Department of Genetics, University of Technology Dresden, c/o Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany
* Author for correspondence (e-mail: brand{at}mpi-cbg.de)
Accepted 5 January 2005
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SUMMARY |
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Key words: Midbrain-hindbrain boundary, MHB/isthmic organizer, Posteriorization, Zebrafish, Neural plate, Morphogen, wnt8, gbx1, otx2, gbx2, nodal, fgf
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Introduction |
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By the end of gastrulation, cell-type diversity of the neural plate is
further refined through activities of local organizing centers. The
midbrain-hindbrain boundary (MHB) organizer mediates positional information in
the neuroectoderm via secreted molecules such as Fgf8 or Wnt1. Mutant analyses
in mouse and zebrafish identified several genes involved in MHB development,
e.g. Wnt1, Pax2 and Fgf8, which are involved in the
formation and organizing activity of the MHB (for reviews, see
Rhinn and Brand, 2001;
Wurst and Bally-Cuif, 2001
;
Raible and Brand, 2004
).
Restricted gene expression in the zebrafish neural plate around the MHB is
first observed during gastrulation stages
(Lun and Brand, 1998
;
Reifers et al., 1998
;
Rhinn et al., 2003
), and is
prefigured by the interface between anterior otx2- and posterior
gbx1-expressing cells. As in other vertebrates, this precise
alignment suggests that forming the otx2/gbx1 interface is a crucial
step for positioning the MHB in the neural plate. Indeed, Otx and
Gbx loss- and gain-of-function experiments in different species
support the notion that these genes are required to correctly position the MHB
in the neural plate (Wassarman et al.,
1997
; Rhinn et al.,
1998
; Rhinn et al.,
2004
; Acampora et al.,
1998
; Broccoli et al.,
1999
; Millet et al.,
1999
; Li and Joyner,
2001
; Martinez-Barbera et al.,
2001
; Kikuta et al.,
2003
) (M.R. and M.B., unpublished).
These findings raise the issue of how the interface itself is positioned in
the early neural plate. Here, we study how the otx2/gbx1 interface is
set up in the zebrafish neuroectoderm. Regionally restricted otx2 and
gbx1 expression occurs very early during gastrulation stages, and
could be linked to the formation of axial mesendoderm. Conflicting results
have been obtained concerning the involvement of the axial mesendoderm in the
AP patterning of the CNS. Studies in Xenopus have indicated that
induction of AP neural patterning might involve signals from the involuted
dorsal mesoderm to the overlying ectoderm
(Ruiz i Altaba and Jessel,
1993). Work in several species suggests that signals from the
anterior mesendoderm or notochord might regulate the expression of
Engrailed genes in the neural plate
(Hemmati-Brivanlou et al.,
1990
; Ang and Rossant,
1993
; Darnell and Schoenwolf,
1997
; Shamim et al.,
1999
). However, these findings are inconsistent with node removal
experiments, which reveal correct AP patterning, including En2
expression, in the absence of axial mesendoderm
(Darnell et al., 1992
), and a
role in refining rostrocaudal pattern has been suggested instead
(Rowan et al., 1999
;
Camus et al., 2000
). Moreover,
in mouse or in zebrafish that lack the notochord, a well-formed neuraxis
develops (Ang and Rossant,
1994
; Weinstein et al.,
1994
; Klingensmith et al.,
1999
; Shih and Fraser,
1996
; Saude et al.,
2000
). To address a possible (even transient) role, we examine the
role of axial mesendoderm in positioning the MHB at the time it is first
formed in zebrafish. We find that axial mesendoderm is not required for AP
positioning of the MHB, but that the likely source of the signal is the
blastoderm margin (non-axial mesendoderm). Thus, MHB positioning is related to
the more general problem of posteriorization of the neuroectoderm
(Woo and Fraser, 1997
).
Through blocking reception for individual signaling pathways, we identify Wnt
signaling, and particularly Wnt8, as being required for initial establishment
of otx2 and gbx1 territories, and present evidence that Wnt8
acts directly. We suggest that initial positioning of the MHB organizer is a
direct consequence of overall posteriorization of the neuroectoderm, and that
Wnt8 protein produced by lateral mesendodermal precursors is necessary to
mediate this process directly.
