Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA
* Author for correspondence (e-mail: alekven{at}mail.bio.tamu.edu)
Accepted 12 May 2004
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SUMMARY |
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Key words: Wnt8, BMP, Dorsoventral, Vent, Vox
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Introduction |
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The organizer influences DV patterning through its secretion of BMP
inhibitors such as Chordin (Chd) or Noggin
(De Robertis et al., 2000).
However, BMP also exerts its own effect on the organizer. The Xvent ventral
homeobox genes were identified as transcriptional targets of BMP in
Xenopus, and were shown to repress organizer gene expression on the
ventral side of the embryo (Gawantka et
al., 1995
; Onichtchouk et al.,
1996
; Onichtchouk et al.,
1998
; Melby et al.,
1999
; Lee et al.,
2002
). Indeed, Xvents repress the transcription of targets such as
chd and goosecoid (gsc)
(Onichtchouk et al., 1996
;
Melby et al., 1999
;
Trindade et al., 1999
).
Analysis of the Xvent1b and Xvent2b promoters revealed the
presence of consensus Lef/Tcf binding sites
(Friedle and Knöchel,
2002
). In addition, the Xvent1b promoter is responsive to
zygotic Wnt activity, suggesting that the expression of Xvent genes in general
may be under the control of Wnt8 (Friedle
and Knöchel, 2002
). In support of this, Hoppler and Moon
found that overexpression of dn-Xwnt8 leads to the reduction of both
Xvent1 and Xvent2 expression in Xenopus
(Hoppler and Moon, 1998
).
Thus, these studies suggest that the expression of transcriptional repressors
required to restrict organizer gene expression may be under the concerted
control of both the BMP and Wnt pathways.
Genetic analysis of zebrafish vent (also known as vega2,
similar to Xvent1) and vox (also known as vega1,
similar to Xvent2) showed that the proteins encoded by these genes
function as redundant transcriptional repressors
(Kawahara et al., 2000;
Melby et al., 2000
;
Imai et al., 2001
). Zebrafish
embryos homozygous for a chromosomal deficiency of the closely linked
vent and vox loci show an expansion of organizer gene
expression and severe DV patterning defects
(Imai et al., 2001
). Further
epistatic analysis suggested that the primary role of Vent and Vox is to
modulate BMP inhibitors secreted by the organizer
(Imai et al., 2001
).
vent and vox are known BMP transcriptional targets in
zebrafish as well, but their dependency on BMP signaling starts at around
70-75% epiboly (Kawahara et al.,
2000
; Melby et al.,
2000
). As a result, zygotic BMP mutants do not have expanded
organizers as vent/vox mutants do at shield stage
(Mullins et al., 1996
;
Miller-Bertoglio, 1997; Imai et al.,
2001
). To date, only two zebrafish zygotic mutants are known to
display significantly expanded organizers: vent/vox mutants
and wnt8 mutants. These data suggest that the relationship between
BMP, Wnt8 and Vent/Vox is an important one for organizer regulation, the
nature of which has been unclear but has been suggested to be complex
(Hoppler and Moon, 1998
;
Marom et al., 1999
).
We have used a loss-of-function approach in zebrafish to study the relationship between Wnt8, zygotic BMP and Vent/Vox regulation and activity, in order to understand the mechanism by which Wnt8 antagonizes the organizer. Our results suggest that Wnt8 directly regulates the transcriptional levels of vent and vox, and that the maintenance of high levels of vent or vox is required for the repression of organizer genes on the ventral side of the embryo. Furthermore, we provide evidence that Vent and Vox are absolutely essential to mediate the organizer repression activity of Wnt8. We also show that organizer repression and the maintenance of ventrolateral mesoderm fates appear to be independent events. Finally, we show that the early regulation of both vent and vox is under Wnt8 and BMP control, but that Wnt8 is the primary regulator; that is, at the onset of gastrulation, the requirement for BMP is only revealed in the absence of Wnt8. Zygotic BMP becomes the primary regulator of vent (but not vox) transcription during mid to late gastrulation. Therefore, Wnt8 and BMP contribute to the repression of the organizer, which will, as a consequence, regulate the distribution of Wnt and BMP inhibitors.
