Howard Hughes Medical Institute, Department of Pharmacology and Center for Developmental Biology, Box 357750, University of Washington School of Medicine, Seattle, WA 98195, USA
* Author for correspondence (e-mail: rtmoon{at}u.washington.edu)
Accepted 15 March 2004
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SUMMARY |
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Key words: Nemo-like kinase, Wnt, Zebrafish, Tcf, Lef
![]() |
Introduction |
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Recently, evidence that canonical Wnt signaling can be regulated by MAP
kinase signaling has emerged. Genetic analysis of endoderm specification in
the early C. elegans embryo demonstrates an essential requirement for
both Wnt signaling and a MAPK-related pathway for proper specification of
endodermal fate (Meneghini et al.,
1999; Rocheleau et al.,
1997
; Rocheleau et al.,
1999
; Thorpe et al.,
1997
). Two of the genes involved in this process, lit-1
and mom-4, encode homologs of the Drosophila MAPK-related
gene nemo and vertebrate TGFß-activated kinase (Tak1),
respectively. Epistasis studies have shown that a C. elegans Tcf/Lef
homolog, pop-1, represses endoderm fates, and that this repression is
relieved by the combinatorial action of Wnt signaling and a parallel pathway
involving lit-1/nemo and mom-4/Tak1, permitting endoderm
development (Meneghini et al.,
1999
; Rocheleau et al.,
1997
; Rocheleau et al.,
1999
; Shin et al.,
1999
; Thorpe et al.,
1997
). Studies of a mouse homolog of lit-1/nemo,
nemo-like kinase (Nlk), in mammalian cell culture also demonstrate
that Nlk and Tak1 can regulate activation of Wnt targets
(Ishitani et al., 2003b
;
Ishitani et al., 1999
).
Biochemical studies indicate that Tak1 activates Nlk, which can then
phosphorylate Tcf, inhibiting the DNA-binding ability of ß-catenin/Tcf
complexes (Ishitani et al.,
2003b
; Ishitani et al.,
1999
; Rocheleau et al.,
1999
).
In different contexts, Nlk proteins can function as either inhibitors or
activators of Wnt target genes. For example, in the early C. elegans
embryo, the eventual outcome of the Wnt signal may be to simply derepress
genes that are inhibited by Tcf proteins (for reviews, see
Behrens, 2000;
Thorpe et al., 2000
).
Following the elimination of the repressive activity of pop-1/Tcf by
lit-1/Nlk and wrm-1/ß-catenin, other elements within
the promoter can drive transcription. For example, two GATA factors,
med-1 and med-2, can initiate transcription of the earliest
endoderm-specific genes when pop-1 repression is relieved
(Maduro et al., 2001
).
In other contexts, where a ß-catenin/Tcf complex is required to
directly activate transcription, phosphorylation of Tcf by Nlk and subsequent
inhibition of DNA binding of the ß-catenin/Tcf complex would block
activation, as is shown in experiments where Nlk blocks activation of the
Wnt-responsive TOPFLASH reporter (Ishitani
et al., 2003b; Ishitani et
al., 1999
). Also, injection of mouse or Xenopus Nlk RNA
into Xenopus embryos blocks duplication of the dorsal axis induced by
Wnt or ß-catenin (Hyodo-Miura et al.,
2002
; Ishitani et al.,
1999
).
A role for Wnt signaling in overcoming Tcf-mediated repression has recently
been described in zebrafish (Kim et al.,
2000; Dorsky et al.,
2003
). A Tcf3 homolog, encoded by the headless (hdl)
gene, is required to repress Wnt signaling during neural patterning
(Kim et al., 2000
).
wnt8 is required during gastrulation to induce posterior neural fates
(Erter et al., 2001
;
Fekany-Lee et al., 2000
;
Lekven et al., 2001
), and this
posteriorizing activity is opposed in prospective anterior neuroectoderm by
hdl/tcf3a (Kim et al.,
2000
). hdl/tcf3a mutant embryos lack anterior neural
structures and have expanded posterior neural fates, consistent with ectopic
Wnt signaling. Inhibition of tcf3a results in expanded posterior
neural fates even in the absence of wnt8 function, supporting a model
in which Wnt signaling functions solely to derepress Tcf-inhibited genes
(Dorsky et al., 2003
).
