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First published online 21 June 2006
doi: 10.1242/dev.02445
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1 Department of Molecular, Cell and Developmental Biology, Mount Sinai School of
Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
2 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.
* Author for correspondence (e-mail: sergei.sokol{at}mssm.edu)
Accepted 15 May 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wnt, Xenopus, Dsh, Midbrain, Morphogenesis, Kinase, JNK, SNF-1
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Before gastrulation, the pathway is used to establish the Spemann organizer, a
major inductive center in the embryo, which is responsible for dorsal
development and neural induction (Harland
and Gerhart, 1997; Moon and
Kimelman, 1998; Sokol,
1999; Tao et al.,
2005). After the initial induction of the central nervous system,
localized sources of Wnt ligands induce and maintain a secondary organizing
center at the midbrain-hindbrain boundary, which mediates regional patterning
of the brain (Chi et al.,
2003; Hidalgo-Sanchez et al.,
2005; Liu and Joyner,
2001). Besides organizer specification and brain patterning, Wnt
signaling is involved in somitogenesis, eye and neural crest development,
cardiogenesis, and endoderm differentiation
(Bainter et al., 2001;
Cavodeassi et al., 2005;
Ciani and Salinas, 2005;
Gamse and Sive, 2000;
Logan and Nusse, 2004;
Wu et al., 2003). Thus, Wnt
signaling is re-used at many developmental stages in different embryonic
tissues.
The pathway leads to two major outcomes: ß-catenin-dependent
(canonical) activation of target genes and the regulation of actin
cytoskeleton and cell polarity via a poorly understood process (noncanonical
signaling). In the canonical pathway, Wnt ligands and their cognate Frizzled
receptors signal through Dishevelled to stabilize ß-catenin and induce
its association with the TCF transcription factors to activate Wnt target
genes. By contrast, Wnt signaling to the cytoskeleton occurs through the
activation of small GTPases (Habas et al.,
2003; Habas et al.,
2001), Rho-associated kinases
(Marlow et al., 2002;
Winter et al., 2001),
Jun-N-terminal kinases (Boutros et al.,
1998; Lisovsky et al.,
2002) and intracellular Ca2+ release
(Sheldahl et al., 2003;
Slusarski et al., 1997).
Noncanonical Wnt signaling regulates changes in cell shape and motility during
gastrulation, neurulation and organogenesis
(Ciani and Salinas, 2005;
Saneyoshi et al., 2002;
Sokol, 1996;
Wallingford et al., 2002;
Winklbauer et al., 2001;
Zohn et al., 2003). Both
canonical and noncanonical branches of pathway involve Dishevelled, a protein
phosphorylated in response to Wnt signaling
(Yanagawa et al., 1995),
although how Dishevelled directs signals to different molecular targets
remains unclear (Wallingford and Habas,
2005).
Here, we report that a SNF1 (sucrose non fermenting 1)-related protein
kinase, known as metastasis-associated kinase (MAK)
(Gardner et al., 2000b;
Korobko et al., 1997),
phosphorylates Dishevelled, inhibits the canonical Wnt pathway and upregulates
non-canonical Wnt signaling during early development. SNF1-related kinases are
involved in the metabolic response to nutritional and environmental stress,
cell cycle, cell polarity and vertebrate development
(Becker and Brendel, 1996;
Hardie et al., 1998;
Ruiz et al., 1994). MAK is
distantly related to the yeast Kin-1 and to the C. elegans PAR-1.
Kin-1 is crucial for growth polarity and cytoskeletal organization in fission
yeast (Drewes and Nurse, 2003;
Levin et al., 1987;
Tassan and Le Goff, 2004).
