1 Molecular and Cellular Biology Program, University of Washington School of
Medicine, Seattle, WA 98195, USA
2 Howard Hughes Medical Institute, Department of Pharmacology and Center for
Developmental Biology, Box 357750, University of Washington School of
Medicine, Seattle, WA 98195, USA
3 Neurobiology and Behavior Graduate Program, University of Washington School of
Medicine, Seattle, WA 98195, USA
Author for correspondence (e-mail:
rtmoon{at}u.washington.edu)
Accepted 27 September 2004
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SUMMARY |
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Key words: Dishevelled, Dapper, Zebrafish, Wnt signaling
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Introduction |
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In addition to a Wnt ligand, Wnt/ß-catenin signaling involves a seven
transmembrane receptor, Fz, and a transmembrane co-receptor, LRP
receptor-related protein-5 or -6 (LRP5/6)
(Mao et al., 2001;
Tamai et al., 2000
). In the
absence of Wnt signaling, ß-catenin is phosphorylated in a complex of
proteins that includes the scaffolding proteins Axin and APC, and the kinases
GSK-3ß and CK1
. Phosphorylation of ß-catenin leads to its
ubiquitination and degradation (Liu et
al., 1999
; Marikawa and
Elinson, 1998
). In response to a Wnt signal, Dishevelled (Dvl) is
activated, promoting the stabilization of ß-catenin. As ß-catenin
accumulates in the cytoplasm and nucleus, it associates with the LEF/TCF
transcription factors to activate transcription of targets genes
(Huelsken and Behrens, 2002
).
In vertebrates, the Wnt/Ca2+-PCP pathway also appears to involve a
Wnt ligand and Fz, but does not involve LRP-5/6 co-receptors. Instead,
stimulation of this pathway leads to the association of Dvl with proteins that
control cytoskeletal remodeling, including Rac and the Rho-interacting protein
Daam (Habas et al., 2003
;
Habas et al., 2001
). Vertebrate
Dvl also interacts with orthologs of Drosophila proteins that
regulate PCP signaling, including Strabismus/Trilobite (Stbm/Tri) and Prickle
(Pk) (reviewed by Wharton,
2003
). However, the general mechanisms of Wnt/Ca2+-PCP
signaling remain unclear (reviewed by
Veeman et al., 2003a
).
The phosphoprotein Dvl is therefore involved in the Wnt/ß-catenin and
Wnt/Ca2+-PCP pathways. Both genetic and biochemical screens have
identified a host of Dvl-interacting proteins (reviewed by
Wharton, 2003). In addition to
those proteins previously mentioned, this list includes Naked cuticle (Nkd)
(Rousset et al., 2001
), Casein
kinase 1
(CK1
) (Peters et al.,
1999
), Protein kinase CK2 (CK2)
(Willert et al., 1997
), Par1
(Sun et al., 2001
), GBP
(Li et al., 1999
), FRODO
(Gloy et al., 2002
) and Dapper
(Dpr; Dact Zebrafish Information Network)
(Cheyette et al., 2002
).
CK1
, CK2, Par1, GBP and FRODO have been described as positive regulators
of Wnt/ß-catenin signaling, while Nkd and Dpr have been characterized as
inhibitors of Wnt signaling. A number of these Dvl-interacting proteins have
been proposed to act as switches that regulate Dvl functions in different
Wnt/Fz signaling pathways. For example, it has been proposed that Par1
promotes Wnt/ß-catenin signaling at the expense of
Wnt/Ca2+-PCP signaling, while vertebrate Nkd has been proposed to
do the opposite (Sun et al.,
2001
; Yan et al.,
2001
). Clearly an understanding of Dvl-associated proteins is
central to understanding how Wnt pathways are regulated.
We have previously reported the characterization of a Dvl-interacting
protein, Xenopus Dpr1 (Cheyette et
al., 2002), and presented evidence that it is required for
notochord formation. More recently, Hikasa and Sokol
(Hikasa and Sokol, 2004
) have
presented evidence that a Dpr homolog, FRODO, is involved in aspects of
organizer formation and neural patterning. To understand better the functions
of the Dpr family in vertebrate embryos, in the present study we have
characterized two zebrafish Dpr paralogs, Dpr1 and Dpr2, which we analyze
through their loss and gain of function. Our data suggest that the Dpr1 and
Dpr2 paralogs have different roles in early vertebrate development.