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Materials and methods |
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Whole-mount in situ hybridization and antibody staining
In situ hybridization, probes and antibody staining against EN4D9 (Ntl) was
performed as described previously (Brand et
al., 1996; Reifers et al.,
1998
). Probes and wild-type expression patterns have been
described elsewhere: otx2
(Mercier et al., 1995
),
wnt8 (Kelly et al.,
1995
), pax2.1 (Krauss
et al., 1991
), eng2
(Ekker et al., 1992
;
Fjose et al., 1988
),
wnt1 (Kelly et al.,
1993
), fgf8 (Reifers
et al., 1998
), and gbx1 and gbx2
(Rhinn et al., 2003
).
DNA construct and RNA synthesis
To generate the C-terminal Wnt8-GFP fusion protein, a gfp-coding
sequence (Clontech) was inserted into pCS2+ vector
(Rupp et al., 1994) via
XbaI/SnabI. wnt8-coding sequence was amplified by
PCR and fused in frame into PCS2+/GFP vector. We synthesized capped mRNA as
described previously (Reifers et al.,
1998
).
RNA and morpholino injections
For RNA injections, embryos were dechorionated using pronase and injected
at the one-cell stage. Morpholinos were designed and synthesized by Gene-Tools
(Corvallis, OR). They were resuspended in sterile water, stored at -20°C
as 10 mg/ml solutions and diluted before use to the appropriate concentration
in water, 0.2% Phenol Red. For morpholino injections, embryos were
dechorionated using pronase and injected in the yolk with 1-15 ng between the
one- and four-cell stage.
Transplantations
Donors embryos were injected with biotin-coupled
tetramethylrhodaminedextran (Mr 10,000, Molecular Probes
D-1817) diluted in 0.25 M KCl. Transplantations of donor cells into host
embryos were carried out at shield stage using trimmed borosilicate
capillaries. Transplanted cells were then visualized by immunochemically
staining using the Vectastain ABC system (VectorLabs) and the DAB system
(Sigma).
Pharmacological inhibition of Fgf signaling
To inhibit Fgfr activity, embryos were treated with Su5402
(Mohammadi et al., 1997)
(Calbiochem) at 16 µM in E3 medium as described
(Reifers et al., 2000
).
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Results |
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Transplantation experiments in zebrafish suggested that the lateral
blastoderm margin is involved in neural posteriorization at pre-gastrula
stages (Woo and Fraser, 1997;
Koshida et al., 1998
). When
cells from this region are transplanted into the animal pole of the embryo,
hindbrain markers such as krox20 and hoxa1, are induced in
the host tissue, suggesting that secreted molecules are involved in this
process (Woo and Fraser, 1997
;
Koshida et al., 1998
;
Momoi et al., 2003
).
In order to analyze a possible involvement of the blastoderm margin in the
establishment of the otx2/gbx1 interface, we transplanted cells from
the blastoderm margin situated at a defined angular distance from the shield
(30-45°), to the animal pole, a region fated to become forebrain.
Expression analysis shows that otx2 is repressed
(Fig. 2A,A';
n=50/50) (Koshida et al.,
1998), whereas gbx1 is induced ectopically
(Fig. 2B,B';
n=25/40) around the transplanted clones as early as 2 hours after
transplantation. The ability of the blastoderm margin to repress otx2
and to induce gbx1 strongly suggests that the signal for correct
positioning of the otx2 and/or the gbx1 expression domain
arises from the blastoderm margin.
|
(1) fgf3, fgf8 and fgf24 are expressed in the margin
during gastrulation (Fürthauer et
al., 1997; Reifers et al.,
1998
; Raible and Brand,
2001
; Draper et al.,
2003
). Indeed, gbx1 and otx2 expression is
altered in wild-type embryos in which Fgf signaling has been blocked
pharmacologically using Su5402 (Mohammadi
et al., 1997
), a potent inhibitor that blocks all Fgf receptor
signaling (Fig. 2D,H). Su5402
treatment causes a posterior shift of otx2 expression mediodorsally
(Fig. 2D), probably reflecting
the enhanced ventralization after loss of Fgf function
(Fürthauer et al., 2004
).
In addition, ventral gbx1 expression is strongly reduced in
Su5402-treated embryos (Fig. 2H
insets), presumably owing to the lack of induction of prospective vegetal
neural fate (Kudoh et al.,
2004
; Rentzsch et al.,
2004
).
(2) Nodal-related factors are required for mesendoderm induction, and also
affect gbx1 and otx2 expression, as studied in
Maternal-Zygotic one-eyed-pinhead (MZoep) mutants that are
unresponsive to Nodal signaling (Gritsman
et al., 1999; Schier,
2001
). MZoep mutants show an expansion of gbx1
expression up to the margin (Fig.