Furthermore, a truncated Tcf3a protein that cannot bind ß-catenin and can
thus only act as a repressor completely rescues the hdl mutant
(Kim et al., 2000
). To
determine whether the cooperative function of Nlk and Wnt signaling in
derepressing Tcf-inhibited genes was conserved from nematodes to teleost fish,
we undertook an analysis of Nlk function in zebrafish embryos.
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Materials and methods |
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Cloning of nlk
We used degenerate PCR on a 24-hour-stage cDNA library, followed by
5' and 3' RACE using the SMART RACE kit (Clontech), to amplify a
cDNA containing the entire nlk ORF, plus 170 base pairs (bp) of
5' UTR and 574 bp of 3'UTR sequence. This sequence was deposited
in GenBank (Accession Number AY562552). For in situ hybridization, a fragment
containing the complete ORF, plus 62 bp of 5' UTR and 130 bp of 3'
UTR was amplified and ligated into pGEM-T (Promega). For mRNA synthesis, we
designed primers corresponding to the 5' and 3' ends of the ORF,
and inserted the full-length product into pCS2+
(Turner and Weintraub, 1994).
The nlk point mutants, nlk (K117M) and nlk (C387Y),
were constructed using standard PCR-based site-directed mutagenesis.
mRNA and morpholino injections
For mRNA injections, sense transcripts were synthesized using the mMessage
mMachine kit (Ambion). For templates, we used full-length cDNA inserted into
pCS2+. mRNA was resuspended in water or Danieau's buffer prior to injection.
Morpholino antisense oligonucleotides (nlk:
5'-GTGTGTGGTACCTTAAGCAGACAGT-3') were obtained from Gene Tools
(Philomath, OR). tcf3b, lef1, wnt8b, wnt11, wnt8 ORF1 and
wnt8 ORF2 morpholinos have been previously described
(Dorsky et al., 2003;
Dorsky et al., 2002
;
Houart et al., 2002
;
Lekven et al., 2001
;
Lele et al., 2001
). The
standard control morpholino provided by Gene Tools was used in some
experiments. Morpholinos were dissolved in Danieau's buffer
(Nasevicius and Ekker, 2000
)
prior to use. For all injections, 2-3 nl of a 1 ng/nl stock was injected at
the one-cell stage, except where otherwise noted.
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Results |
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|
nlk RNA overexpression results in loss of anterior neural fates
As a first test to assess the activity of nlk during early
development, we injected in vitro-transcribed nlk mRNA into one-cell
zebrafish embryos. Injection of 200 pg nlk RNA results in embryos
with variable loss of eyes and forebrain
(Fig. 2D and data not shown),
and no other obvious morphological abnormalities at 24 hpf (59% of embryos
have small or missing eyes; n=510). Changing a conserved cysteine
residue to tyrosine at amino acid 425 in mouse Nlk eliminates its ability to
bind Tcf (Ishitani et al.,
1999). We made the analogous change in zebrafish nlk
(C387Y) and found that injection of this mutated RNA had almost no effect on
development (1% of embryos had small eyes; n=190), suggesting that
the nlk overexpression phenotype is dependent on interaction with Tcf
proteins. Furthermore, we constructed nlk (K117M), analogous to mouse
Nlk (K155M), which is kinase dead and unable to block Tcf binding to
DNA (Ishitani et al., 1999
).
Injection of nlk (K117M) RNA has no effect on development (2% of
embryos had small eyes; n=109), indicating that the kinase activity
of nlk is required to induce the loss of anterior neural
structures.
|
Injection of tcf3a MO results in a loss of telencephalon and eyes at 24 hpf (Fig. 2G, compare with wild type in Fig. 2A), and caused an expansion of the midbrain/hindbrain boundary (MHB) domain of pax2 (Fig. 2H) and a decrease in the diencephalic expression of pax6 (Fig. 2I), reflecting the increase in caudal character of the neurectoderm. Injection of tcf3b MO caused only a disorganization of the hindbrain (Fig. 2J); both pax2 and pax6 expression was normal (Fig. 2K,L). Co-injection of both tcf3 morpholinos strongly enhanced the tcf3a phenotype, eliminating the morphological MHB at 24 hpf (Fig. 2M), greatly expanding pax2 expression, and eliminating pax6 expression (Fig. 2N,O).
