1 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
2 Laboratory of Molecular Genetics, National Institute of Child Health and Human
Development, National Institute of Health, Bethesda, MD 20892, USA
3 MRC Centre for Developmental Neurobiology, New Hunt's House, Kings College
London, London SE1 9RT, UK
* Authors for correspondence (e-mail: t.kudoh{at}ucl.ac.uk or s.wilson{at}ucl.ac.uk)
Accepted 14 April 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Zebrafish, Default model, Neural induction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the Bmp signalling pathway is likely to play a role in neural and
epidermal development in all vertebrates, a variety of studies have challenged
the idea that antagonism of Bmp activity is sufficient to induce neural tissue
(Stern, 2001;
Stern, 2002
;
Wilson and Edlund, 2001
).
Indeed, several other signals may have roles in neural induction
(Bally-Cuif and Hammerschmidt,
2003
) and among these, Fgfs are perhaps the most extensively
studied candidates (Akai and Storey,
2003
). Fgfs can induce neural (or prospective neural) identity
both in stem cells (e.g. Ying et al.,
2003
) and when ectopically expressed in vivo (e.g.
Bertrand et al., 2003
;
Furthauer et al., 1997
;
Kengaku and Okamoto, 1995
;
Koshida et al., 2002
;
Lamb and Harland, 1995
;
Sheng et al., 2003
;
Streit et al., 2000
;
Wilson et al., 2000
); in
addition, abrogation of Fgf activity can in some circumstances disrupt neural
induction (e.g. Bertrand et al.,
2003
; Hongo et al.,
1999
; Streit et al.,
2000
; Wilson et al.,
2000
).
In this study, we attempt to resolve the relative contributions of organiser-derived Bmp antagonists and Fgf signals to the initial steps of neural induction in zebrafish embryos. We show that Fgf activity, rather than Bmp antagonism, initiates development of prospective vegetal neural tissue that contributes to trunk and tail CNS. In vegetal prospective neural tissue, Bmp activity does not antagonise the induction of prospective neural markers, rather it promotes the ability of cells to contribute to caudal neural ectoderm. Therefore, at gastrula stages, the role of Bmp activity in the animal and vegetal ectoderm is different. In animal ectoderm, high levels of Bmp activity push cells towards a non-neural rather than neural fate whereas in vegetal ectoderm, differential levels of Bmp activity influence the regional (rostral to caudal) character of the neural tissue.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fate mapping
Zebrafish embryos were injected with caged fluorescein (Molecular Probes)
at the one-cell stage. Photoactivation of fluorescent dye was performed as
previously described (Rohr and Concha,
2000) at around the 70-80% epiboly stage. Following
photoactivation, DIC and fluorescent images were acquired using Openlab
software (Improvision) with a cooled CCD camera (Hammamatsu) attached to an
Axioplan microscope (Zeiss).
mRNA synthesis, injection of mRNA or morpholinos and cell transplantations
Capped RNA was synthesised by mMessage mMachine SP6 kit (Ambion) according
to the manufacturer's instructions. RNA concentrations used for injection
were: bmp2b (50 pg); fgf3 (50 pg); XFD (300 pg);
chordin (50 pg); and eGFP (100 pg). mRNAs were injected into cells at
one- to two-cell stage in all blastomeres while the morpholino for the
chordin gene (500 pg) (Nasevicius
and Ekker, 2000) was injected at the one- to two-cell stage in the
yolk. More than 15 embryos were examined in each injection experiment.
For transplantation, donor embryos were injected with various RNA
constructs and cells removed at blastula or early gastrula stages using a
microelectrode connected to a Hamilton syringe as previously described
(Houart et al., 2002). Donor
cells were gently aspirated into various positions of similar stage host
embryos. To detect donor cells expressing GFP, embryos were labelled by
peroxidase conjugated anti-GFP antibody (x1000 dilution) (TP401, Torrey
Pines Biolabs) and detected by DAB substrate reaction after the in situ
hybridisation protocol. In cases where two populations of cells were
co-transplanted, donors were distinguished by injection with either
fluorescein dextran (green) or rhodamine dextran (red).
Donor embryos injected with constructs encoding both Fgf3 and the truncated Fgf receptor XFD, did not show an Fgf gain-of-function phenotype and instead showed a phenotype similar to embryos expressing XFD alone. This suggests that cells from these embryos produce, but are compromised in their ability to respond to, Fgf signals. In induction assays, it is likely that Fgf3 from these cells acts directly on host cells as opposed to acting indirectly by inducing the expression of other signals within the donor cells.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Bmp antagonists and other organiser-derived signals are not essential for induction of markers of prospective neural tissue in vegetal regions of the ectoderm
Most models suggest that neural induction is dependent upon secreted
molecules, notably Bmp antagonists, emanating from the dorsal organiser
(Kodjabachian et al., 1999;
Munoz-Sanjuan and Hemmati-Brivanlou,
2002
); but the presence of prospective neural tissue in the
ventrovegetal region of the gastrula challenges this idea. We therefore
performed several analyses to test if Bmp antagonists or other
organiser-derived signals are necessary for the expression of markers of the
vegetal, prospective neural ectodermal domain. Vegetal expression of the
prospective neural markers sox3 and zic2.2 is unaffected
following overexpression of bmp2b (n=55), in mutant embryos
lacking the activity of the Bmp antagonist, Chordin (din)
(n=9), and in ichabod-/- embryos (n=20)
that are defective in ß-catenin signalling and entirely lack the
organiser (Hammerschmidt and Mullins,
2002
; Kelly et al.,
2000
) (Fig. 3A-J).