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Materials and methods |
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In situ hybridization
In situ hybridizations were performed as described
(Oxtoby and Jowett, 1993).
Probes used were gsc (Stachel et
al., 1993
), chd
(Miller-Bertoglio et al.,
1997
), wnt8 ORF1 and wnt8 ORF1+ORF2
(Lekven et al., 2001
),
eve1 (Joly et al.,
1993
), vent/vox
(Melby et al., 2000
),
bmp2b (Kishimoto et al.,
1997
), opl (Grinblat
et al., 1998
), pax2a
(Krauss et al., 1991
) and
tbx6 (Hug et al.,
1997
).
Genotyping of embryos
wnt8 mutants were genotyped as described
(Lekven et al., 2001).
vent;vox mutants were genotyped using vox R1
(5'-GATATTGCACACCAGCGTGA-3') and vox L1
(5'-GTTCCAGAACCGAAGGATGA-3') primers. swr mutants were
genotyped as described (Wagner and
Mullins, 2002
). Embryos were classified according to their
phenotype, photographed and genotyped. For wnt8;swr double mutants,
at least 85 embryos from an intercross were examined in the same fashion.
Embryo microinjection, morpholinos, constructs
MOs (Genetools, LLC), RNA or DNA were injected into one- to four-cell stage
embryos. Approximately 3 nL was injected per embryo. Capped mRNAs were
synthesized using mMESSAGE mMACHINE (Ambion) and diluted in water. MOs were
diluted in Danieau's buffer as recommended (Genetools). wnt8 MOs
(targeting ORF1 and ORF2), and vent and vox MOs, have been
described (Lekven et al.,
2001; Imai et al.,
2001
). GR-LEF
N-ßCTA RNA was injected at 300 ng/µL
into one-cell stage embryos. Embryos were dechorionated manually in fish water
(Westerfield, 2000
) prior to
treatment. Dexamethasone (DEX; Sigma) treatments were performed for one hour
at 1, 2, 3, 4 or 5 hours post-fertilization (HPF). DEX (100 mM stock solution
in 100% ethanol) was used at a final concentration of 10 mM in
0.3xDanieau's solution. Treated embryos were fixed at 6 HPF. For the
Cycloheximide (CHX; Calbiochem) treatment, embryos were first injected with
GR-LEF
N-ßCTA RNA then treated with CHX (10 µg/mL), with or
without DEX. For vent induction analysis, n(CHX)=37 and 55,
n(DEX)=44, 37 and 11, and n(CHX+DEX)=28, 34 and 28, where
n=total number of embryos analyzed in each experiment. For
vox induction, n(CHX)=16, 17 and 12, n(DEX)=5, 12
and 20, and n(CHX+DEX)=9, 14 and 19. As a control for CHX treatments,
uninjected embryos were treated with CHX from 1.5 HPF to sphere stage, then
fixed and stained for gsc (Leung
et al., 2003
). No treated embryos expressed gsc
(n=34). The
2 test was used to determine statistical
significance.
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Results |
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In zebrafish, wnt8 contains two open reading frames (ORF1 and
ORF2) (Lekven et al., 2001).
The two Wnt8 proteins were shown to function redundantly in anteroposterior
(AP) and DV patterning, as the Df w8 phenotype is
phenocopied only by co-injection of both ORF1 and ORF2 MOs
(Lekven et al., 2001
).
Similarly, the Df st7 phenotype is phenocopied by the
co-injection of vent and vox MOs
(Imai et al., 2001
).