Injection of 200 pg of nlk RNA results in anterior truncations like those seen in tcf3a morphants (Fig. 2D). Similar to tcf3a morphants, pax2 expression is expanded and pax6 expression is reduced (Fig. 2E,F). nlk overexpression, then, phenocopies inhibition of tcf3a function, suggesting that nlk can negatively regulate the repression of posterior neural genes by tcf3a. Co-injection of 100 pg tcf3a RNA resulted in significant rescue of the nlk overexpression phenotype [62% (n=63) of nlk/tcf3a RNA-injected embryos had normal pax2 expression at 3 somites versus 31% (n=51) of nlk/GFP RNA-injected embryos], further indicating that the nlk overexpression phenotype is due to downregulation of tcf3a. Although most nlk-injected embryos closely resemble tcf3a morphants, when we injected high doses (800 pg) of nlk RNA, we saw embryos with a slightly more severe phenotype, but not to the degree seen in tcf3a/tcf3b double morphants (data not shown). This suggests that although nlk may preferentially interact with tcf3a, it may also be able to inhibit tcf3b to some degree. To test this, we co-injected nlk RNA with either tcf3a or tcf3b morpholinos. If nlk can downregulate tcf3b in addition to tcf3a, then co-injection of nlk RNA and tcf3a MO should phenocopy tcf3a/tcf3b morphants, showing both the dramatic expansion of pax2 and the loss of diencephalic pax6 expression. Indeed, nearly every embryo shows a more severe phenotype than that seen by injection of nlk RNA alone, with most phenocopying tcf3a/tcf3b morphants (82%; n=73; Fig. 2P,Q,R, compare with Fig. 2M,N,O). Most nlk RNA/tcf3b MO embryos also show the more severe phenotype characteristic of tcf3a/3b morphants (89% of embryos completely lack pax6; n=54; Fig. 2S,T,U). These results indicate that nlk is capable of interacting with both tcf3a and tcf3b to relieve their repression of posterior neural genes.
nlk requires wnt8 signaling to posteriorize neurectoderm
The nlk phenotype is similar to that seen with injection of low
levels (5-10 pg) of zebrafish wnt8 RNA
(Kelly et al., 1995).
wnt8 is required to induce posterior fates within the neurectoderm,
and overexpression of wnt8 expands posterior neural identity at the
expense of anterior fates (Erter et al.,
2001
; Lekven et al.,
2001
). To test whether nlk inhibits expression of
anterior neural markers during gastrulation, we fixed nlk- or
wnt8-injected embryos at 90% epiboly and performed in situ
hybridization using probes for opl, a telencephalic marker, and
tbx6, a marker of ventrolateral mesoderm
(Fig. 3). Like
wnt8-injected embryos, nlk-injected embryos show a dramatic
reduction of opl expression, indicative of a loss of anterior neural
identity (compare the loss of opl in
Fig. 3B,C with wild-type
expression in 3A, indicated by
arrowheads).
|
Morpholino antisense inhibition of nlk
To assess the function of endogenous nlk, we used morpholino
antisense oligonucleotides to interfere with the splicing of nlk mRNA
(Draper et al., 2001). We designed a morpholino to anneal to the splice donor
site between exon 5 and intron 5 (see Fig.