In all these situations, rostral neural tissue is reduced or absent, whereas
expression of the epidermal marker p63 extends further dorsally
consistent with previous observations (Fig.
3K-O) (Hammerschmidt and
Mullins, 2002
; Kelly et al.,
2000
). By contrast, irrespective of the severity of the
ventralised phenotype, p63 expression does not expand vegetally,
always retaining a gap between its expression domain and that of ntl
in the nascent mesoderm (Fig.
3O). These results indicate that expression of markers of
prospective vegetal neural tissue is not dependent upon the organiser. They
also suggest that induction of these markers is either unaffected by high
levels of Bmp signalling or that Bmp activity in vegetal regions of the
gastrula can be antagonised by mechanisms independent of the organiser.
|
Expression of vegetal ectodermal markers was examined in embryos that had
been treated from the 1024-cell stage with SU5402, an inhibitor for Fgf
receptor activity (Mohammadi et al.,
1997) that should abrogate activity of zygotically encoded Fgf
signals emanating from the germ ring. In embryos treated with 60 µM SU5402,
vegetal sox3 expression is variably decreased, whereas the animal
(anterior) domain of sox3 expression expands vegetally on the dorsal
side of the embryo (Fig. 4A-D).
With a higher dose of SU5402 (90 µM), more than 90% of embryos showed a
severe phenotype equivalent to that shown in
Fig. 4D (n>25). In
these embryos, the vegetal ectodermal markers sox31 and
zic2.2 behaved similarly to sox3, showing loss of
ventrovegetal expression and retention of dorsal expression
(Fig. 4E-H). Supporting the
notion that the remaining prospective neural tissue on the dorsal side of
these embryos has anterior character, expression of otx2, which is
restricted to the prospective anterior neural tissue of gastrula stage
wild-type embryos, is expanded vegetally on the dorsal side of the embryo
(Fig. 4I,J) (see also
Koshida et al., 1998
;
Kudoh et al., 2002
).
Complementing the changes in prospective neural genes, expression of the
prospective epidermal marker, p63, was expanded ventrovegetally into
the territory from which prospective neural marker gene expression is lost
(Fig. 4K,L). Similar results
(Fig. 5A-C), were obtained
through overexpression of a truncated, dominant-negative Fgf receptor, XFD
(Amaya et al., 1991
;
Griffin et al., 1995
). These
data support the idea that induction of vegetal prospective neural tissue is
dependent on Fgf activity and that the residual neural tissue that remains
after Fgf receptor blockade has anterior character.
|
|
Exogenous Fgf activity is able to promote expression of prospective neural markers even when the Bmp signalling pathway is active
Complementing the Fgf loss-of-function studies, increasing Fgf3 activity
leads to induction of ubiquitous ectodermal expression of sox3, sox31
and zic2.2 (Fig.
5E,H,K) (Koshida et al.,
2002; Furthauer et al.,
1997
) and this induction is unaffected by overexpression of
bmp2b (Fig. 5F,I,L)
(>90%, n>15). This suggests either that Fgf can induce
expression of prospective neural genes in the presence of high levels of Bmp
activity or that Fgf activity can block Bmp signalling.
Previous studies have shown that one mechanism by which Fgf signalling can
indirectly induce neural tissue is by antagonising Bmp activity
(Furthauer et al., 1997;
Koshida et al., 2002
;
Pera et al., 2003
;
Wilson et al., 2000
). We
consequently asked if abrogation of Bmp signalling by Fgf activity is
necessary for Fgf activity to induce sox3 expression. Overexpression
of fgf3 leads to widespread induction of chd expression,
which contributes to suppression of bmp4 expression [and presumably
lowered Bmp activity (Koshida et al.,
2002
); Fig. 5N,Q;
>90%; n>30]. In this situation, the expanded expression of
prospective neural markers (Fig.
5E,H,K) could, therefore, be due to suppression of Bmp activity.
However, co-expression of bmp2b in embryos overexpressing
fgf3 suppresses chd expression and expands bmp4
expression (Fig. 5O,R)
(>80%, n>30), and yet despite this, the ectoderm still
ubiquitously expresses prospective neural markers
(Fig. 5F,I,L). Therefore, the
Bmp pathway can be activated in the presence of Fgf signalling without
affecting induction of prospective neural gene expression. These data indicate
that although Fgf activity can suppress Bmp signalling by promoting expression
of Bmp antagonists [and by other means (e.g.