Expression analysis of the dorsal markers chd, gsc, floating head
(flh) and dharma (bozozok) at shield stage shows
that they are expanded ventrally in wnt8 mutants
(Fig. 1B,F)
(Lekven et al., 2001) (and
data not shown) as well as in vent;vox mutants
(Fig. 1C, inset, and
Fig. 1G)
(Imai et al., 2001
).
swr mutants, however, do not exhibit a similar expansion at shield
stage (Fig. 1D,H)
(Mullins et al., 1996
;
Miller-Bertoglio et al.,
1997
). Importantly, the expansion of dorsal markers is stronger in
vent;vox mutants than in wnt8 mutants. For instance,
gsc encircles the margin of vent;vox mutants
(Fig. 1C, inset) but extends
over a
90° arc in wnt8 embryos at the same
stage (Fig. 1B). This
comparative analysis shows that Wnt8 and Vent/Vox, but not BMP, are normally
required ventrally during gastrulation to restrict the size of the organizer,
which is in agreement with previous reports
(Mullins et al., 1996
;
Miller-Bertoglio et al., 1997
;
Imai et al., 2001
;
Lekven et al., 2001
).
|
Wnt8 is also required to promote ventral fates. eve1, a ventral
mesodermal marker, is reduced in wnt8 mutants
(Fig. 1B). It is similarly
reduced in swr mutants (Fig.
1D) (Mullins et al.,
1996). By contrast, eve1 is less reduced in
vent;vox mutants (Fig.
1C) than in wnt8 and swr mutants
(Fig. 1B,D), despite the fact
that the dorsal markers gsc (Fig.
1C, inset) or chd
(Fig. 1G) encircle the margin
of the same embryos. Hence, Wnt8 and BMP are required in the ventral mesoderm
for the maintenance of eve1, a ventral-specific gene, and this
function is separable from repression of the organizer.
Wnt8 regulates vent and vox mRNA levels
Because Wnt8 and Vent/Vox share the function of repressing dorsal genes, we
analyzed their epistatic relationship. We first examined vent and
vox mRNA levels in wild-type versus wnt8
backgrounds (Fig. 2). In
zebrafish, vent is expressed at the mesodermal margin during
gastrulation, whereas vox displays both ventral mesoderm and ectoderm
expression (Melby et al.,
2000).
|
To determine the correspondence between vent and vox
reduction and the onset of an observable phenotype in wnt8 mutants,
we examined chd expression at these early stages. At 30% epiboly, no
visible difference in the chd expression domain was observed in
wnt8 mutants (data not shown), but we did detect an expansion of
chd expression at 40% epiboly, the timepoint at which both
vent and vox are reduced in wnt8
embryos (Fig. 2M-O). Hence, our
results suggest that a reduction in both vent and vox levels
may be required to observe the expanded organizer phenotype at 40% epiboly,
which is consistent with Vent and Vox functioning redundantly
(Imai et al., 2001).
During the rest of gastrulation, vent and vox mRNA levels
stay reduced in wnt8 mutants/morphants compared with in wild type
(Fig. 2D-F,J-L; data not
shown). By comparison, vent and vox levels are unchanged in
swr mutants at shield stage
(Kawahara et al., 2000;
Melby et al., 2000
), which
explains the lack of an organizer phenotype
(Mullins et al., 1996
;
Miller-Bertoglio et al.,
1997
). Indeed, Bmp2b is only required at mid to late gastrulation
for the maintenance of vent and ectodermal vox expression
(Melby et al., 2000
).
Therefore, Wnt8 regulation of vent and vox starts at the
blastula/gastrula transition (30/40% epiboly), whereas Bmp2b regulation of
these genes occurs later (70% epiboly).
To test the reciprocal possibility of wnt8 being regulated by Vent
and Vox, we looked at the expression of wnt8 in
vent;vox mutants (Fig.
3). As zebrafish wnt8 produces transcripts for both
protein coding regions, we used probes to detect either the ORF1/ORF2
bicistronic transcript (ORF1), or both the bicistronic transcript and the ORF2
transcript (ORF1+ORF2) (Lekven et al.,
2001). No differences from wild-type expression were observed in
30% or 40% epiboly vent;vox mutants
(Fig. 3A,B,G,H). Because
vent;vox mutants are affected prior to 30% epiboly
(Imai et al., 2001
), this
suggests that a change in wnt8 expression is not responsible for the
vent;vox mutant phenotype. The dorsal domain lacking ORF1 expression
is slightly expanded in vent;vox mutants at shield stage
(Fig. 3C,D; confirmed with MOs)
and is more pronounced at 75% epiboly (Fig.