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Materials and methods |
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Expression constructs
All genes used except for ß-galactosidase, CK1, CK1D
N,
Drosophila Par1 and Par1-KN were cloned into pCS2P+. The
Xenopus CK1
and CK1D
N in pCS2 were gifts from J. Graff
(Peters et al., 1999
).
CK2
and ß subunits, gifts from D. Seldin, were subcloned into
pCS2P+. Drosophila Par1 and Par1-KN were in pCS2+, as reported
previously (Sun et al., 2001
).
GBP RNA was a gift of D. Kimelman and H. Gist Farr. The zebrafish
Dvl2
PGB construct deletes amino acids 231-252. Capped mRNAs for
injection were prepared using the Message Machine kit (Ambion, Austin, TX) and
dissolved in water.
RT-PCR analysis of Wnt/ß-catenin target genes
For analysis of the Wnt/ß-catenin target genes siamois
(sia) and Xenopus nodal-related 3 (Xnr3),
Xenopus embryos were injected, animal caps isolated, and RT-PCR
conducted essentially as described
(Cheyette et al., 2002).
Controls omitting RT were conducted for all experiments (not shown).
Morpholino injections of zebrafish
Stock morpholinos (MO; Genetools, Philomath, OR) were dissolved in 1
xDanieu's buffer to a concentration of 10 ng/nl
(Nasevicius and Ekker, 2000).
The diluted morpholino stocks (2 nl) were injected into one-cell zebrafish
embryos. strabismus/trilobite MO
(Park and Moon, 2002
),
wnt11/silberblick MO (Lele et
al., 2001
), wnt8-orf1 and wnt8-orf2 MOs
(Lekven et al., 2001
) have
been reported previously. Morpholino sequences for dpr1 and
dpr2 are as follows:
Immunocytochemistry
For immunocytochemistry, Xenopus embryos were injected, and animal
caps were isolated, fixed in phosphate-buffered saline and processed by
standard methods (Cheyette et al.,
2002). The anti-Myc antibody was the monoclonal 9E10 and the
secondary antibody was the goat-anti-mouse Alexa Fluor 488 (Molecular Probes,
Eugene, OR).
Xenopus embryo lysates and immunoprecipitation
Embryos injected with 3 ng of each RNA were lysed with 150 mM NaCl, 20
mM Tris-HCl pH 7.5, 0.5% CHAPS, 1 mM EDTA, 1 mM EGTA and Complete protease
cocktail (Roche, Indianapolis, IN). For immunoprecipitation (IP), the lysis
buffer did not include EDTA and EGTA. Anti-HA antibody was a commercial
monoclonal antibody.
Cell culture and reporter assays
Human embryonic kidney 293T cells were transfected using Lipofectamine Plus
(Invitrogen) with the reporter construct, Super(8X)TOPFLASH
(Veeman et al., 2003b), an
internal control construct encoding Renilla luciferase and the indicated
plasmids and empty vector to maintain constant amounts of DNA. Luciferase
activity was measured 24 hours after transfection, using the Dual-Luciferase
Reporter Assay System (Promega).
GenBank Accession numbers
Zebrafish Dpr1, AY545443; zebrafish Dpr2, AY545444; zebrafish Dvl2,
AY552332.
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Results |
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At tailbud stage, dpr2 is maintained in the axial mesoderm and in
the anterior brain. This brain expression is transient and is predominantly
lost by three somites (not shown). dpr2 expression is maintained in
the lateral mesoderm, and occurs in a banding pattern that is reminiscent of
wnt11 (arrowhead; Fig.