4). Translation of the improperly spliced message would result in
a truncated, probably non-functional, protein, with the 229 amino acids
encoded by exons 1-5 appended to 19 non-conserved amino acids encoded by the
5' end of intron 5, followed by a stop codon.
|
|
|
wnt8 likely functions through lef1 to specify
ventrolateral mesoderm, as lef1 morphants also show decreased
tbx6 expression during gastrulation
(Dorsky et al., 2002). In
contrast to tcf3a and tcf3b, lef1 appears to function
predominantly as an activator of Wnt target genes
(Kengaku et al., 1998
;
Merrill et al., 2001
). We
tested whether nlk and lef1 cooperate to activate
tbx6 by co-injecting both morpholinos and examining tbx6
expression (Fig. 6). When
nlk MO is injected at a low dose (0.6 ng), no effect on tbx6
is seen at 60% epiboly (Fig.
6B, compare with wild type in
6A). lef1 MO, as
previously reported, reduces tbx6 expression
(Fig. 6C). When 0.6 ng
nlk MO is co-injected with lef1 MO, tbx6 expression
is almost completely eliminated (Fig.
6D). It has recently been reported that tcf3a/tcf3b
double morphants have elevated levels of lef1 transcripts during
early gastrulation, suggesting that one role of tcf3a and
tcf3b is to negatively regulate lef1 expression
(Dorsky et al., 2003
). Thus,
one explanation for the synergy between nlk and lef1 could
be that nlk is required for relieving tcf3-mediated
repression of lef1 transcription. To test this hypothesis, we
examined lef1 expression in nlk MO-injected embryos. At
shield stage, lef1 expression is reduced in nlk morphants
compared with wild-type embryos (compare
Fig. 6F with 6E). If
nlk activates lef1 expression by inhibiting tcf3a
and tcf3b, then injection of tcf3a and tcf3b MOs
should rescue lef1 expression in nlk morphants. Indeed, we
found that nlk/tcf3a/tcf3b MO embryos
(Fig. 6H), like
tcf3a/tcf3b morphants (Fig.
6G), showed significantly elevated lef1 expression
compared with wild type, suggesting that nlk is not directly required
for lef1 transcription, but functions indirectly by modulating
tcf3a/tcf3b activity. Expression of tcf3a and tcf3b
is unaffected in nlk morphants (data not shown), indicating that the
interaction between nlk and tcf3a/3b is not due to
upregulation of tcf3a/3b transcription in the absence of
nlk.
To confirm that these phenotypes were due to loss of nlk function, we attempted to rescue the reduction of tbx6 and lef1 expression in nlk morphants by co-injecting nlk RNA. As a control RNA, we co-injected nlk (C387Y) RNA (shown as nlk* RNA in Fig. 6). nlk RNA significantly rescues the reduction in tbx6 expression (compare Fig. 6K with 6J) induced by nlk MO injection [80% show wild-type levels of expression at 60% epiboly (n=65) versus 29% (n=63) when nlk (C387Y) RNA was co-injected). lef1 RNA was also able to restore normal tbx6 expression in nlk MO embryos (Fig. 6L; 77% show wild-type expression, n=60), further suggesting that the reduction in tbx6 expression in nlk morphants is due to a reduction in lef1. nlk RNA was able to restore normal lef1 expression in a significant fraction of nlk morphants. Although only 27% (n=62) of nlk MO/nlk (C387Y) RNA-injected embryos had wild-type levels of lef1 at 60-70% epiboly (compare Fig. 6N with wild type in 6M), 70% (n=43) of nlk MO/nlk RNA-injected embryos (Fig. 6O) had normal levels of lef1. Taken together, these results show that nlk MO specifically interferes with nlk function, and that nlk is required for normal lef1 expression and subsequent ventrolateral mesoderm formation.
nlk morphants are defective in later aspects of AP brain patterning
Wnt signaling late in gastrulation has been proposed to regulate regional
identity within the anterior neural plate, apart from the earlier action of
wnt8 ORF1 and ORF2 (Heisenberg et
al., 2001; Houart et al.,
2002
; Houart et al.,
1998
; Kim et al.,
2002
; van de Water et al.,
2001
). Knockdown of either wnt8b or fz8a results
in a reduction in the expression of markers in the midbrain and posterior
diencephalon, suggesting that these two genes play key roles in promoting
development of these regions of the brain
(Houart et al., 2002
;
Kim et al., 2002
). We tested
whether nlk plays a role in this process by injecting embryos with
nlk MO, wnt8b MO, or both morpholinos together, and staining
embryos with several neural markers (Fig.