3F). Although there is an observable difference dorsally, ORF1
levels ventrally seem to be unaffected in vent;vox mutants
(Fig. 3C-F), suggesting that
the reduction in dorsal wnt8 ORF1 expression is an indirect
consequence of an enlarged organizer. Analysis of ORF2 expression at later
stages revealed that it is not affected by the loss of Vent and Vox
(Fig. 3I-L). This is not
unexpected as wnt8 ORF2 accumulates dorsally during gastrulation
(Fig. 3K) and is therefore
insensitive to molecules present in the organizer. Thus, only wnt8
ORF1 expression depends on Vent and Vox, but this dependency is restricted
dorsally and may be indirect. By comparison, wnt8 ORF2 expression
does not depend on Vent and Vox.
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Wnt8 repression of the organizer requires Vent/Vox
As vent and vox transcription is regulated by Wnt8, we
hypothesized that Vent and Vox function downstream of Wnt8 to repress dorsal
genes, and that the wnt8 organizer phenotype is due
to reduced vent and vox levels. If this is correct,
injection of vent or vox RNA or DNA into wnt8
mutants would suppress the expanded organizer phenotype. We first established
amounts of injected Vox or Vent that are sufficient to reduce the expression
of dorsal markers (gsc, chd, flh) in wild-type embryos
(Fig. 5A, panels a,c; data not
shown). When injected into wnt8 mutants, Vox was able to reduce the
expression of dorsal genes (Fig.
5A, compare panels b and d;
Table 2). Similar results were
obtained with either DNA or RNA injection for both vent and
vox (Table 2, and data
not shown). Thus, Vent and Vox expression can bypass wnt8
loss-of-function in repressing organizer genes, thus supporting the placement
of vent and vox genetically downstream of wnt8.
These results suggest that the difference in severity of the
wnt8 and
vent;vox organizer
phenotypes (see Fig. 1) could
be explained by residual Vent and Vox activity in wnt8 mutants. In
agreement with this, further reduction of Vent and Vox in wnt8
mutants by injection of sub-maximal concentrations of vent and
vox MOs enhances the severity of the wnt8
phenotype (Fig. 5B).
|
|
To confirm that the wnt8 transcripts in vent;vox mutants
produce functional proteins, we used two assays of Wnt8 function. First, we
examined the expression of the Wnt/ß-catenin activity reporter TOPdGFP
(Dorsky et al., 2002;
Lewis et al., 2004
). We
analyzed the expression of TOPdGFP mRNA at 100% epiboly in embryos homozygous
for the transgene after injection of wnt8 or vent+vox MOs
(Fig. 6A-D). As expected, and
confirming previous results (Phillips et
al., 2004
), wnt8 MOs severely reduce TOPdGFP expression
in 90% of injected embryos to almost undetectable levels (n=20;
Fig. 6B). In vent/vox
morphants, three phenotypic classes were observed: the first class displayed
wild-type TOPdGFP expression (50%, n=22;
Fig. 6C); the second class
showed moderate reduction in TOPdGFP (14%, not shown); and the third class
displayed a stonger reduction in staining (36%;
Fig. 6D), but this class had
significantly more TOPdGFP expression than wnt8 morphants (compare
Fig. 6D to
Fig. 6B). As a control for the
strength of the vent+vox MO injections, a sample of the
injected embryos was examined at 24 HPF and all showed a strong
vent/vox loss-of-function phenotype (n=23)
(Imai et al., 2001
). Thus,
TOPdGFP is a reporter of Wnt8 activity and is still expressed in
vent+vox morphants. Reduced levels of TOPdGFP expression in
some vent+vox morphants could reflect the fact that
expression of the Wnt antagonists Dickkopf 1 and Frzb is significantly
expanded (Imai et al., 2001
)
(and our own observations).
|
To further show that Wnt8 requires Vent and Vox in organizer repression, we tested whether exogenous Wnt8 can repress organizer genes in vent;vox mutants. We injected a wnt8 ORF1 expression plasmid (20 ng/µL) into one-cell stage vent;vox mutants and assayed gsc expression at shield stage. No injected vent;vox mutant embryos (n=25; genotyped by PCR) displayed reduced gsc expression, although this treatment did result in decreased gsc expression in wild-type siblings (n=54). As a control, we checked that the injected wnt8 DNA was sufficient to induce ectopic vent and vox expression in wild-type embryos (64% ectopic expression for vent, n=25; 42.8% ectopic expression for vox, n=35). Thus, repression of the organizer by exogenous Wnt8 requires Vent or Vox.