2I, arrowhead) and a posterior shift of otx2 expression
mediodorsally (Fig. 2E),
presumably owing to the involvement of Nodal in the specification of dorsal
mesendoderm.
(3) dickkopf-1 (dkk1) is a secreted inhibitor of Wnt
signaling (Glinka et al.,
1998; Hashimoto et al.,
2000
). dkk1 mRNA injection causes complete absence of
gbx1 expression at 60% of epiboly
(Fig. 2J). However, by 80% of
epiboly, gbx1 expression recovers to nearly its normal size, although
its location shifts more posteriorly (data not shown). Simultaneously, a
posterior shift of otx2 expression is observed in
dkk1-injected embryos, severely reducing the gap between the
posterior border of otx2 and the blastoderm margin
(Fig. 2F). In summary,
establishment of gbx1 and otx2 expression is strongly
affected when Nodal, Fgf or Wnt signals are inhibited in whole embryos, but
these experiments do not distinguish direct from indirect action of these
pathways.
Wnt signaling directly defines the posterior border of otx2
To test how direct the action of these factors is, we examined whether
clones of cells that are `blind' to Fgf, Nodal or Wnt proteins - owing to
disrupted signal reception - can respond to AP positional information in an
otherwise wild-type environment. A dominant-negative Xenopus Fgf
receptor, XFD (Amaya et al.,
1991) makes cells unresponsive to Fgf signaling when
overexpressed. XFD mRNA-injected donor embryos show the same defects
in otx2 and gbx1 expression as Su5402-treated embryos and
fail to activate the Fgf8-target gene sprouty4
(Fürthauer et al., 2001
)
(Fig. 3O,P). We transplanted
cells from XFD-injected donors into wild-type host embryos and
monitored their fate. The transplanted cells showed otx2 and
gbx1 expression appropriate to their location
(Fig. 3A-D; otx2,
40/40; gbx1, 30/30) [for otx2 see also Koshida et al.
(Koshida et al., 1998
)],
demonstrating that Fgf signaling is not directly involved in defining the
otx2/gbx1 interface. Likewise, MZoep donor-derived
cells that are unresponsive to Nodal signaling show
otx2/gbx1 expression appropriate to their location
(Fig. 3E-H; otx2,
70/70; gbx1, 60/60), showing that Nodals are also not directly
involved.
|
Expression of gbx1 and otx2 is regulated by Wnt8
Among several Wnt genes expressed in the blastoderm margin, wnt8
shows the closest correlation in spatiotemporal expression to the expected
signal with an onset in the blastoderm margin at 50% of epiboly and exclusion
from the shield (Kelly et al.,
1995; Lekven et al.,
2001
; Erter et al.,
2001
). To test a possible role of wnt8 for activation of
gbx1 expression, we expressed wnt8 mRNA in all cells by
injecting into one-cell stage zebrafish embryos. Depending on the amount of
injected wnt8 mRNA, different levels of gbx1 expression are
induced. Compared with the normally posteriorly restricted domain of
gbx1 expression, low doses (1-5 pg) enlarge the gbx1 domain,
but leave the animal pole area free of gbx1 expression
(Fig. 4A-B). Intermediate doses
of wnt8 mRNA (10-50 pg) induce high levels, whereas high doses
(200-400 pg) induce lower levels of gbx1 expression throughout the
neuroectoderm (Fig.
4A,C,D). Conversely, otx2 repression is apparent at low
doses (1-5 pg) but more complete at intermediate (10-50 pg) and high doses
(200-400 pg) of wnt8 mRNA (Fig.