Pera et al., 2003
)], this is
not necessary for Fgf signalling to be able to induce markers of prospective
neural identity.
Cells unable to receive Fgf signals fail to contribute to caudal neural tissue
To further explore the epistatic relationships between the Fgf and Bmp
pathways, we examined the consequences of locally activating or suppressing
Fgf signalling. fgf3-expressing ectodermal cells transplanted into
animal pole regions of host embryos induce sox3 non-autonomously in
surrounding host cells (Fig.
6A; >80%; n>20). This induction still occurs if the
donor cells are from embryos co-expressing a truncated Fgf receptor
(Fig. 6B), suggesting that the
Fgf signal from the donor cells acts directly on the host (100%;
n=19). When both donor and host cells are overexpressing bmp2b,
fgf3-expressing cells still induce sox3 and suppress expression
of the epidermal marker gene, foxi1
(Fig. 6C,D; 100%;
n=22), again suggesting that exogenous Bmp activity does not block
induction of prospective neural marker genes by Fgf.
|
To directly assess if Fgf signals are essential for vegetal ectoderm to
form neural tissue, we traced the eventual fate of XFD-expressing donor cells
transplanted to wild-type hosts. In these experiments, labelled wild-type
cells were co-transplanted with XFD-expressing cells to the same locations in
the vegetal ectoderm of unlabelled host embryos at the end of blastula stage.
When the donor cells were transplanted to dorsal side, wild-type cells
primarily contributed to the hindbrain, whereas the XFD-expressing cells
localised more anteriorly, mainly in the midbrain (n=14). However,
when transplanted to ventral vegetal ectoderm, wild-type donor cells
contributed to spinal cord and muscle (see also
Kimmel et al., 1990) whereas
XFD-expressing cells were excluded from the CNS and found in tissues such as
the epidermis and fin (n=8). These results suggest that Fgf
signalling is required for vegetal ectoderm to contribute to caudal neural
tissue. They also suggest that the consequences of suppression of Fgf
signalling in cells in dorsal and ventral domains of the vegetal ectoderm are
different: dorsally, cells with compromised Fgf signalling frequently move
into anterior neural tissue; ventrally, cells move into the prospective
epidermis and are excluded from neural tissue. These results are consistent
with analyses of embryos in which XFD is expressed ubiquitously (e.g.
Griffin et al., 1995
)
(Fig. 3) and which show loss of
posterior neural structures and anteriorisation of remaining CNS tissue on the
dorsal side of the embryo.
Bmp and Fgf activities in ventrovegetal regions of the ectoderm cooperatively promote prospective caudal neural fate
If Bmp signalling can still occur in vegetal ectoderm without suppressing
sox3 expression, then this raises the issue of what Bmp activity is
doing in this domain. Within animal pole ectoderm, Bmp activity promotes
expression of markers of ventral, non-neural, ectodermal fates (such as
p63 and foxi1) at the expense of expression of dorsal neural
markers [such as otx2
(Hammerschmidt and Mullins,
2002); Fig. 7B,
parts i,iv; C, parts i,iv]. In vegetal ectoderm, DV organisation does not
correspond to neural versus non-neural fates, but rather is predictive of the
anterior-to-posterior (AP) position that neural cells will occupy within the
caudal CNS (Fig. 2). This
raises the possibility that in vegetal ectoderm, Bmp activity promotes
regional specification within the prospective neural ectoderm rather than
neural/non-neural fate specification. Supporting a global role for Bmp
signalling in promoting ventral fates in the gastrula, irrespective of their
neural or non-neural destiny, expression of the ventral vegetal marker
eve1 is induced by overactivation of Bmp signalling, whereas the
dorsal vegetal marker hoxb1b is suppressed
[Fig. 7B, parts ii,iii; C, part
ii,iii; similar results have been observed in mutants or embryos with other
modulations affecting Bmp signalling (e.g.
Bakkers et al., 2002
;
Hammerschmidt et al., 1996
;
Mullins et al., 1996
)]. As
cells within the domain of eve1 expression of wild-type embryos give
rise to more posterior neural fates than cells within the ectodermal domain of
hoxb1b expression, then this suggests that graded Bmp activity in
vegetal ectoderm contributes to the allocation of fates along the AP axis of
the neural tube rather than to a neural versus non-neural fate choice.