7). In one experiment, we fixed embryos at tailbud stage and
probed with a cocktail of opl, pax2 and tbx6 to mark the
telencephalon, MHB and margin (Fig.
7A-C), or with en2 to mark the prospective MHB and
midbrain (Fig. 7D-F). We
observed no changes in opl expression in wnt8b MO
(Fig. 7B) or nlk MO
(Fig. 7C) embryos, confirming
published results for wnt8b MO
(Kim et al., 2002
) and
indicating that wnt8-dependent posteriorization was occurring
normally. pax2 expression was normal in wnt8b morphants, but
reduced in nlk MO embryos, similar to what is observed in
fz8a MO embryos (Kim et al.,
2002
). en2 is substantially reduced in most
wnt8b morphants (Fig.
7E, compare with control in
7D), as previously reported,
and is eliminated in most nlk MO embryos
(Fig. 7F). When we examined
en2 expression at the 7-somite stage, en2 was present but at
a much reduced level in wnt8bMO- (76%; n=45;
Fig. 7H) or nlk MO
(81%; n=42; Fig.
7I)-injected embryos. Co-injection of a high dose (1 ng each) of
both morpholinos does not result in a stronger phenotype, but does increase
the penetrance somewhat (95%; n=17). When low doses (0.5 ng) are
injected, both nlk MO and wnt8bMO still cause a reduction of
en2 staining at the 7-somite stage, but at a lower penetrance [48%
(n=27) and 51% (n=31), respectively]. Co-injection of both
MOs at the lower doses results in a significant increase in the penetrance of
the en2 phenotype (91%; n=35), suggesting that nlk
and wnt8b are functioning together to regulate en2
expression.
|
nlk interacts with non-canonical Wnt signals
The data above indicate that nlk can function with canonical Wnts
in regulating aspects of mesoderm and neurectoderm development. Recently
published work using mammalian cell culture supports a potential role for Nlk
as a downstream effector of non-canonical Wnts such as Wnt5a
(Ishitani et al., 2003a). In
vertebrates, non-canonical Wnts, together with homologs of Drosophila
planar cell polarity (PCP) signaling pathway components such as
strabismus and prickle, regulate convergence/extension
movements during gastrulation (reviewed by
Tada et al., 2002
;
Veeman et al., 2003
). As
nemo mutants in Drosophila exhibit some defects in PCP
signaling, it is possible that the cell culture data describes a conserved
role for Nlk proteins in non-canonical Wnt signaling
(Choi and Benzer, 1994
;
Verheyen et al., 2001
).
Therefore, we attempted to detect genetic interactions between nlk
and non-canonical Wnt signaling in zebrafish.
In zebrafish, mutations in two non-canonical Wnts, pipetail
(ppt)/wnt5 and silberblick
(slb)/wnt11, have been characterized.
ppt/wnt5 embryos are defective in tail extension, whereas
slb/wnt11 mutations cause partial cyclopia due to a defect
in migration of the anterior axial mesoderm, the prechordal plate
(Heisenberg and Nusslein-Volhard,
1997; Heisenberg et al.,
2000
; Kilian et al.,
2003
; Rauch et al.,
1997
; Ulrich et al.,
2003
). To test for genetic interactions between nlk and
ppt/wnt5, we injected nlk MO into embryos from a
cross of ppt heterozygotes. nlk MO/ppt embryos
display only an additive phenotype, with a moderately more severe tail defect
(data not shown).