4G-J). In these experiments, we observed that gbx1 can be
induced both in prospective neural and non-neural ectoderm
(Fig. 4A-D). We therefore asked
if neural induction precedes gbx1 induction by wnt8 ectopic
expression in prospective non-neural ectoderm, by investigating whether the
non-neural markers foxi1 and p63, or the pan-neural markers
sox31 and zic2.2 (Kudoh
et al., 2004
; Rentzsch et al.,
2004
) are induced or suppressed, respectively. We observe that the
non-neural markers tested are repressed, and that neural markers are expanded
throughout the ectoderm (see Fig. S1 in supplementary material). This is also
true when Wnt8 is expressed from transplanted clones of cells (see
Fig. 6O-Q''). These
findings suggest that wnt8 gain-of-function is associated with
induction of neural tissue. Further studies will need to address how direct
this effect is and/or which other molecules may cooperate in promoting
induction of prospective neural fate under these conditions (see also
Baker et al., 1999
). We also
found that Wnt8 can act independently of Nodal signaling
(Fig. 4F,L), but does require
Fgf signaling for gbx1 expansion, though not for otx2
repression (Fig. 4E,K),
probably reflecting the ventralization of the neuroectoderm following Fgf
inhibition (Fürthauer et al.,
2004
). As expected, inhibition of Fgf signaling through Su5402, or
of Nodal signaling in MZoep embryos, eliminates or strongly
diminishes wnt8 mRNA expression dorsally
(Fig. 4M-O).
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Wnt8 can repress otx2 expression independently of gbx1 and mesendoderm
To reveal how direct the effect of Wnt8 on gbx1 and otx2
is, we transplanted wnt8-overexpressing cells into wild-type host
embryos at pre-gastrula stages, and analyzed by in situ hybridization for
induction of gbx1 and repression of otx2. Host embryos
carrying such clones showed ectopic gbx1 expression
(Fig. 6A-F,R), both in the
transplanted cells and in the surrounding host tissue
(Fig. 6B,D,E). Fig. 6F summarizes all
locations in which Wnt8-expressing clones were able to induce gbx1
expression in host cells. Although the Wnt8-expressing clones were distributed
essentially randomly, it appears to be more `difficult' to activate
gbx1 expression close to the margin (zone II in
Fig. 6F), probably because the
level of endogenous Wnt8 in zone II is too high to allow gbx1
induction (akin to the suppression observed at high doses of injected
wnt8 in Fig. 4D).
Similarly, fewer clones were found dorsoanteriorly, probably owing to the
activity of extracellular Wnt inhibitors in the anterior neural plate
(Wilson and Houart, 2004) (see
below). Within the otx2 domain, transplanted Wnt8-expressing cells
were able to repress otx2 expression in the host over about five cell
diameters (Fig. 6G,H,R). This
repression was never observed in control embryos transplanted with cells from
donor embryos injected with lacZ mRNA
(Fig. 6G'). Contrary to a
previous report (Agathon et al.,
2003
), we never observed wnt8 induction in neighboring
non-transplanted host cells, arguing that wnt8 expression does not
`self-induce' (see Fig. S2 in supplementary material). To further confirm this
result, we showed that repression of otx2 around a
wnt8-expressing transplanted clone of cells does not require
wnt8 function in the host embryo, as tested in embryos homozygous for
Df(wnt8) (see Fig. S2 in supplementary material).
Considering the importance of mutual repression between otx2 and
gbx1 at later stages, we sought to investigate if the repression of
otx2 by Wnt8-expressing clones requires gbx1 function. In
embryos transplanted with wnt8-overexpressing cells we do not observe
gbx1 induction when otx2 is suppressed around the
transplanted clone (Fig. 6I,J),
arguing that the repression of otx2 by Wnt8 is not mediated through
gbx1. Given the previously reported Wnt8-function in specifying
ventrolateral mesodermal fate (Christian
et al., 1991; Lekven et al.,
2001
), could Wnt8 indirectly alter otx2 and gbx1
expression through inducing mesoderm secondarily? Probably not, because
gbx1 expression is induced throughout the ectoderm in
wnt8-mRNA injected embryos, whereas Ntl expression is only slightly
broadened at the blastoderm margin (Fig.
6M,N) (Kelly et al.,
1995
). Moreover, otx2 inhibition or gbx1
induction is not linked with mesendoderm induction, as seen in double in situ
hybridization on transplanted embryos with a ntl and an otx2
probe (Fig. 6K,L,R), in which
we never detected ectopic ntl expression around the transplanted
clone. Therefore, otx2 inhibition and gbx1 activation around
clones of Wnt8-expressing cells are more likely due to a direct effect of
Wnt8.