|
Alterations to Bmp activity in embryos lacking Chordin function affects the allocation of trunk versus tail fates
To explore the possibility that in vegetal regions of the ectoderm, Bmp
signalling promotes the adoption of caudal neural fate, we examined marker
gene expression and fates of vegetal ectoderm in din-/-
mutants/morphants in which Bmp activity is enhanced. In
din-/- embryos, the caudal marker, eve1 is
expanded to lateral vegetal ectoderm
(Hammerschmidt et al., 1996)
(Fig. 8B), whereas vegetal
sox3 expression is not significantly altered
(Fig. 3B,
Fig. 8D). Furthermore, while
exogenous Fgf3 induces sox3 throughout the ectoderm both in
din-/- and in wild-type embryos
(Fig. 8G,H), eve1 is
suppressed in wild-type embryos (n=37), whereas expression expands
throughout most of the sox3-expressing ectoderm in
din-/- mutants (n=13)
(Fig. 8E,F) with the exception
of the dorsal most ectoderm which retains hoxb1b expression in this
condition (Koshida et al.,
2002
). These data suggest that a key role for Chordin in the
vegetal ectoderm is to suppress eve1 and tail formation, and raise
the possibility that cell fate in the lateral vegetal ectoderm may be changed
from trunk neural to tail neural fate in din-/- mutants.
Indeed, the size of trunk may be smaller and tail bud larger in
din-/- embryos compared with wild-type embryos
(Hammerschmidt et al., 1996
;
Myers et al., 2002
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Bmp signalling may promote caudal neural fate by regulating cell movements
Given that Bmp activity may not directly specify cell identity (as it
promotes several distinct fates for ventrally positioned cells), it is
intriguing to consider how this pathway can promote the adoption of caudal
neural fate. An attractive possibility is that it does so by regulating the
movements of gastrulating cells. Solnica-Krezel and her colleagues
(Gonzalez et al., 2000;
Myers et al., 2002
) have
analysed movements of gastrulating cells in embryos with different levels of
Bmp activity. They have shown that laterally positioned cells in the wild-type
gastrula move dorsally, whereas the ventral-most cells fail to converge
dorsally and consequently end up in the tail bud area. In
din-/- embryos, the domain of non-convergence is expanded
laterally with the likely consequence that more cells end up in the tail bud.
Therefore, if Bmp-dependent regulation of dorsal convergence also occurs
within the ectoderm, then this model provides an attractive explanation of why
laterally positioned prospective neural cells have a higher likelihood of
ending up in the tail spinal cord in din-/- embryos.
If the role of the Bmp pathway in caudal development is primarily to
regulate cell movements, then other pathways may cooperate with Bmps to impose
caudal cell fates. Several candidates are well documented
(Munoz-Sanjuan and Hemmati-Brivanlou,
2001), and in fish, Agathon and colleagues
(Agathon et al., 2003
) have
recently shown that entire tail structures could be induced by simultaneous
activation of Bmp, Nodal and Wnt pathways. Although a role for Wnt signalling
in posterior development is supported by many other studies (e.g.
Erter et al., 2001
;
Hashimoto et al., 2000
;
Marlow et al., 2004
), a
requirement for Nodal signalling to form tail tissue is less clear given that
fish embryos lacking Nodal activity do still form tails that contain neural
tissue (Feldman et al., 2000
).
Nevertheless, it is likely that cooperation between several signalling
pathways (e.g. Haremaki et al.,
2003
) is required for ventrally positioned cells to move and adopt
appropriate fates within the tail.
To our knowledge, a source of tail neural tissue distant from the dorsal organiser has not been shown for other model species and it will be of interest to determine if there are similar roles for Bmp activity in promoting caudal neural fates in these species.
Fgf signalling promotes both induction of prospective neural fate and posteriorisation of the ectoderm
We show that Fgf signalling promotes expression of prospective neural
markers and is required for ectodermal cells to contribute to caudal CNS
structures. This is consistent with data in other species that suggest Fgfs
are important regulators of neural induction
(Bertrand et al., 2003;
Kengaku and Okamoto, 1995
;
Lamb and Harland, 1995
;
Streit et al., 2000
;
Wilson et al., 2000
;
Ying et al., 2003
). However,
there is a larger body of literature that indicates Fgf signalling promotes
caudalisation of neural tissue (Bally-Cuif
and Hammerschmidt, 2003
; Cox
and Hemmati-Brivanlou, 1995
;
Koshida et al., 1998
;
Kudoh et al., 2002
) and it
will be important to determine if these two activities are separable.
In this study, we show that abrogation of Fgf activity leads to loss of
prospective neural tissue on the ventral side of the embryo (supporting a role
for Fgf signalling in an early step of neural induction) and also
anteriorisation of the residual neural tissue on the dorsal side of the embryo
(supporting a role for Fgf in posteriorisation) (see also
Koshida et al., 1998;
Kudoh et al., 2002
). Some of
our data and other reports suggest that Fgf-dependent induction of prospective
neural fate and posteriorisation of induced neural tissue are separable
events. For example, induction of expression of the posterior neural gene,
hoxb1b by Fgf3 depends on Chordin function
(Koshida et al., 2002
) and is
probably mediated indirectly by retinoic acid
(Kudoh et al., 2002
). By
contrast, induction of the prospective neural markers sox3, sox31 and
zic2 is independent of Chordin activity (this study). Furthermore,
Koshida et al. (Koshida et al.,
2002
) have shown that XFD-expressing cells transplanted to
wild-type hosts are still able to express hoxb1b, supporting an
indirect role for Fgf on hoxb1b expression. However, our results
suggest that ventral vegetal ectodermal cells may directly need to receive Fgf
signalling to specify neural fate (to induce sox3, to suppress
foxi1 and to contribute to caudal CNS). Therefore, at least in part,
the roles of Fgf signalling in neural induction and posteriorisation by Fgf
seem to be separable.