By contrast, we observe a strong interaction between nlk and
wnt11/slb. We quantified the severity of the phenotype by
calculating the Cyclopia Index (CI)
(Marlow et al., 1998) (see
Fig. 8). Injection of 0.5 ng
wnt11 MO causes a mild phenotype, with the large majority of embryos
being either unaffected or having slightly more closely set eyes (CI=1.59).
nlk MO has no cyclopia phenotype on its own, but strongly enhances
the wnt11 MO phenotype, resulting in many embryos with complete eye
fusion (CI=3.98). As a control, we saw no effect on the wnt11 MO
phenotype upon co-injection of wnt8 ORF1 MO (CI=1.54 versus 1.59 in
wnt11 MO alone). We directly analyzed migration of the prechordal
plate by fixing embryos at 95-100% epiboly and staining with probes for
tbx6 (to mark the margin) and gsc (to mark the prechordal
plate). Although all nlk MO (n=45), and most wnt11
MO (85%, n=41) embryos showed normal migration of the prechordal
plate to near the animal pole (Fig.
8C,D, arrowheads; compare with wild type in B), most
nlk/wnt11 MO embryos showed a dramatic defect in migration of the
prechordal plate (Fig. 8E).
This effect of nlk MO on prechordal plate migration is specific to
wnt11, as co-injection of nlk MO does not enhance the mild
cyclopia induced by injection of low doses of strabismus/trilobite MO
(Fig. 8), nor does it enhance
the phenotype of knypek/glypican4 mutants (data not shown), which
very rarely exhibit partial cyclopia, but which do strongly enhance the
cyclopic phenotype of both wnt11 and strabismus mutants
(Henry et al., 2000
;
Jessen et al., 2002
;
Park and Moon, 2002
;
Sepich et al., 2000
;
Topczewski et al., 2001
).
|
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Discussion |
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nlk inhibits tcf3a and tcf3b, not lef1
The interplay between caudalizing wnt8 and rostrally localized
tcf3 homologs functioning as inhibitors of Wnt signaling helps
establish AP polarity within the neural plate. Overexpression of nlk
in zebrafish phenocopies the effects of ectopic wnt8 signaling on AP
patterning, and enhances the phenotype associated with loss of tcf3
function. In light of the well-established ability of Nlk homologs in other
species to phosphorylate Tcfs and inhibit the DNA binding ability of
ß-catenin/Tcf complexes, we suggest that Nlk and ß-catenin can
function together to derepress genes inhibited by Tcf3a and Tcf3b. This
provides the first evidence in vertebrates that Nlk can function as a positive
regulator of canonical Wnt target genes.
Mouse Nlk has been shown to directly interact with both mammalian Lef1 and
Tcf4, as well as Xenopus Tcf3
(Ishitani et al., 2003b;
Ishitani et al., 1999
;
Meneghini et al., 1999
).
Mutational analysis has demonstrated that Nlk phosphorylates conserved serine
and threonine residues on Lef1 and Tcf4, and that these residues are required
for Nlk to inhibit the DNA-binding ability of Tcf/ß-catenin complexes
(Ishitani et al., 2003b
).
Zebrafish tcf3a, tcf3b and lef1 all possess serine or
threonine residues at analogous sites to their mammalian homologs, suggesting
that they are all potential substrates for nlk. Interestingly,
overexpression of zebrafish nlk only affects AP patterning in the
neurectoderm, consistent with an inhibition of only tcf3a and
tcf3b, not of lef1. Also, overexpression of nlk
does not cause ventralization in either zebrafish or Xenopus (J.
Waxman, unpublished), suggesting that nlk does not interact with a
(as yet unknown) Tcf/Lef protein required for transducing maternal
ß-catenin signaling. By contrast, mouse Nlk can block dorsal axis
formation in Xenopus (Ishitani et
al., 1999
), and can block formation of dorsal mesoderm when
injected into zebrafish (data not shown), suggesting that it can inhibit
maternal ß-catenin signaling. Although little is known regarding which
regions of Nlk are important for interaction with ß-catenin or Tcf/Lef
proteins, it is possible that the substantial differences between mammalian
and zebrafish nlk over the first 90-100 amino acids could contribute
to the differences in phenotypes when overexpressed. In early zebrafish
development, nlk seems to interact specifically with two Tcf3 genes
known to play a role in the repression of Wnt target genes, but not with
lef1, which has been proposed to function primarily as an
activator.
nlk morphants are defective in canonical Wnt signaling
Mutational analyses in Drosophila and in mice have been
inconclusive in distinguishing whether Nlk functions as an activator or an
inhibitor of the Wnt pathway. For example, nemo mutant flies show,
among other phenotypes, a variable segment polarity phenotype that can include
both excess naked cuticle, indicative of excess wingless
(wg)/Wnt signaling, and loss of denticle diversity, which is
suggestive of a reduction of wg signaling
(Mirkovic et al., 2002;
Verheyen et al., 2001
).