Wnt8 does not act via a relay mechanism to inhibit otx2 expression
To test this notion further, we examined if Wnt8 directly inhibits
otx2 and activates gbx1, rather than doing so through other
factors (`relay mechanism'), using three different approaches. First, we
transplanted Wnt8-expressing cells ectopically into host ectoderm that cannot
receive Wnt8 signaling, because they express Fzb1-gpi
(Momoi et al., 2003), a
competitive secreted inhibitor that binds Wnts and thus should block the
effects of Wnt8 clones on otx2 and gbx1 expression if
signaling is direct. The gpi anchor of Fzb1-gpi prevents Fzb1 from diffusing
into the transplanted clone, and should thus allow them to produce Wnt8. In
such embryos, otx2 repression is limited to the transplanted cells,
and is not observed in surrounding host cells
(Fig. 7A-A'', 10/15).
Likewise, gbx1 is induced only in transplanted cells and not in host
cells (Fig. 7B-B'',
75/80). In a few (5/80) cases, gbx1 is expressed in one-two cells
around the transplanted clone, probably reflecting a slight variability in the
Fzb1-gpi inhibition.
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Third, we tested if Wnt8 protein is found around expressing clones at an
appreciable distance. We visualized Wnt8 protein around a transplanted clone
by tagging it with GFP. A C-terminal Wnt8-GFP fusion protein is biologically
active, as judged by its ability to induce gbx1 and repress
otx2 following global injection (data not shown). We transplanted
clones of wnt8-gfp overexpressing cells at pre-gastrula stages into
wild-type hosts that were previously injected at the one-cell stage with a
palmitoylated RFP mRNA (Iioka et
al., 2004) to label all cell membranes
(Fig. 7I), and analyzed Wnt8
localization by confocal microscopy. We observed GFP-fluorescent puncta in
host cells around the transplanted cells, representing accumulated Wnt8-GFP
protein (Fig. 7J, white
arrows), mostly associated with the cell surface or extracellular matrix
(Fig. 7K). Members of the Wnt
family, including Wingless, are thought to tightly associate with membranes
and heparan sulfate proteoglycan (Nusse,
2003
). We can visualize accumulated Wnt8-GFP at a distance of
about two cell diameters away from the donor cell clones, which is generally
consistent with the genetically determined non-autonomous characteristics of
Wnt8 (see Discussion). Because we observe otx2 inhibition two to five
cell diameters away from transplanted cell clones expressing Wnt8, it is
likely that levels of Wnt8 protein below the detection threshold act at
distances further away.
Taken together, our findings support a model where Wnt8 regulates, in a concentration-dependent manner, the location of the otx2/gbx1 interface and hence, the position of the MHB organizer, via direct action of secreted Wnt8 emanating from the endomesodermal primordium at the blastoderm margin (Fig. 7L).
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Discussion |
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The wealth of inductive interactions in early gastrulation stages makes it
difficult to dissect the contribution of the various signaling pathways
involved in posteriorization of the neural plate. This is clearly evident also
for the formation of the gbx1 and otx2 expression domains in
our loss-of-function experiments of the Nodal, Fgf or Wnt signaling pathways,
all of which are active in the early blastoderm margin
(Fig. 2). Specific interference
with signal reception for individual signaling pathways in combination with a
transplantation assay allowed us, however, to distinguish their contribution.
Examination of cell clones that are `blind' to any of the three signals
enabled us to address whether such cells can respond with correct
otx2/gbx1 expression to positioning cues
(Fig. 3). We find that Wnt
signaling is crucially involved in setting up the otx2 and
gbx1 expression domains, because cells that are `blind' to Wnt
signaling do not respond to AP positional information, causing otx2
to be ectopically expressed in more posterior domains of the embryo. This is
not the case when Nodal or Fgf signal reception is blocked, arguing that these
signals have a different role. A role for Wnt molecules in repressing the
otx2 domain has been suggested previously in different experimental
systems. Caudal chick neural plate cells revert to a rostral forebrain
character when grown in vitro in the absence of a Wnt signal
(Nordstrom et al., 2002). In
Xenopus, treatment of animal caps with XWnt8 leads to a progressive
posteriorization and a repression of anterior markers, including otx2
(Kiecker and Niehrs, 2001
). By
contrast, Fgf and Nodal signaling seem to act in a more global context,
because individual cells that cannot receive Fgf or Nodal signals respond
correctly to AP positional signals. We suggest that Fgf and Nodal signaling
are more indirectly involved in generating the otx2/gbx1 interface:
Nodal proteins, through their role in mesoderm formation (reviewed by
Schier, 2001
); and Fgfs,
through their involvement in the induction of neural fate of vegetal ectoderm
(Kudoh et al., 2004
;
Rentzsch et al., 2004
) and in
ventralization of the neuroectoderm
(Fürthauer et al.,
2004
).