Other signals may cooperate with Fgfs during formation and differentiation of neural tissue
Although the regulation of early neural/non-neural marker gene expression
in vegetal ectoderm is independent of Bmp antagonism, this does not discount
the possibility that Bmp antagonists may be contributing to the maintenance
and/or further differentiation of neural plate. Although there has not been
any detailed analysis of mature neural tissue in ichabod mutant or
other severely ventralised embryos, it appears that relatively few neurones
are present in these fish (Gonzalez et
al., 2000) (T.K., unpublished), despite the early expression of
vegetal prospective neural markers. This suggests that exposing ectodermal
cells to Fgf signalling alone at blastula and gastrula stage is not sufficient
to maintain and promote further differentiation of the neural tube. Bmp
antagonists such as Chordin and Noggin 1 may therefore contribute to later
steps in the induction, maturation and/or maintenance of neural tissue.
Indeed, the idea that Chordin is not necessary for the initial phase of neural
induction but rather functions at a later step of neural development has been
suggested from neural induction assays in chick
(Stern, 2002
;
Streit et al., 1998
).
In our studies, we noticed that although exogenous Fgf induced early
prospective neural marker genes it actually appeared to inhibit the expression
of markers of mature neuronal identity such as huC (data not shown).
This is of interest in light of recent observations that, in chick, levels of
Fgf activity are believed to be high in immature neural tissue and low in
mature neural tissue (Diez del Corral et
al., 2003). Indeed Diez del Corral et al. propose that inhibition
of Fgf activity is a necessary step in the maturation of neural tissue that
allows neuronal differentiation to occur. Thus, although exogenous Fgf
activity may promote early steps in neural development (induction of
expression of prospective neural markers and suppression of prospective
epidermal markers), it may inhibit this prospective neural tissue from
progressing to a fully differentiated state. Furthermore, it has recently been
shown that Sox protein activity in undifferentiated neural tissue inhibits
neurogenesis, and that this activity needs to be suppressed through the
activity of proneural bHLH proteins
(Bylund et al., 2003
).
Therefore as expression of Sox genes is promoted by Fgf signalling (this
study) (Streit et al., 2000
),
this may provide an explanation of how Fgf can promote early neural fate yet
inhibit later neuronal differentiation.
The molecular mechanisms by which Fgfs and Bmp antagonists interact to
regulate prospective neural/non-neural gene expression are undoubtedly
complex. In our study, we have shown that Fgf3 can induce sox3 in
ectodermal cells without suppressing the ability of the Bmp pathway to induce
bmp4 and suppress chordin expression. This indicates that
Fgf activity does not lead to a comprehensive block of Bmp signalling.
However, data from other studies have shown intracellular crosstalk between
Fgf and Bmp pathways by which Fgf pathway activation can antagonise Bmp
pathway activity. For example, in frogs, Map kinase, a downstream effector of
Fgf signalling, phosphorylates a linker domain in Smad1 thereby suppressing
Bmp signalling (Pera et al.,
2003). Assuming similar Fgf-dependent regulation of Smad1 activity
occurs in fish, then either this is insufficient to abrogate Bmp signalling,
or there may be several independently regulated intracellular responses
downstream of Bmp receptor activation. In addition to intracellular crosstalk
between these two pathways, Fgf signalling can profoundly affect Bmp
signalling activity through the regulation of expression of secreted Bmp
antagonists such as Chordin (Koshida et
al., 2002
; Furthauer et al.,
1997
) (this study).
Is Fgf signalling required for induction of anterior prospective neural tissue?
In our study and in related experiments (e.g.
Griffin et al., 1995;
Koshida et al., 1998
), XFD
injection and SU5402 treatment both suppressed posterior neural induction,
whereas anterior neural fate was retained in both situations. This is
consistent with some experiments in other species showing a requirement for
Fgf activity only in posterior neural development
(Munoz-Sanjuan and Hemmati-Brivanlou,
2001
). By contrast, in chick and ascidian (and from some
experiments in frogs), it has been proposed that Fgf activity is crucial for
induction of neural tissue, including anterior domains
(Bertrand et al., 2003
;
Hongo et al., 1999
;
Hudson and Lemaire, 2001
;
Streit et al., 2000
;
Wilson et al., 2000
). One
possibility is that in fish, Fgf activity may be required in prospective
anterior neural tissue at lower levels or at earlier stages than in more
caudal neural tissue. If so, then sufficient early Fgf activity might still be
present in manipulated embryos in our experiments to allow development of
rostral neural tissue. Indeed, very recent studies have shown that early
activation of Fgf signalling plays an important role in the regulation of the
Bmp signalling pathway in prospective anterior neural tissue
(Furthauer et al., 2004
) and
preliminary data suggest that abrogation of Fgf activity at stages earlier
than reported here leads to more severe depletion of anterior neural tissue
(T.K., unpublished).