Our results show that in the absence of Vent and Vox, wnt8 is expressed and is active, as assayed by TOPdGFP reporter expression, tbx6 expression and embryonic AP patterning. Furthermore, ectopic Wnt8 cannot repress gsc in vent;vox mutants. These data strongly support a linear model in which Wnt8 acts directly upstream of Vent and Vox to repress the organizer.
Both Wnt8 and Bmp2b are required at different timepoints for the maintenance of vent and vox
Two pathways are required for the maintenance of vent and
vox expression in zebrafish: the zygotic BMP pathway
(Melby et al., 2000;
Imai et al., 2001
) and the Wnt
pathway (this work). To understand the combined regulation of vent
and vox during gastrulation by the Wnt8 and BMP pathways, we analyzed
the phenotype of wnt8;swr double mutants
(Fig. 7). Using swr
(bmp2b) mutants is sufficient to assess the influence of zygotic BMP
signaling, as it was previously shown that loss of Bmp2b produces a zygotic
bmp null phenotype
(Schmid et al., 2000
). The
requirement for both BMP and Wnt8 inputs towards vent and
vox expression would be revealed if wnt8;swr double mutants
exhibit a phenotype similar to the
vent;vox phenotype. We
found that gsc and chd are expressed in a broader domain
around the mesodermal margin in shield stage wnt8;swr double mutants
compared with either single mutant (Fig.
7B, compare with Fig.
1; data not shown), and thus they phenocopy vent;vox
mutants (Fig. 7A). The same
results were obtained when using the wnt8 deficiency or wnt8
MO knockdown (Fig. 7G),
confirming the specificity of the interaction.
|
The fact that double mutants appear to be worse than wnt8 or swr single mutants suggests that Wnt8 and BMP function in parallel to regulate vent and vox. Consistent with this, bmp2b expression in wnt8 mutants/morphants is close to wild type (Fig. 7M-O), and wnt8 expression in swr mutants is normal at shield stage (Fig. 7K,L). Hence, both Wnt8 and Bmp2b are early regulators of vent and vox, but Wnt8 has a more prominent role until mid-gastrula stages.
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Discussion |
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Interestingly, it has recently been observed that overexpression of a
conditional dominant repressor form of Tcf (hs-Tcf) leads to a more
severe phenotype than the loss of Wnt8
(Lewis et al., 2004
). Lewis et
al. found that gsc expression encircles the margin of transgenic
hs-
Tcf embryos heat-shocked at 4 HPF, a phenotype similar to
vent;vox or wnt8;swr double mutants. Why would
overexpression of a dominant-negative Tcf produce a more severe phenotype than
loss of Wnt8 signaling? This could be explained if
Tcf not only
abolishes Wnt8 function but also prevents other factors from positively
regulating vent and vox. One such factor could be the Smads
that mediate Bmp2b function, as we have shown that zygotic BMP signaling is
essential for maintaining vent and vox expression in the
absence of Wnt8. In other words,
Tcf may prevent Smad-dependent
regulation of vent and vox.
Regulation of vent and vox by Wnt8: comparison between zebrafish and Xenopus
The transcriptional regulation of Xvent genes has been studied
quite extensively in Xenopus, where most were found to be direct
targets of Bmp4 signaling (Rastegar et
al., 1999; Henningfeld et al.,
2000
; Henningfeld et al.,
2002
; Lee et al.,
2002
). However, the analysis of their regulation by Xwnt8 is less
complete. It was found that zygotic Wnt signaling is necessary and sufficient
for Xvent1 and Xvent2 expression
(Hoppler and Moon, 1998
;
Marom et al., 1999
), in
agreement with our findings for zebrafish Wnt8. Analysis of Xenopus
embryos overexpressing dominant-negative Xvent1 and Xvent2 revealed that
Xwnt8 expression is not affected by the loss of Xvent activity
(Onichtchouk et al., 1998
).