PAR-1 regulates cell polarity in C. elegans and Drosophila
embryos and mammalian cells (Bohm et al.,
1997; Pellettieri and Seydoux,
2002; Shulman et al.,
2000; Tomancak et al.,
2000) and has been implicated in Wnt signaling
(Ossipova et al., 2005;
Sun et al., 2001). Our
experiments demonstrate that MAK regulates morphogenetic movements, eye
development and midbrain patterning in Xenopus embryos and suggest
that MAK may function as a molecular switch between the canonical and
noncanonical Wnt pathways.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
expression pattern of the second gene (Accession Number AY318878)
(Ruzov et al., 2004) differs
from what we report here for xMAK. To generate pXT7-GFP-xMAK, xMAK
cDNA was amplified by PCR with 5'-AAGAATTCGATGCCGGCTGCCGCTG-3' and
5'-GCCTCGAGGCAAGGACAAATCTGTT-3', and subcloned into pXT7-GFP
(Itoh et al., 2000) that has
been digested with EcoRI and SalI. Flag-xMAK constructs were
generated by PCR and subcloned into pCS2-Flag. Untagged mMAK cDNA in the pCTX
vector (S.Y.S., unpublished) was used as a transcription template. Myc-MAK
constructs were generated from the wild-type or kinase-dead (K91>R) mMAK
cDNA (Korobko et al., 2004) by
PCR with the primers 5'-AACTCGAGATGCCGGCAGCGG-3' and
5'-AGGTTAACACTGGCCCTTGACACCGTC-3' and subcloned into the
XhoI and EcoRV sites of pCTX-Myc. Details of cloning are
available upon request. Other expression constructs were pCS2-nßgal
(Turner and Weintraub, 1994),
Xfz3 (Shi et al., 1998), rFz2,
membrane-tethered DsRed (Unterseher et
al., 2004), Myc-Xdsh and ß-catenin
(Sokol, 1996), HA-Xdsh
(Itoh et al., 2000), Xwnt8
(Sokol et al., 1991) and
N-Fz8 (Lisovsky et al.,
2002).
Embryo culture, microinjection, axis induction and extension assays
In vitro fertilization, culture and microinjections of Xenopus
eggs were essentially as described (Sokol,
1996). Stages were determined according to Nieuwkoop and Faber
(Nieuwkoop et al., 1967).
Morpholino oligonucleotides were from GeneTools (Oregon) and had the following
sequences: MAK MO, 5'-CGGCATCCCCAGTGGTGTAGATCTC-3'; ß-Cat MO,
5'-TTTCAACCGTTTCCAAAGAACCAGG-3'; control MO,
5'-ATCGACTTCCTCCGAAACGGACATG-3'. RNAs for microinjection were
synthesized using mMessage mMachine kit (Ambion). Axis induction assays were
carried out by injecting HA-Xdsh, Xwnt8 or ß-catenin RNA into a single
vegetal ventral blastomere at the four- to eight-cell stage at indicated doses
and assessed when the injected embryos reached stage 36-40. To monitor axis
extension defects, MAK RNA, MAK-KD or GFP RNA were injected into two
dorsovegetal blastomeres of four-cell embryos (2 ng each injection) and the
injected embryos were allowed to develop until sibling embryos reached stage
32. In other cases, sites of injection are specified in corresponding figure
legends. For lineage tracing, 20-40 pg of RNA encoding nuclear
ß-galactosidase (pCS2-nßgal) was injected together with morpholinos
or mRNAs. ß-Galactosidase activity was visualized with the Red-Gal
substrate (Research Organics).
For animal cap elongation assay, four-cell embryos were injected four times in the animal region of each blastomere with mMAK or mMAK-KD RNA (2 ng per injection), animal caps were dissected at stage 8 to 9, and cultured with or without 50 ng/ml of human recombinant activin A until the sibling embryos reached stage 15.
Transcriptional reporter assays
Embryos were injected into animal pole region of one ventral blastomere at
the four-cell stage with 20 pg of the -833pSia-Luc reporter DNA
(Fan et al., 1998) per embryo,
alone or with the indicated amounts of mRNAs. Lysates were prepared from
embryos at stage 10.5 and assayed for luciferase activity as previously
described (Fan et al., 1998).
For every experimental group, measurements were carried out for triplicate
samples, each consisting of five embryos. Values shown are
averages±s.d., which are representative of at least three different
experiments.
Subcellular localization of MAK-GFP
For subcellular localization of MAK-GFP constructs, RNAs encoding MAK-GFP
(500 pg) and mDsRed (2 ng) were co-injected with or without Fz3 or Fz2 RNA (1
ng) into the animal pole region of two- to four-cell embryos. Animal cap
explants were dissected at stage 9-9.5, fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 35-45 minutes, washed and mounted for
observation in the Vectashield mounting medium with DAPI (Vector) as described
(Itoh et al., 2005).
Fluorescence was visualized on a Zeiss Axiophot microscope with Apotome
attachment and images were acquired with Axiocam HR camera.
Whole-mount in situ hybridization
Digoxigenin-labeled antisense RNA probes were synthesized from linearized
plasmids, encoding En2 (Brivanlou
and Harland, 1989), Krox20
(Bradley et al., 1993),
Otx2 (Pannese et al.,
1995), Gbx2 (von
Bubnoff et al., 1996) and xMAK, using a
digoxigenin-labeling mixture (Boehringer Mannheim). Whole-mount in situ
hybridization was carried out according to Harlan
(Harland, 1991) with
modifications as described previously
(Hikasa and Sokol, 2004). For
in situ hybridization, embryos were rehydrated in 1xPBS, 0.1% Tween 20.
The staining reaction was carried out for sense and anti-sense probes for the
same duration. 5-Bromo-4-chloro-3-indolyl phosphate (Sigma) and Nitro blue
tetrazolium (Sigma) were used for chromogenic reactions. After staining, some
embryos were dehydrated, embedded into paraplast and sectioned at 10 µm for
image analysis at higher resolution.
RNA isolation and RT-PCR
Total RNA was extracted from embryos or animal caps by proteinase Kphenol
extraction as described (Itoh and Sokol,
1997). cDNAs were made from DNase-treated RNA using the
Superscript first strand synthesis system (Invitrogen). RT-PCR was performed
on total RNA isolated from stage 10.5 embryos as previously described
(Itoh and Sokol, 1997).
Primers for RTPCR were: xMAK, 5'-ACCAGAAGATGGTCGA-3',
5'-TTCCAACTGATGAAACT-3'; chordin,
5'-AACTGCCAGGACTGGATGGT-3',
5'-GGCAGGATTTAGAGTTGCTTC-3'; Xnr3,
5'-CGAGTGCAAGAAGGTGGACA-3',
5'-ATCTTCATGGGGACACAGGA-3'; siamois,
5'-CTCCAGCCACCAGTACCAGATC-3',
5'-GGGGAGAGTGGAAAGTGGTTG-3'; gsc,
5'-TTCACCGATGAACAACTGGA-3',
5'-TTCCACTTTTGGGCATTTTC-3'; MyoD,
5'-AGCTCCAACTGCTCCGACGGCATGAA-3',
5'-AGGAGAGAATCCAGTTGATGGAAACA-3'; Dkk1,
5'-CACCAAGCACAGGAGGAA-3', 5'-TCAGGGAAGACCAGAGCA-3';
EF-1
, 5'-CAGATTGGTGCTGGATATGC-3',
5'-ACTGCCTTGATGACTCCTAG-3'; FGFR,
5'-TTGAAGTCTGATGCGAGTGA-3',
5'-GGGTTGTAGCAGTACTCCAT-3'. One quarter of each PCR reaction was
electrophoresed in a 5% polyacrylamide gel, stained with ethidium bromide and
photographed under ultraviolet light.
Immunoprecipitation and western analysis
Immunoprecipitation and western analyses were carried out with embryo
lysates as described (Gloy et al.,
2002). To prepare embryo lysates at stage 10+, each blastomere of
four-cell embryos was injected with different mRNAs. For immunoprecipitation,
20 µl of 9E10 (anti-Myc) or 12CA5 (anti-HA) hybridoma supernatants (ATCC)
were used per sample. Protein amount equivalent to one half embryo was loaded
per lane for embryo lysates, and the equivalent of four to nine embryos for
immunoprecipitated proteins. Monoclonal M2 antibodies (anti-Flag) and
polyclonal anti-ß-tubulin antibodies were from Sigma, secondary
HRP-conjugated antibodies were from Jackson ImmunoResearch.
In vitro translation, MAK and JNK kinase assays
In vitro translation reactions were performed using rabbit reticulocyte
lysates from the Retic Lysate IVT kit (Ambion) or TnT system (Promega)
according to manufacturer's instructions. MAK kinase reactions were performed
essentially as described (Ossipova et al.,
2005). For in vitro blocking studies, 600 pM of MAK MO or COMO
were incubated with 0.3 µg of xMAK RNA or mMAK RNA in 10 µl of distilled
water for 30 minutes at room temperature, following by in vitro translation
reaction for 90 minutes. Half of each reaction was loaded on an
SDS-polyacrylamide gel. Radiolabeled lysates were electrophoresed as described
(Sambrook et al., 1989), gels
were fixed in 50% methanol and 10% acetic acid, dried and exposed to Kodak
XAR-5 film for autoradiography.
In vitro JNK kinase assays were performed essentially as described
(Lisovsky et al., 2002).
Four-cell stage embryos were injected into each cell with GFP, mMAK, or
mMAK-KD RNA at 1.5 ng per injection, or N-Fz8 RNA (0.5 ng). Embryos
were lysed at stage 14 in 100 µl of buffer containing 40 mM HEPES (pH 7.5),
50 mM KCl, 5 mM EDTA, 5 mM EGTA, 50 mM ß-glycerophosphate, 2 mM DTT, 1 mM
sodium vanadate, 50 mM sodium fluoride, 1% Triton X-100, 10 M PMSF, 10
µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin and 1 µg/ml
antipain. The reaction mixture (25 µl) contained 1 µl of total embryo
lysate, 0.5 µg of GST-Jun(1-135) and the kinase buffer (20 mM HEPES, pH
7.5, 10 mM MgCl2, 1 mM sodium vanadate, 2 mM DTT, 25 mM
ß-glycerophosphate, 100 M cold ATP). The reactions were allowed to
proceed for 30 minutes at 30°C. Proteins were separated by 12% SDS-PAGE,
phosphorylated Jun-GST was detected with anti-phospho-Jun-specific antibodies
(Cell Signaling Technology).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gain-of-function phenotype in embryos overexpressing MAK
To evaluate activity of MAK in a gain-of-function assay, mRNAs encoding
tagged MAK or its kinase-dead mutant (MAK-KD) have been injected in the dorsal
margin of four-cell embryos (Fig.
2A). We observed that MAK RNA, but not the control GFP RNA, caused
strong morphogenetic abnormalities, eye and anterior brain deficiencies in
injected embryos. In a typical experiment, MAK RNA produced axis extension
defects in 82% of injected embryos (n=27) at stage 38. MAK-KD RNA did
not have a significant effect on gastrulation or neurulation movements,
indicating that MAK enzymatic activity is necessary for the morphogenetic
defects observed. The majority (93%) of MAK RNA-injected embryos had
incomplete or absent retinal pigmentation, and some were missing anterior head
structures (Fig. 2A). MAK-KD
RNA injections also produced mild eye deficiencies at lower frequency
(Fig. 2A). These observations
suggest that MAK may be involved in the control of morphogenetic movements,
head and eye development.
To evaluate whether MAK selectively interferes with morphogenetic movements
or cell fate specification, we analyzed elongation of animal cap explants
treated with activin, a common model for convergent extension
(Sokol, 1996;
Symes and Smith, 1987).
Whereas MAK-KD RNA-injected and control uninjected explants elongated in
response to activin, explants expressing wildtype MAK failed to elongate under
identical culture conditions
(Fig.2B). Both proteins were
expressed equally well in injected embryos, demonstrating that the difference
in activity is not due to different expression levels
(Fig. 2C).
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MAK is an activator of Jun N-terminal kinase
The strong effect of the wild-type MAK on convergent extension movements
suggested that MAK may be involved in non-canonical Wnt signaling. Several
noncanonical Wnt signaling components, including Dsh, Frizzled, Prickle and
Strabismus, have been reported to activate JNK in mammalian cells and
Xenopus embryonic ectoderm
(Boutros et al., 1998;
Lisovsky et al., 2002;
Park and Moon, 2002;
Takeuchi et al., 2003). As JNK
activation is often associated with non-canonical Wnt signaling, we measured
Jun phosphorylation in lysates of embryos injected with wild type MAK or
MAK-KD RNA. We observed that MAK, but not MAK-KD, activated JNK in injected
embryos (Fig. 3), suggesting a
role for MAK in noncanonical Wnt signaling. Both constructs were expressed at
equal levels (data not shown). An activated form of Fz8, previously reported
to induce JNK activity (Lisovsky et al.,
2002) served as a positive control in this assay. The effects of
MAK on morphogenetic movements and the ability to activate JNK are consistent
with the postulated function of MAK in noncanonical signaling.
|
Dsh is another protein reported to be recruited by Frizzled to the cell
membrane, and this recruitment has been considered to be crucial for
non-canonical signaling (Axelrod et al.,
1998; Boutros et al.,
1998; Rothbacher et al.,
2000; Yang-Snyder et al.,
1996). Therefore, we tested whether MAK can physically associate
with Dsh. We found that MAK co-immunoprecipitated with Dsh in lysates of
injected embryos (Fig. 4B). To
assess whether this association is functional, we performed an immune complex
kinase assay in vitro and demonstrated that MAK can phosphorylate Dsh
(Fig. 4C). To exclude a
possibility that another kinase is responsible for Dsh phosphorylation, we
used MAK-KD as a control. As expected, the MAK-KD mutant did not have any
kinase activity towards Dsh. This finding shows that Dsh may represent a
molecular substrate for MAK in vivo.
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MAK plays essential roles in axis elongation and eye development
To further investigate a role for MAK in development in loss-of-function
studies, an antisense morpholino oligonucleotide (MAK MO) has been designed to
suppress MAK RNA translation in a sequence-specific manner. Indeed, MAK MO,
but not a control MO (COMO), inhibited in vitro translation of xMAK
RNA in rabbit reticulocyte lysates (Fig.
5A). MAK MO did not suppress the translation of mMAK RNA
that lacks MO target sequence. More importantly, MAK MO caused a reduction of
xMAK protein levels in vivo in injected embryos
(Fig. 5B).
To assess a developmental role for xMAK, we examined the phenotype of embryos injected with MAK MO into both dorsal blastomeres at the four-cell stage. At stage 38, the majority of MAK-depleted embryos, but not those injected with the same dose of COMO, exhibited shortened trunks and tails and severe retinal defects (Fig. 5C, Table 1). The requirement of MAK for eye development correlates with the observed expression of MAK in the eye field (Fig. 1D). The effect of MAK MO on retinal development was cell-autonomous (Fig. 5D,E). The eye defect was dose dependent, as lower doses of MAK MO had a less pronounced effect on eye pigmentation (data not shown), and it was partially rescued by co-injection of mMAK mRNA, lacking MO target sequence (Fig. 5G,H). These observations indicate that the effect of MAK MO is specific and demonstrate a role for MAK in axis elongation and eye development. Thus, the same processes are affected in both gain-of-function and loss-of-function experiments, indicating that MAK levels are under a tight control during normal development.