In the animal (prospective anterior) gastrula stage ectoderm, Bmp antagonists emanating from the organiser, its derivatives and other tissues are crucial for neural development, whereas in prospective caudal regions, prospective neural fate appears to be specified by Fgfs emanating radially from the germ ring (Fig. 9). Therefore, we suggest that the organiser and the germ ring constitute two distinct and at least partially independent sources of signals that promote neural development. We predict that the combined activity of germ ring and organiser signals establishes prospective neural tissue from the dorsal animal ectoderm through to the ventral vegetal ectoderm. One consequence of this view of early neural induction is that the neural to non-neural ectodermal fate choice should not only be considered as occurring between dorsal and ventral ectoderm but also, on the ventral side of the gastrula, between animal and vegetal ectoderm.
Note added in proof
Two very recent papers examine early activity and function of the Fgf
pathway in zebrafish (Furthauer et al.,
2004; Tsang et al.,
2004
). We briefly cite the Furthauer paper in the Discussion, but
it contains additional data pertinant to our study.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agathon, A., Thisse, C. and Thisse, B. (2003). The molecular nature of the zebrafish tail organizer. Nature 424,448 -452.[CrossRef][Medline]
Akai, J. and Storey, K. (2003). Brain or brawn: how FGF signaling gives us both. Cell 115,510 -512.[CrossRef][Medline]
Amacher, S. L., Draper, B. W., Summers, B. R. and Kimmel, C. B. (2002). The zebrafish T-box genes no tail and spadetail are required for development of trunk and tail mesoderm and medial floor plate. Development 129,3311 -3323.[Medline]
Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66,257 -270.[Medline]
Aybar, M. J. and Mayor, R. (2002). Early induction of neural crest cells: lessons learned from frog, fish and chick. Curr. Opin. Genet. Dev. 12,452 -458.[CrossRef][Medline]
Bakkers, J., Hild, M., Kramer, C., Furutani-Seiki, M. and Hammerschmidt, M. (2002). Zebrafish DeltaNp63 is a direct target of Bmp signaling and encodes a transcriptional repressor blocking neural specification in the ventral ectoderm. Dev. Cell 2,617 -627.[Medline]
Bally-Cuif, L. and Hammerschmidt, M. (2003). Induction and patterning of neuronal development, and its connection to cell cycle control. Curr. Opin. Neurobiol. 13, 1-10.[CrossRef]
Barth, K. A., Kishimoto, Y., Rohr, K. B., Seydler, C.,
Schulte-Merker, S. and Wilson, S. W. (1999). Bmp activity
establishes a gradient of positional information throughout the entire neural
plate. Development 126,4977
-4987.
Bertrand, V., Hudson, C., Caillol, D., Popovici, C. and Lemaire, P. (2003). Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors. Cell 115,615 -627.[CrossRef][Medline]
Bylund, M., Andersson, E., Novitch, B. G. and Muhr, J. (2003). Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat Neurosci. 6,1162 -1168.[CrossRef][Medline]
Cox, W. G. and Hemmati-Brivanlou, A. (1995).
Caudalization of neural fate by tissue recombination and bFGF.
Development 121,4349
-4358.
Diez del Corral, R., Olivera-Martinez, I., Goriely, A., Gale, E., Maden, M. and Storey, K. (2003). Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron 40, 65-79.[CrossRef][Medline]
Draper, B. W., Stock, D. W. and Kimmel, C. B.
(2003). Zebrafish fgf24 functions with fgf8 to promote posterior
mesodermal development. Development
130,4639
-4654.
Erter, C. E., Wilm, T. P., Basler, N., Wright, C. V. and Solnica-Krezel, L. (2001). Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo. Development 128,3571 -3583.[Medline]
Feldman, B., Dougan, S. T., Schier, A. F. and Talbot, W. S. (2000). Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish. Curr. Biol. 10,531 -534.[CrossRef][Medline]
Furthauer, M., Thisse, C. and Thisse, B.
(1997). A role for FGF-8 in the dorsoventral patterning of the
zebrafish gastrula. Development
124,4253
-4264.
Furthauer, M., Lin, W., Ang, S., Thisse, B. and Thisse, C. (2002). Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat. Cell Biol. 4, 170-174.[CrossRef][Medline]
Furthauer, M., Van Celst, J., Thisse, C. and Thisse, B.
(2004). Fgf signalling controls the dorsoventral patterning of
the zebrafish embryo. Development
131,2853
-2864.