Again, our data agree as wnt8 is expressed in
vent;vox mutants. The inability of Xwnt8 to rescue the
dominant-negative Xvent phenotype was interpreted to mean that Xwnt8
functions in a different pathway than Bmp4/Xvent
(Onichtchouk et al., 1998
).
However, we propose that, as in zebrafish, Xwnt8 functions upstream of
Xvent genes, and that apparent differences between our model and
Xenopus models may be due to the different experimental approaches.
For example, concomitant reduction of Xwnt8, and Xvent1 and Xvent2, activities
using dominant-negative proteins results in a more severe phenotype than
reducing Xvent1 and Xvent2 alone
(Onichtchouk et al., 1998
).
This is also what we observed when injecting vent and vox
MOs in a wnt8 background. Thus, our results agree
with data obtained in Xenopus, although our interpretation of the
Wnt8/Vent/Vox relationship is somewhat different.
Wnt8 and zygotic BMP are required during gastrulation to maintain vent and vox expression at different timepoints
Our results show that both Wnt8 and Bmp2b (hence zygotic BMP) are required
to maintain vent and vox levels during gastrulation, but
that Wnt8 regulation of those genes occurs earlier at the blastula/gastrula
transition (Fig. 8). The lack
of an expanded organizer in swr mutants can be explained by the late
regulation of vent and vox by zygotic BMP after the
organizer has been formed. In addition, mesodermal vox levels are
unchanged in swr mutants (only ectodermal vox levels are
reduced at 70%) (Melby et al.,
2000). Hence, mesodermal Vox can repress dorsal genes in
swr mutants. Consistent with this, injection of vox MO in
swr mutants results in expanded gsc expression at 70%
epiboly (M.-C.R. and A.C.L., unpublished).
There are two known BMP signaling pathways in Xenopus and
zebrafish (Dale and Jones,
1999; Wilm and Solnica-Krezel,
2003
). In zebrafish, the maternal BMP pathway is thought to
establish ventral identity in a manner analogous to the establishment of a
dorsal axis by maternal ß-catenin activity
(Kramer et al., 2002
;
Sidi et al., 2003
).
Understanding the regulation of Wnt8 by maternal and zygotic BMP may explain
apparently contradictory results from Xenopus and zebrafish. For
instance, whereas it was found that regulation of zebrafish vent and
vox by zygotic BMP occurs at mid to late gastrulation
(Melby et al., 2000
),
Xenopus Xvent2 regulation by BMP signaling occurs during early
gastrulation (stage 10.5) (Ladher et al.,
1996
). Xvent2 regulation was observed in embryos
overexpressing a truncated Bmp2/4 receptor that does not distinguish between
Bmp2 or Bmp4 ligands (Suzuki et al.,
1994
). However, Bmp2 is both maternally provided and zygotically
expressed (Dale and Jones,
1999
). It has therefore been suggested that Xvent2
expression may be under the influence of a maternal BMP signal
(Ladher et al., 1996
).
Interestingly, the use of the same BMP-knockdown approach also results in
decreased Xwnt8 expression
(Schmidt et al., 1995
;
Hoppler and Moon, 1998
). In
zebrafish, it has been reported that loss of maternal BMP (Radar) signaling
does not interfere with the induction of vent and vox at MBT
(Sidi et al., 2003
), although
embryos homozygous for maternal smad5 display slightly expanded
gsc and chd expression
(Kramer et al., 2002
). Thus,
the elucidation of the relationship between Wnt8 and maternal or zygotic BMP
in zebrafish using a loss-of-function approach may address whether the
regulation of vent and vox is fundamentally different
between zebrafish and Xenopus.
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ACKNOWLEDGMENTS |
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