The posteriorizing molecule Wnt8 mediates positioning of the MHB
Both our loss- and gain-of-function studies show that Wnt8, normally
expressed in the blastoderm margin, is involved in the onset and correct
positioning of the gbx1 expression domain, and for the establishment
of the posterior border of the otx2 expression domain (Figs
4,
5,
6). Although we have focused
here on gbx1 and otx2 as the most critical components for
MHB development, it is likely that other target genes with posterior-specific
expression would respond similarly to Wnt8, e.g. the Cdx genes. Our
findings raise the issue of whether Wnt8 is directly involved in positioning
the MHB primordium. We addressed this issue by injecting increasing amounts of
wnt8 mRNA, and by employing wnt8-overexpressing clones,
which we find can repress otx2. Our clonal analysis in a Fzb1-gpi
background, the cell-autonomous activation of the Wnt pathway by
Xgsk-3KR clonal analysis and our visualization of
Wnt8-GFP all support the argument that Wnt8 regulates otx2 and
gbx1 directly in a non-cell-autonomous manner
(Fig. 7).
As in other vertebrates, mutually repressive interactions are thought to
exist in zebrafish between Otx and Gbx genes
(Rhinn and Brand, 2001;
Rhinn et al., 2003
) (M.R. and
M.B., unpublished). Importantly, several findings argue that feedback
regulation is preceded by a phase of direct regulation of both otx2
and gbx1 via Wnt8. First, we found that Wnt8 can regulate both genes
independently, because it can induce gbx1 and regulate otx2
prior to the establishment of the feedback loop between them around 70%
epiboly. In the Df(wnt8) mutants and in wnt8 morphants,
posterior expansion of otx2 is evident prior to the 70% stage when
these mutually repressive interactions become evident. Second,
wnt8-overexpressing clones can repress otx2 without inducing
gbx1, and Wnt `blind' cells ectopically express otx2.
Analysis of wnt8 showed that the gene is crucially involved in the
patterning of mesoderm and neural ectoderm
(Christian et al., 1991
;
Lekven et al., 2001
;
Erter et al., 2001
).
gbx1 activation coincides with the involution of the forming
mesendoderm (Rhinn et al.,
2003
), raising the possibility that vertical signaling from the
involuted mesendoderm to the overlying ectoderm could also be involved in this
process. Our results argue against this possibility. First, in MZoep
embryos, where no dorsolateral mesendoderm involution occurs, gbx1 is
induced and otx2 expansion does not extend further posteriorly,
towards the lateral margin. Second, gbx1 expression throughout the
complete epiblast is observed when wnt8 is expressed ectopically with
a limited upregulation of mesendodermal markers
(Kelly et al., 1995
)
(Fig. 6M,N). This suggests that
the whole embryo can respond to wnt8 signaling to induce
gbx1 in the absence of mesendoderm. Third, we found that in
transplantations of wnt8-expressing cells, gbx1 is induced
and otx2 is repressed without new mesoderm induction, consistent with
previous findings that Wnt signaling can induce posterior neural markers in
the absence of mesendoderm (McGrew et al.,
1997
; Domingos et al.,
2001
; Kiecker and Niehrs,
2001
). Altogether these findings suggest that wnt8 plays
a key role in the activation of gbx1 and repression of otx2,
independent of its role in mesoderm patterning.
Graded activity of Wnt8 signaling in the early neural plate
How does Wnt8 participate in positioning of the MHB organizer?
wnt8 is expressed in the marginal cells and hypoblast and two
receptors, fz8c and fz9, are detected in both hypoblast and
epiblast (Momoi et al., 2003).