Girard, F., Cremazy, F., Berta, P. and Renucci, A. (2001). Expression pattern of the Sox31 gene during zebrafish embryonic development. Mech. Dev. 100, 71-73.[CrossRef][Medline]
Gonzalez, E. M., Fekany-Lee, K., Carmany-Rampey, A., Erter, C.,
Topczewski, J., Wright, C. V. and Solnica-Krezel, L. (2000).
Head and trunk in zebrafish arise via coinhibition of BMP signaling by bozozok
and chordino. Genes Dev.
14,3087
-3092.
Griffin, K., Patient, R. and Holder, N. (1995).
Analysis of FGF function in normal and no tail zebrafish embryos reveals
separate mechanisms for formation of the trunk and the tail.
Development 121,2983
-2994.
Hammerschmidt, M. and Mullins, M. (2002). Dorsoventral patterning in the zebrafish: bone morphogenic proteins and beyond. In Pattern Formation in Zebrafish, Vol.40 (ed. L. Solnica-Krezel), pp.72 -95. Berlin, Heidelberg, New York: Springer.
Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., van
Eeden, F. J., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). dino and mercedes, two genes
regulating dorsal development in the zebrafish embryo.
Development 123,95
-102.
Haremaki, T., Tanaka, Y., Hongo, I., Yuge, M. and Okamoto,
H. (2003). Integration of multiple signal transducing
pathways on Fgf response elements of the Xenopus caudal homologue Xcad3.
Development 130,4907
-4917.
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu, T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000). Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation. Dev. Biol. 217,138 -152.[CrossRef][Medline]
Hongo, I., Kengaku, M. and Okamoto, H. (1999). FGF signaling and the anterior neural induction in Xenopus. Dev. Biol. 216,561 -581.[CrossRef][Medline]
Houart, C., Caneparo, L., Heisenberg, C., Barth, K., Take-Uchi, M. and Wilson, S. (2002). Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron 35,255 -265.[Medline]
Hudson, C. and Lemaire, P. (2001). Induction of anterior neural fates in the ascidian Ciona intestinalis. Mech. Dev. 100,189 -203.[CrossRef][Medline]
Itoh, M., Kudoh, T., Dedekian, M., Kim, C. H. and Chitnis, A. B. (2002). A role for iro1 and iro7 in the establishment of an anteroposterior compartment of the ectoderm adjacent to the midbrain-hindbrain boundary. Development 129,2317 -2327.[Medline]
Joly, J. S., Joly, C., Schulte-Merker, S., Boulekbache, H. and
Condamine, H. (1993). The ventral and posterior expression of
the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant
embryos. Development
119,1261
-1275.
Kelly, C., Chin, A. J., Leatherman, J. L., Kozlowski, D. J. and
Weinberg, E. S. (2000). Maternally controlled
(beta)-catenin-mediated signaling is required for organizer formation in the
zebrafish. Development
127,3899
-3911.
Kengaku, M. and Okamoto, H. (1995). bFGF as a
possible morphogen for the anteroposterior axis of the central nervous system
in Xenopus. Development
121,3121
-3130.
Kimmel, C. B., Warga, R. M. and Schilling, T. F. (1990). Origin and organization of the zebrafish fate map. Development 108,581 -594.[Abstract]
Kodjabachian, L., Dawid, I. B. and Toyama, R. (1999). Gastrulation in zebrafish: what mutants teach us. Dev. Biol. 213,231 -245.[CrossRef][Medline]
Koshida, S., Shinya, M., Mizuno, T., Kuroiwa, A. and Takeda,
H. (1998). Initial anteroposterior pattern of the zebrafish
central nervous system is determined by differential competence of the
epiblast. Development
125,1957
-1966.
Koshida, S., Shinya, M., Nikaido, M., Ueno, N., Schulte-Merker, S., Kuroiwa, A. and Takeda, H. (2002). Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish. Dev. Biol. 244,9 -20.[CrossRef][Medline]
Kudoh, T., Tsang, M., Hukriede, N. A., Chen, X., Dedekian, M.,
Clarke, C. J., Kiang, A., Schultz, S., Epstein, J. A., Toyama, R. et al.
(2001). A gene expression screen in zebrafish embryogenesis.
Genome Res. 11,1979
-1987.
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129,4335 -4346.[Medline]
Kumano, G. and Smith, W. C. (2002). Revisions to the Xenopus gastrula fate map: implications for mesoderm induction and patterning. Dev. Dyn. 225,409 -421.[CrossRef][Medline]
Lamb, T. M. and Harland, R. M. (1995).
Fibroblast growth factor is a direct neural inducer, which combined with
noggin generates anterior-posterior neural pattern.
Development 121,3627
-3636.
Lane, M. C. and Sheets, M. D. (2002). Rethinking axial patterning in amphibians. Dev. Dyn. 225,434 -447.[CrossRef][Medline]
Marlow, F., Gonzalez, E. M., Yin, C., Rojo, C. and
Solnica-Krezel, L. (2004). No tail co-operates with
non-canonical Wnt signaling to regulate posterior body morphogenesis in
zebrafish. Development
131,203
-216.