2D, part d) (Kilian et al.,
2003). At the three somite stage, dpr2 is expressed
posterior to a sharp boundary in the posterior hindbrain and anterior spinal
cord. Double staining with dpr2 (blue) and krox20 (red)
reveals this sharp boundary is adjacent to r5 at three somites (red arrow;
Fig. 2D, part e). Similar to
dpr1, dpr2 expression is quite specific during somitogenesis
(Fig. 2D, part f). By the 12
somite stage, dpr2 is upregulated in the anterior of the older
somites (Fig. 2D, part f,
arrow), but there is little dpr2 expression in the nascent somites
(arrowhead; Fig. 2D, part f).
dpr2 is also expressed in the posterior presomitic mesoderm and
tailbud. Later expression patterns for dpr2 are presented in Fig. S3
in the supplementary material. The data of Gillhouse et al.
(Gillhouse et al., 2004
),
published during revision of this manuscript, support these patterns of
expression of dpr1 and dpr2.
Dpr1 and Dpr2 have unique functions in development
In order to study the requirements for endogenous Dpr1 and Dpr2 in
zebrafish development, we used morpholino antisense oligonucleotides (MOs)
(Draper et al., 2001;
Nasevicius and Ekker, 2000
).
The dpr1 MO was designed to block the proper splicing of the second
intron/exon boundary. The dpr1 MO was able to significantly abrogate
proper splicing by the appearance of an
1700 bp band as analyzed by
RT-PCR (Fig. 3A). The
morpholino to dpr2 was designed to the 5'-UTR. To test if this
specifically blocked translation, the 5'-UTR was cloned upstream of
luciferase (5'Dpr2-luc). The dpr2 MO specifically blocked the
in vitro translation of the 5'-UTR Dpr2-luciferase construct, relative
to an internal control ß-galactosidase
(Fig. 3B).
|
dpr2 morphants (n=258/289 89%) have a different phenotype that is indicative of aberrant convergent extension movements during gastrulation (Fig. 3C, part c; 3D, parts c,d). The body axes are short and wide, and there are no major changes in cell fates. Consistent with this observation, the anterior of the embryos does not extend as far around the yolk (compare arrowheads in Fig. 3D, part c with part a), the notochord is wider and somites are elongated (compare distance between small, posterior arrowheads in Fig. 3C, part c with those in 3C, parts a,b; note elongated somites in Fig. 3D, part d compared with those in 3D, part b). Similar results were obtained during somitogenesis with a different MO, dpr2 MO(2), that abrogates proper splicing of this gene (data not shown).
To see if Dpr1 and Dpr2 are functionally redundant we injected both dpr morpholinos. The phenotype (n=121/137 88%) was strictly additive (Fig. 3C, compare part d with parts a-c). We conclude that dpr1 morphants have a subtle phenotype leading to reduction of the midbrain and diencephalon, while dpr2 appears to regulate convergent extension movements. However, repeating the morpholino injections in sensitized, hypomorphic, backgrounds proved to be informative, as discussed below.
Dpr1 is an enhancer of Wnt/ß-catenin signaling
Although dpr1 morphants do not have a severe phenotype, the
pattern of dpr1 expression could be consistent with it playing a role
in early Wnt-8/ß-catenin signaling that is required for ventralization
and posteriorization of embryos (Lekven et
al., 2001). To determine if dpr1 functionally interacts
with the Wnt8 signal during early zebrafish development, we injected the
dpr1 MO with a low amount of two wnt8 morpholinos
(MO2, ORF1 MO+ORF2 MO), a dose that produces no phenotype or a weak
hypomorphic phenotype (Lekven et al.,
2001
). Embryos were first examined at 24 hours, and scored on a 4
point scale with 0 being wild type, 1 having modest enlargement of the
telencephalon, 2 having enlargement of the anterior and reduction of the
posterior, and 3 having severe enlargement of the anterior and reduction of
the posterior (Fig. 4A).
Injection of wnt8 MO2 plus control MO yielded embryos with
a slightly larger telencephalon than controls
(Fig. 4A, arrow in panel 1 and
graph). dpr1 MO plus control morpholino injected embryos are
equivalent to dpr1 MO alone (Fig.
4A, graph). However, when the dpr1 MO was injected with
the wnt8 MO2, the morphants phenocopied strong loss of
Wnt8 function (Fig. 4A, graph).
These embryos had enlarged telencephalons (arrowhead in panel 3), eyes, and
reduced posterior (trunk and tail). We confirmed these results using a second
dpr1 morpholino (dpr1 MO2, not shown).
|
Given that dpr2 morphants have convergence extension problems, we would not expect to see the same interaction with Wnt8. To test this hypothesis, we injected the hypomorphic dose of wnt8 MO2 plus a maximal dose of the dpr2 MO. In dpr2 MO plus control morpholino, the embryos did not extend as far around the yolk and their bodies are wider (Fig. 4B, parts k,l). Injection of dpr2 MO plus wnt8 MO2 is strictly additive (compare Fig. 4B, parts m,n with parts k,l and parts c,d). The embryos are shorter and wider, coupled with modest expansion of opl, like wnt8 MO2 alone. We conclude that Dpr2 does not play a role in Wnt8 signaling in early zebrafish development.
Dpr2 functionally interacts with the Wnt/Ca2+-PCP pathway(s)
The dpr2 morphant phenocopies loss of PCP components like
strabismus/trilobite (stbm), kynpek (kny)
and prickle (pk) (Fig.
5A) (Jessen et al.,
2002; Park and Moon,
2002
; Topczewski et al.,
2001
; Veeman et al.,
2003b
). At 24 hours dpr2 morphants are noticeable
shorter, with block-like somites (Fig.
5A, arrow) and they have eyes that have not properly separated
owing to improper migration of underlying mesodermal tissues
(Fig. 5A, arrowhead). To test
if dpr2 functionally interacts with stbm, we injected a low
hypomorphic dose of the stbm MO with a low hypomorphic dose of the
dpr2 MO. Embryos were then scored on a scale of 0 to 2, with 0 being
wild-type, 1 having mild convergence extension (CE) problems and 2 having
strong CE problems (Fig. 5B).
Injection of either morpholino alone (plus control morpholino) yielded 10% and
15% of the embryos with mild CE problems for stbm MO and
dpr2 MO, respectively (Fig.
5C). None of the embryos had strong CE problems
(Fig. 5C). However,
co-injection of the stbm MO and dpr2 MO yielded 30% of the
embryos having mild CE problems and 55% of the embryos with strong CE defects.
Thus, we conclude there is a functional interaction between Dpr2 and Stbm.
|
To test if these functional interactions with stbm/tri and wnt11/slb are specific to Dpr2 and not Dpr1, we co-injected embryos with a higher dose of dpr1 MO(2) and either the stbm MO or wnt11 MO (Fig. 5C,E). The scoring of these embryos was almost identical to the stbm MO or wnt11 MO plus control morpholino. Therefore, there is not a functional interaction between stbm or wnt11 and dpr1.
Association with zebrafish Dvl2 and subcellular localization of Dpr orthologs
After injection of dpr1-myc or dpr2-myc RNA with
zebrafish dvl-HA RNA into Xenopus embryos, followed by
immunoprecipitation with anti-HA antibodies, both Dpr1-myc and Dpr2-myc
proteins were detected by western blot
(Fig. 6A,B). Thus, both
zebrafish Dpr family members can associate either directly or indirectly with
Dvl2, as reported for Xenopus Dpr family members
(Cheyette et al., 2002;
Gloy et al., 2002
).
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Gain of function of Dpr1 and Dpr2 in Xenopus and HEK293T cell assays
Overexpression of signaling proteins is useful in establishing the activity
of a protein in a specific context, and thus learning its potential
involvement in a given pathway. We asked whether gain-of-function of Dpr1 and
Dpr2 might affect Wnt/ß-catenin signaling. Unfortunately the
overexpression studies could not be conducted in zebrafish, as injection of
Dpr RNAs into embryos leads to a high percentage of early embryonic lethality
and non-specific toxicity. Therefore, the gain-of-function assays were
conducted in Xenopus embryos and in HEK293T cells, both of which are
responsive to Wnt signals.
We first tested whether Dpr1 and Dpr2 induce Wnt/ß-catenin target genes in Xenopus animal caps and found that dpr1 but not dpr2 RNA induces sia and Xnr3 (Fig. 6E, compare lane 1 with lanes 2 and 3). Consistent with this result, ventral injection of dpr1 RNA induced partial secondary axes in 36% of Xenopus embryos (n=78), while dpr2 RNA induced partial secondary axes in only 5% of Xenopus embryos (n=52) (not shown). Injection of less than 4 ng of dpr1 or dpr2 RNA did not induce Wnt/ß-catenin target genes or secondary axes (not shown).
While injecting embryos for these animal cap experiments we observed that injection of dpr2 RNA into the animal pole resulted in aggregation of pigment around the site of injection, whereas injection of dpr1 RNA and ß-gal control RNA did not (see Fig. S4 in the supplementary material). This result, which we do not understand in terms of mechanisms, further suggests functional differences in the activities of the two Dpr proteins.
Despite differences in the ability of injected dpr1 and dpr2 RNAs to induce Wnt/ß-catenin target genes, we found that both Dpr orthologs synergize with zebrafish Dvl2 to induce Wnt/ß-catenin target genes in Xenopus animal caps (Fig. 6F, compare lanes 2-7 with control lane 8; see Fig. S5 in the supplementary material for similar results with Xenopus Dpr orthologs). However, dpr1 synergizes more strongly (n=6 experiments). This is probably not due to preferential expression of Dpr1, as Dpr1 and Dpr2 translate at similar levels in vitro (Fig. 6G) and tagged proteins Dpr1-myc and Dpr2-myc, which are functional equivalent to untagged Dpr1 and Dpr2, are expressed similarly in frog embryos (not shown and Fig. 6G). This synergistic activation of Wnt/ß-catenin target genes by Dpr1 and Dvl2 was blocked by co-expression with Axin (Fig. 6H), consistent with its being a ß-catenin-dependent synergy.
As Dpr1 and Dpr2 synergize with Dvl2, a component of Wnt signaling
pathways, we next asked if either would synergize with Wnt itself to activate
Wnt/ß-catenin target genes. Neither zebrafish ortholog synergized with
Wnt8, nor did either inhibit activation of target genes
(Fig. 6I). We then turned to an
independent assay system to investigate effects of co-expression of Dpr
orthologs with Dvl2 or Wnt. HEK293T cells are responsive to Wnt/ß-catenin
signaling, which can be monitored by the luciferase activity of
Super(8X)TOPFlash, a ß-catenin-responsive reporter construct
(Veeman et al., 2003b).
Consistent with what we observed in Xenopus animal cap experiments,
both zebrafish Dpr1 and Dpr2 synergize with zebrafish Dvl2 after transient
transfection in HEK293T cells (Fig.
6J). However, both Dpr orthologs were inhibitory to the
Wnt-mediated activation of the luciferase reporter in this cell line (see Fig.
S6 in the supplementary material).
As the Dvl-associated protein Dpr1 synergizes with Dvl2, and does so to a
greater extent than Dpr2, we next investigated whether Dpr1 could synergize
with other proteins that interact with Dvl. We focused on CK1, protein
kinase CK2 (here referred to as CK2), Par1 and GBP/FRAT-1. We expected that
these Dvl-interacting proteins should synergize with Dvl2 in animal cap assays
to activate Wnt/ß-catenin target genes, which is what we observed
(Fig. 7A, compare lanes 2-6
with lane 1).
|
We then tested whether Dpr1 could synergize with a second Dvl-associated
kinase, CK2, to induce Wnt/ß-catenin target genes. As CK2 functions as a
hetero-tetramer, `CK2' indicates that we injected RNA encoding both
and ß subunits (Pinna,
2002
) unless otherwise noted. Although ck2 alone did not
induce the target genes, co-injection of ck2 RNA with dpr1
RNA induced ß-catenin target genes
(Fig. 7C, compare lanes 2 and 3
with lane 1). As CK2 functions as a hetero-tetramer, with the ß subunit
being required to stabilize the
subunit
(Pinna, 2002
), we tested the
specificity of the Dpr1-CK2 synergy by co-injecting just the
ck2
subunit RNA with dpr1 RNA. No synergy was
observed (data not shown).
We next tested whether there is a functional interaction between Dpr1 and the Dvl-associated kinase Par1. At any dose tested, par1 alone did not activate Wnt/ß-catenin target genes in animal caps. However, when par1 RNA was co-injected with dpr1 RNA, there was a synergistic activation of Wnt/ß-catenin target genes (Fig. 7D, compare lanes 2 and 3 with lane 1). To determine whether this functional interaction was dependent upon Par1 kinase activity, we co-injected dpr1 RNA with a higher dose of par1-KN (a kinase dead form of Par1) RNA. Dpr1 did not synergize with Par1-KN (Fig. 7D, compare lanes 4 and 5 with lanes 2 and 3).
Finally, we tested whether GBP/FRAT-1 and Dpr1 synergize to activate
Wnt/ß-catenin target genes. We only observed a mild enhancement between
Dpr1 and GBP/FRAT-1 when co-injected (Fig.
7E). As Par1 synergizes with Dpr1, while the GBP/FRAT-1-mediated
gene activation was weakly enhanced by Dpr1, we deleted the
Par1-GBP/Frat-1-binding domain (Hino et
al., 2003) in zebrafish Dvl2, resulting in the construct
Dvl2
PGB (
Par1 and GBP Binding domain). In principle, this should
result in a Dvl2 protein that would be unable to interact with endogenous GBP
or Par1. Like Hino et al, (Hino et al.,
2003
), we found this construct could activate Wnt/ß-catenin
target genes alone (not shown). However, we found that Dvl2
PGB did not
synergize with Dpr1 in the Xenopus animal cap assay
(Fig. 7F, compare lane 2 with
lane 1).
In Xenopus explant assays, the above data show that Dpr1 can
functionally interact with Dvl2 and the Dvl-interacting proteins CK1,
Par1 and CK2, but it does not functionally interact as well with GBP. We next
turned to prospective loss-of-function studies using the CK1
inhibitor
CKI-7 (Peters et al., 1999
).
CKI-7 had no apparent effect on gene induction by stabilized ß-catenin, a
positive control for inducing sia and Xnr3
(Fig. 8A, compare lane 5 with
lane 6). Whether gene activation by Dvl requires CK1
has not been
explored. In addressing this, we found that Dvl-mediated induction of target
genes was not affected by CKI-7 injection
(Fig. 8A, compare lane 3 with
lane 4), though it has been reported that Dvl-mediated axis duplication in
Xenopus can be inhibited by CKI-7
(Peters et al., 1999
). We then
tested if Dpr1-Dvl2 synergy requires CK1
. Dpr1 and Dvl2 did not
synergize in gene induction when CKI-7 was co-injected
(Fig. 8B, compare lane 1 with
lane 2). Thus, Dpr1 requires endogenous CK1
to enhance Dvl activity. We
also found that the synergy of Dpr1-Par1 requires endogenous CK1
activity (Fig. 8B, compare lane
5 with 6). Thus, Dpr1 is dependent on CK1
for synergistic interactions
with both Dvl2 and Par1.
|
Potential Dpr mechanisms
We further attempted to determine any mechanisms by which Dpr1 and Dpr2
might affect Wnt pathways. With respect to the Wnt/Ca2+-PCP
pathway, Rho activation has been proposed to be a potential indicator of this
poorly understood pathway(s) (Habas et al.,
2001). However, neither Dpr1 nor Dpr2 affected Rho activation in
HEK 293T cells or in Xenopus embryo lysates (not shown). With respect
to Wnt/ß-catenin signaling, CK1
has been shown to increase GBP
association with Dvl-1 (Hino et al.,
2003
). However, we did not find that CK1
enhanced Dpr1 or
CK2 association with Dvl2, nor did we find that Dpr increased the association
of other Dvl-interacting molecules with Dvl2 (not shown). Increased CK2
activity is correlated with increased Dvl-2 stability
(Song et al., 2000
). However,
we did not find that Dpr or any other Dvl-interacting protein (including CK2)
could enhance the stability of Dvl (not shown). Finally, Dvl has been proposed
to undergo an electrophoretic mobility shift in correlation with Wnt or Fz
activation (Lee et al., 1999
;
Rothbacher et al., 2000
;
Sun et al., 2001
). We also did
not find that Par1 or CK2, in the presence or absence of ectopic Dpr,
increased the phosphorylation state of Dvl, as monitored by electrophoretic
mobility (not shown). With the caveats associated with any negative data, we
suspect that these potential mechanisms of action of Dpr orthologs are not the
most likely to explain how Dpr family members participate in Wnt signaling
pathways.
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Discussion |
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Gain- and loss-of-function of Dvl, as well as structure-function analysis
are consistent with the idea that specific Wnt and Fzs somehow activate Dvl in
both vertebrate Wnt pathways. How Dvl regulates multiple Wnt pathways is not
known. One hypothesis, based primarily on gain-of-function assays in cell
culture, predicts that Dvl-interacting proteins direct Dvl function into a
specific Wnt/Fz pathway (Sun et al.,
2001; Yan et al.,
2001
). Although this is an attractive hypothesis, there is yet
little evidence to support it. Most loss-of-function perturbations of the
Wnt/Ca2+-PCP pathway do not produce gain-of-function phenotypes of
the Wnt/ß-catenin pathway and vice versa. Examples include dsh
mutants in Drosophila, wnt11/slb and stbm/tri mutants in
zebrafish, and daam1 Xenopus morphants
(Axelrod et al., 1998
;
Habas et al., 2001
;
Heisenberg et al., 2000
;
Jessen et al., 2002
). In
addition, Drosophila nkd is not required for the PCP pathway
(Rousset et al., 2001
).
Nevertheless, a few recent reports provide some evidence that in specific
contexts there may be opposition of these pathways, though they do not point
to the opposition occurring at Dvl (Topol
et al., 2003
; Westfall et al.,
2003
). Thus, in some contexts there might be mechanisms by which
Dvl is free to regulate either ß-catenin or Wnt/Ca2+-PCP
pathway without affecting the other.
In the current study, we provide evidence that the zebrafish Dpr paralogs represent examples of Dvl-interacting paralogs that appear to be functionally associated with separate Wnt pathways. Importantly, they do not appear to have redundant functions, yet they have largely overlapping expression patterns in the early zebrafish development and both can interact with zebrafish Dvl2. Therefore, the Dpr morphant phenotypes are consistent with a hypothesis whereby the Wnt/ß-catenin and Wnt/Ca2+-PCP pathways can co-exist independently of each other.
Zebrafish Dprs are enhancers of Wnt signaling
The loss-of-function data in zebrafish support the general conclusion that
Dpr orthologs are enhancers of multiple Wnt pathways. The Dpr1 morphants have
subtle phenotypes that do not lead to any discernable loss of cell fates, yet
they have strong Wnt loss-of-function phenotype in a mildly hypomorphic Wnt8
background. These results are reminiscent of those obtained in CK1 RNAi
experiments, which also acts as a signaling enhancer in nematodes
(Peters et al., 1999). Alone,
RNAi of CK1 gives a low percentage of worms with the more-mesoderm (MOM)
phenotype. However, when combined with loss of another component of the Wnt
pathway, there is a strong MOM phenotype. Likewise, while loss of Dpr2 has a
phenotype similar to Wnt/Ca2+-PCP mutants, the strongest affected
dpr2 morphants are not as severe as the strongest affected
stbm/tri alleles or morphants
(Jessen et al., 2002
;
Park and Moon, 2002
).
Nevertheless, there is a strong functional interaction between dpr2
and stbm/tri or wnt11/slb, indicating that dpr2 may
act to promote Wnt/Ca2+-PCP pathway activity.
How is it possible that the zebrafish dpr2 morphant indicates a
loss of Wnt/Ca2+-PCP activity, but when overexpressed it can
synergize with Dvl2 to activate Wnt/ß-catenin targets? We do not
interpret these results as being mutually exclusive. Wnt components have been
demonstrated to be context-dependent activators of Wnt/ß-catenin or
Wnt/Ca2+-PCP pathways. Wnt5a/pipetail is probably the best
example. Wnt5a overexpression in Xenopus leads to improper convergent
and extension movements (Du et al.,
1995). Similarly, a loss-of-function mutation of wnt5/ppt
in zebrafish leads to improper convergence extension movements in the tail
(Rauch et al., 1997
;
Westfall et al., 2003
). Thus,
the overt phenotype of loss or gain of Wnt/Ca2+-PCP components is
similar. However, overexpression of Wnt5a along with Fz5 or LRP6 activates
Wnt/ß-catenin target genes in animal caps and duplicates axes in
Xenopus embryos (He et al.,
1997
; Tamai et al.,
2000
). Furthermore, expression of high levels of Wnt11 and some
Fzs can weakly duplicate axes and induce ß-catenin target genes,
emphasizing a caveat for overexpression of paralogous proteins
(Du et al., 1995
;
Ku and Melton, 1993
;
Sheldahl et al., 1999
).
Comparison with the Xenopus Dpr family
We have previously reported that Xenopus Dpr1a could function as
an inhibitor of Wnt/ß-catenin signaling
(Cheyette et al., 2002). Gloy
et al. (Gloy et al., 2002
)
initially reported that the related FRODO/XDpr1b acted like an activator of
Wnt signaling. In an effort to resolve the apparent discrepancies between the
first two studies, the Sokol laboratory has compared the signaling activities
of the two Dpr family members side by side in gain- and loss-of-function
studies. They conclude that both XDpr1a and FRODO/XDpr1b can act as either
activators or inhibitors, depending on the level at which the pathway is
activated, and the doses of the proteins
(Hikasa and Sokol, 2004
). To
understand better the normal functions of Dpr family members we undertook the
present study in zebrafish in part to exploit the ability to conduct
loss-of-function of Dpr orthologs in sensitized backgrounds. Although our new
experiments support the hypothesis that endogenous Dpr orthologs are positive
modulators of Wnt signaling in zebrafish under the conditions assayed, we do
not dispute the conclusions of Cheyette et al.
(Cheyette et al., 2002
) and
Hikasa and Sokol (Hikasa and Sokol,
2004
) that Xenopus Dpr family members can act as
inhibitors in some contexts. We propose that further analysis of Dpr family
members in other contexts besides frog and fish embryos may help determine the
mechanisms of action of these interesting proteins.
Conclusions
In conclusion, we provide loss-of-function evidence that Dpr1 but not Dpr2
is required for proper dorsoventral and anteroposterior patterning in
zebrafish that are mildly hypomorphic for Wnt8, indicating that Dpr1 probably
acts as an enhancer of Wnt8/ß-catenin signaling in early development.
Dpr2 but not Dpr1 acts as an enhancer of stbm/tri and
wnt11/slb in the Wnt/Ca2+-PCP pathway(s). Thus, in early
zebrafish development, loss-of-function evidence indicates Dpr1 and Dpr2 are
not redundant, and they function to regulate Wnt/ß-catenin and
Wnt/Ca2+-PCP pathways, respectively. Regarding mechanisms of
action, how Dpr2 functions is unclear, largely because of the lack of
understanding of the Wnt/Ca2+-PCP pathway and the lack of robust
assays. With regard to Dpr1 orthologs, in the majority of contexts they
function as positive regulators of Wnt/ß-catenin signaling, though they
can inhibit this pathway in various overexpression assays. We extend our
analysis of the Dvl-associated Dpr1 orthologs by demonstrating that zebrafish
Dpr1 synergizes in gene induction with three Dvl-associated kinases,
CK1, Par1 and CK2. Moreover, we demonstrate that gene regulation by Dpr1
in some contexts is dependent upon CK1
. We look forward to genetic and
proteomic approaches being employed to further test these findings on the
functions and possible mechanisms of action of Dapper family members.
Note added in proof
During the final preparation of this manuscript, Zhang et al.
(Zhang et al., 2004) reported
dpr2 morphants have a similar phenotype as reported here.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/23/5909/DC1
* Present address: Skirball Institute of Biomolecular Medicine, New York
University School of Medicine, New York, NY 10016, USA
Present address: Department of Surgery, University of Washington School of
Medicine, Seattle, WA 98195, USA
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