Conceivably, Wnt8 is transmitted in a planar fashion through the
neuroectoderm. This idea is supported by the clonal analysis of wnt8
overexpressing cells: gbx1 is activated in the host tissue one or two
cells distant from the transplanted cells, and otx2 is repressed four
or five cells distant from the transplanted cells
(Fig. 6). In unmanipulated
neuroectoderm, the onset of gbx1 expression occurs close to the
wnt8 domain with little or no overlap, and the otx2
expression domain is situated eight to ten cell diameters away from the
wnt8 domain at 60% of epiboly. Thus, the wnt8 expression
domain is appropriately located to generate a graded morphogenetic Wnt8 signal
that regulates the expression of gbx1 and otx2 genes in
vivo. This finding is more generally consistent with the ability of Wnt
molecules to form gradients and to activate target genes in a
concentration-dependent manner, as in the Drosophila wing imaginal
disc, where expression of wingless target genes like neuralized,
distalless and vestigial depends on the distance from
wingless-expressing cells (Zecca et al.,
1996
; Strigini and Cohen,
2000
). Similarly, in the unmanipulated zebrafish neuroectoderm,
the otx2 and the gbx1 domains are located at different
distances from the Wnt8 source at the lateral blastoderm margin. Following
global misexpression experiments, different Wnt8 doses can differentially
regulate otx2 and gbx1 expression: wnt8 ectopic
expression can induce gbx1 expression at low/intermediate doses, but
represses at high doses. Conversely, otx2 is increasingly repressed
with increasing wnt8 concentration. Similarly, around
wnt8-expressing clones, gbx1 is induced at a distance of one
or two cells around the clone, whereas otx2 is repressed at a
distance of four or five cells. This suggests that a lower Wnt8 concentration
is needed to repress otx2 than to induce gbx1. Altogether,
these observations suggest that Wnt8 has properties of a morphogen whose
activity is required to correctly position the otx2/gbx1 interface,
and probably other target genes in the forming neural plate. Our observation
of secreted Wnt8-GFP protein emanating from clones of producing cells is
generally consistent with this possibility. Distribution of another signaling
molecule in the early neural plate, Fgf8, is carefully controlled by
endocytosis (Scholpp and Brand,
2004
). It will be interesting to determine if Wnt8 protein is
indeed distributed in a graded fashion, and which mechanisms control this
distribution. In mice, Wnt8 is expressed in the posterior epiblast of
early primitive streak-stage embryos
(Bouillet et al., 1996
);
although its function is unknown, Wnt8 may therefore serve a similar function
as proposed here.
Other studies also suggest that a Wnt/ß-catenin signaling gradient may
underlie AP patterning in the neuroectoerm. In Xenopus gastrula, Wnt
activity declines in the neural plate from high caudal to low rostral levels
(Kiecker and Niehrs, 2001). In
favor of graded Wnt activity are also observations in chicken where neural
plate explants express different regional markers in response to different
concentrations of Wnt-conditioned medium
(Nordstrom et al., 2002
). In
zebrafish, results by Momoi and collaborators
(Momoi et al., 2003
) did not
detect graded Wnt activity. These authors observed at 70% of epiboly stage a
nuclear localization of ß-catenin along the AP axis, but only in
wnt8-expressing cells in the blastoderm margin. However, given the
timing of gbx1/otx2 onset and the genetic requirement for Wnt8
documented here, we predict that the Wnt gradient is generated in the neural
plate already prior to 70% of epiboly. Conceivably, a low level of
ß-catenin nuclear translocation might suffice to transmit the Wnt8 signal
that was not detectable in the assay by Momoi et al.
(Momoi et al., 2003
). In
summary, our data argue that Wnt8 acts without a relay mechanism in the
regulation of otx2 and gbx1, and we hence strongly favor the
idea that during the early and mid-gastrulation stages in zebrafish, similar
to Xenopus and Drosophila, there is a graded Wnt8 signal
that is generated from the blastoderm margin (see also
Dorsky et al., 2003
). The
recent description of Tcf4-binding sites in the enhancers driving
Otx2 expression in the mouse anterior neural plate suggests that one
level of this regulation occurs at the level of the Otx2 promoter
(Kurokawa et al., 2004
).
The suggested Wnt8 action from the lateral blastoderm margin, located at
the posterior edge of the forming neural plate, may be antagonized from the
anterior side forming at the animal pole, through inhibition that is required
for assigning cell fate in the forebrain neural plate
(Kim et al., 2000;
Onai et al., 2004
;
Wilson and Houart, 2004
). One
possibility is that these interactions start quite early when the distances
between cells of the future anterior and posterior neural plate are still
small. The Wnt8 signal is then integrated to process neural AP patterning and
positioning of the MHB primordium at 60% of epiboly
(Fig. 7L). Positioning of the
future MHB organizer is thus achieved by Wnt8 setting up the complementary
gbx1 and otx2 expression domains before mutual interaction
between them sharpens the interface, around 70-80% of epiboly
(Rhinn et al., 2003
), and
before the establishment of the complex regulatory cascade conferring to the
MHB cells their organizing capacity (Fig.
7L).
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Supplementary material |
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ACKNOWLEDGMENTS |
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