Mathieu, J., Barth, A., Rosa, F. M., Wilson, S. W. and
Peyrieras, N. (2002). Distinct and cooperative roles for
Nodal and Hedgehog signals during hypothalamic development.
Development 129,3055
-3065.
Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh,
B. K., Hubbard, S. R. and Schlessinger, J. (1997). Structures
of the tyrosine kinase domain of fibroblast growth factor receptor in complex
with inhibitors. Science
276,955
-960.
Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J.,
Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). Genes establishing
dorsoventral pattern formation in the zebrafish embryo: the ventral specifying
genes. Development 123,81
-93.
Munoz-Sanjuan, I. and Hemmati-Brivanlou, A. (2001). Early posterior/ventral fate specification in the vertebrate embryos. Dev. Biol. 237, 1-17.[CrossRef][Medline]
Munoz-Sanjuan, I. and Hemmati-Brivanlou, A. (2002). Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3, 271-280.[CrossRef][Medline]
Myers, D. C., Sepich, D. S. and Solnica-Krezel, L. (2002). Bmp activity gradient regulates convergent extension during zebrafish gastrulation. Dev. Biol. 243, 81-98.[CrossRef][Medline]
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Nguyen, V. H., Trout, J., Connors, S. A., Andermann, P.,
Weinberg, E. and Mullins, M. C. (2000). Dorsal and
intermediate neuronal cell types of the spinal cord are established by a BMP
signaling pathway. Development
127,1209
-1220.
Pera, E. M., Ikeda, A., Eivers, E. and de Robertis, E. M.
(2003). Integration of IGF, FGF, and anti-BMP signals via Smad1
phosphorylation in neural induction. Genes Dev.
17,3023
-3028.
Roehl, H. and Nusslein-Volhard, C. (2001). Pea3 and erm are general targets of Fgf8 signaling. Curr. Biol. 11,503 -507.[CrossRef][Medline]
Rohr, K. B. and Concha, M. L. (2000). Expression of nk2.1a during early development of the thyroid gland in zebrafish. Mech. Dev. 95,267 -270.[CrossRef][Medline]
Sheng, G., dos Reis, M. and Stern, C. D. (2003). Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation. Cell 115,603 -613.[CrossRef][Medline]
Shih, J. and Fraser, S. E. (1995). Distribution
of tissue progenitors within the shield region of the zebrafish gastrula.
Development 121,2755
-2765.
Shih, J. and Fraser, S. E. (1996).
Characterizing the zebrafish organizer: microsurgical analysis at the
early-shield stage. Development
122,1313
-1322.
Solomon, K. S., Kudoh, T., Dawid, I. B. and Fritz, A.
(2003). Zebrafish foxi1 mediates otic placode formation and jaw
development. Development
130,929
-940.
Stern, C. D. (2001). Initial patterning of the central nervous system: how many organizers? Nat. Rev. Neurosci. 2,92 -98.[CrossRef][Medline]
Stern, C. D. (2002). Induction and initial patterning of the nervous system - the chick embryo enters the scene. Curr. Opin. Genet. Dev. 12,447 -451.[CrossRef][Medline]
Streit, A., Lee, K. J., Woo, I., Roberts, C., Jessell, T. M. and
Stern, C. D. (1998). Chordin regulates primitive streak
development and the stability of induced neural cells, but is not sufficient
for neural induction in the chick embryo. Development
125,507
-519.
Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A. and Stern, C. D. (2000). Initiation of neural induction by FGF signalling before gastrulation. Nature 406, 74-78.[CrossRef][Medline]
Toyama, R., Gomez, D. M., Mana, M. D. and Dawid, I. B. (2004). Sequence relationships and expression patterns of zebrafish zic2 and zic5 genes. Gene Exp. Patt. 4, 345-350.[CrossRef]
Tsang, M., Friesel, R., Kudoh, T. and Dawid, I. B. (2002). Identification of Sef, a novel modulator of FGF signalling. Nat. Cell Biol. 4, 165-169.[CrossRef][Medline]
Tsang, M., Maegawa, S., Kiang, A., Habas, R., Weinberg, E. and
Dawid, I. B. (2004). A role for MKP3 in axial patterning of
the zebrafish embryo. Development
131,2769
-2779.
Wilson, S. I. and Edlund, T. (2001). Neural induction: toward a unifying mechanism. Nat. Neurosci. 4,1161 -1168.[Medline]
Wilson, S. I., Graziano, E., Harland, R., Jessell, T. M. and Edlund, T. (2000). An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo. Curr. Biol. 10,421 -429.[CrossRef][Medline]
Woo, K. and Fraser, S. E. (1995). Order and
coherence in the fate map of the zebrafish nervous system.
Development 121,2595
-2609.
Ying, Q., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21,183 -186.[CrossRef][Medline]
Related articles in Development: