the complementation reported
here between these compounds is unlikely to be simply additive, and suggests
that FGF/ras provide an independent information. Second, the loss of neural
tissue because of FGFR inhibition in whole embryos cannot be rescued by
increased BMP/SMAD1 inhibition (Fig.
8C,D,H). As this issue is crucial to propose a
BMP/SMAD1-independent function of FGF, we attempted to block the BMP pathway
simultaneously at multiple levels, outside the cell (Noggin), at the
cell-surface (tBR) and inside the cell (SMAD6). Even in this case, no neural
tissue was recovered in the complete absence of FGF activity. Although it is
impossible to rule out that some very weak residual BMP signalling may persist
in this assay and explain the lack of neural tissue, this possibility appears
unlikely. Thus, we conclude that FGFR may function during neural induction via
both BMP-dependent and BMP-independent routes
(Fig. 9). The latter response
is probably playing a crucial role directly on the future neurectoderm, as we
show that FGFR-deficient animal caps cannot be neuralised by organiser tissue
or BMP antagonists. Future work will aim to understand the nature of this
response. Indeed, final proof for a BMP-independent action of FGF signalling
requires the identification of a direct transcriptional target of the pathway
in the neurectoderm, the activity of which would be required for neural
specification, a situation that has so far been reached only in Ciona
intestinalis (Bertrand et al.,
2003
).
|
An important aspect of this work is that both gain- and loss-of-function
experiments indicate the existence of a gradient of FGF activity that patterns
the early embryo. The formation of prospective dorsal mesoderm expressing
Xbra occurs at a higher level of FGF signalling than that of the
presumptive neural tissue expressing Sox2
(Fig. 2). Likewise, posterior
neural tissue requires higher levels of FGF signalling than anterior neural
tissue (Fig. 3). Thus,
incomplete inhibition of the pathway in the embryo may be sufficient to
prevent mesoderm and posterior neural tissue formation, without interfering
with anterior neural induction, providing a likely explanation for the
maintenance in previous studies of head features in FGFR-compromised embryos
(Amaya et al., 1991;
Holowacz and Sokol, 1999
;
Kroll and Amaya, 1996
;
Ribisi et al., 2000
). Our
results indicate that weak FGF signalling is sufficient, in a BMP negative
context, to promote neural identity, which makes complete inactivation
particularly difficult to obtain and to ascertain.
We attempted to represent some of the most important genetic relationships
relating to Xenopus neural induction in a schematic model
(Fig. 9). In this model,
presumptive mesoderm, neural ectoderm and epidermis are positioned at blastula
stages in response to the maternal activities of VegT and ß-catenin,
relayed by the FGF and Nodal-related pathways. ß-catenin and FGF
signalling repress Bmp4 and activates BMP inhibitors (chordin,
noggin), which generates a BMP-free region in the ectoderm, a condition
necessary for neural tissue formation
(Baker et al., 1999;
Wessely et al., 2001
).
Consistently, ß-catenin is broadly activated in dorsal mesoderm and
ectoderm, where it activates FGF/ERK signalling
(Schohl and Fagotto, 2002
;
Schohl and Fagotto, 2003
). The
epidermis forms where BMP function is maintained in the ectoderm. While
high-level FGF signalling combined with Nodal-related activity specify
mesoderm in marginal cells, weak FGF signalling induces neural tissue in
BMP-negative animal cells. This dose-dependent effect is consistent with the
graded pattern of ERK activation seen in the embryo
(Schohl and Fagotto, 2002
).
Further evidence for the ability of vertebrate embryonic cells to decide
between a mesodermal and a neural fate include the conversion of mesoderm into
neural cells in mouse and fish mutants of the FGF, Wnt and Nodal pathways, and
in mutants of the T-box family (Chapman and
Papaioannou, 1998
; Ciruna et
al., 1997
; Feldman et al.,
2000
; Yoshikawa et al.,
1997
).
In conclusion, our work uncovers the crucial role played by pre-gastrula FGF signalling during Xenopus neural induction, in addition to the well-documented inhibition of BMP. The evidence presented here appears incompatible with the leading default model, and instead provides support for a shared use of FGF signalling in neural induction across the chordate phylum.
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ACKNOWLEDGMENTS |
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