Similarly, the mouse knockout causes a pleiotropic phenotype that may or may
not reflect an underlying role in the regulation of Wnt signaling
(Kortenjann et al.,
2001a
).
Our loss-of-function studies support a role for nlk in regulating canonical Wnt signaling. The enhancement of wnt8 and lef1 loss-of-function phenotypes suggests that nlk is functioning as an activator of Wnt targets in mesoderm patterning. As nlk is required for lef1 expression in the presence of tcf3, our data support a model in which nlk relieves tcf3-mediated repression of lef1 transcription during gastrulation. ß-catenin stabilized by wnt8 could then interact with Lef1 to activate tbx6 and other genes.
Our observation that nlk MO causes defects in en2 and
pax6 expression similar to those seen in wnt8b and
fz8a morphants suggests that nlk may be functioning in this
later AP patterning event. A simple hypothesis is that nlk relieves
the repression of wnt8b targets by tcf3a and/or
tcf3b. Supporting a possible role for tcf3a in regulating
wnt8b signaling, injection of lower doses of tcf3a MO (0.5
ng) or nlk RNA (100 pg) results in embryos with normal pax2
expression and an expanded pax6 domain, as would be expected from
ectopic wnt8b signaling (Kim et
al., 2002) (and data not shown). This phenotype indicates that
tcf3a may act to limit wnt8b signaling within the rostral
neurectoderm, and suggests that nlk could function in
wnt8b-mediated patterning by inhibiting tcf3a.
Given that overexpression of nlk expands posterior neural fates in
a wnt8-dependent manner, we were somewhat surprised that we did not
observe any affects of nlk MO on the expression of neural genes known
to be regulated by wnt8, such as opl. Although we note that
a comprehensive search of sequenced mammalian and invertebrate genomes
indicates that only one Nlk homolog is present, an ancient gene duplication
event in the ancestry of teleost fish has led to the presence of multiple
copies of many genes that are present only once in tetrapods, raising the
possibility that additional Nlk homolog(s) in zebrafish could be functioning
in this process (Kortenjann et al.,
2001b; Taylor et al.,
2001
).
nlk and wnt8 ORF2 interact with non-canonical Wnt11 signaling
The strong enhancement of the slb/wnt11 phenotype by nlk
MO indicates that nlk has an important role in regulating cell
movements during gastrulation. Our data do not necessarily place nlk
downstream of a non-canonical Wnt, as has been demonstrated in cell culture
with mouse Nlk and Wnt5a. Although components of non-canonical Wnt pathways
are clearly important for controlling cell movements, canonical Wnt signaling
can also play a role. For example, in zebrafish, maternal ß-catenin
signaling is known to activate Stat3, which is required for normal
convergence/extension movements (Yamashita
et al., 2002). Maternal ß-catenin signaling has also been
shown to regulate gastrulation movements in Xenopus, probably through
activation of Xenopus nodal-related 3 (Xnr3)
(Kuhl et al., 2001
). Thus, the
observed interaction between nlk and wnt11 could reflect a
defect in the regulation of a canonical Wnt target caused by loss of
nlk, particularly in light of the observation that nlk
morphants do not show any obvious convergence-extension defects.
Given the potent affects on cell proliferation and fate specification by Wnts, it is essential to restrict activation of Wnt target genes to only the appropriate time and place within an organism. It is likely, then, that many Wnt targets are actively repressed in the absence of Wnt ligand, and a significant function of Wnts in these contexts is to derepress them, permitting activation by other factors. Nlk is an important component of the mechanism by which some Wnt targets are activated in C. elegans, and this activity appears to be conserved in zebrafish.
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ACKNOWLEDGMENTS |
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