|
). To test this possibility, we assessed the
effect of MAK on secondary axis induction by Dsh
(Sokol et al., 1995). In a
representative experiment, Dsh RNA induced complete body axes with eyes and
head structures in 81% of injected embryos (n=27,
Fig. 6A,B). By contrast, when
MAK RNA was co-injected, only 16% embryos (n=32) had
complete secondary axes with head structures. Coinjection of the control
nßgal RNA or MAK-KD RNA did not significantly
alter the axis-inducing activity of Dsh
(Fig. 6B). Consistent with
these results, MAK, but not MAK-KD, RNA dose-dependently
suppressed Dsh-mediated induction of the pSiaLuc reporter, a direct
readout of the canonical pathway (Fig.
6C) (Fan et al.,
1998).
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MAK is involved in regional brain patterning
At neurula stages, xMAK transcripts are present in the isthmus,
suggesting a role for MAK in regional brain patterning. To test this
possibility, we analyzed embryos with altered levels of MAK by in situ
hybridization with region-specific brain markers. MAK MO or MAK RNA was
injected into a single dorsal-animal blastomere of the four to eight-cell
embryo. At stage 20, the injected embryos were fixed and hybridized with
probes for the anterior brain marker Otx2
(Blitz and Cho, 1995;
Pannese et al., 1995), the
midbrain-hindbrain boundary marker En2
(Brivanlou and Harland, 1989;
Joyner and Martin, 1987) and
the hindbrain marker Gbx2 (Millet
et al., 1999; von Bubnoff et
al., 1996). Embryos were scored based on the difference in marker
intensity between injected and uninjected sides
(Fig. 7A-O). We have found that
MAK MO, but not the control MO, caused unilateral suppression or loss of
Otx2 and En2 in 71% (n=58) and 28% (n=50)
of injected embryos, respectively (Table
2). This effect was accompanied by the upregulation of the
posterior neural marker Gbx2 in 71% (n=62) of injected
embryos. These results demonstrate a posteriorizing effect of MAK MO on the
developing midbrain-hindbrain boundary.
|
|
;
Wurst and Bally-Cuif,
2001).
|
) in
injected embryos at the beginning of gastrulation. RT-PCR analysis did not
reveal any significant changes for Chordin (Chd),
Goosecoid (Gsc), Xenopus nodal-related 3
(Xnr3), Siamois (Sia), Dickkopf1
(Dkk1) and Cerberus (Cer) in embryos depleted of
MAK or overexpressing MAK (Fig.
7P). By contrast, ß-catenin MO significantly downregulated
organizer markers, demonstrating high sensitivity of this assay. These
observations suggest that MAK functions to establish the midbrain-hindbrain
boundary (MHB) by influencing neural tissue directly, rather than by affecting
the organizer.
MAK regulates the midbrain-hindbrain boundary by inhibiting ß-catenin signaling
The formation of the isthmic organizer at the midbrain-hindbrain boundary
is known to require canonical Wnt signaling
(Hidalgo-Sanchez et al., 2005;
Kunz et al., 2004).
Interestingly, the observed effect of MAK on the midbrain-hindbrain boundary
closely resembles the effect of ß-catenin MO
(Wu et al., 2005). This
similarity and the potential inhibitory role for MAK in Wnt signaling
(Fig. 6) indicate that MAK may
regulate midbrain-hindbrain boundary by inhibiting the canonical pathway.
To test this hypothesis, we attempted to rescue the effect of MAK MO by depleting ß-catenin. We studied the expression of Otx2 and Gbx2 by in situ hybridization in embryos co-injected into a single animal dorsal blastomere with MAK MO and ß-catenin MO (Fig. 8). Marker expression was scored by comparing injected and uninjected sides of stage 20 embryos, which have been identified by lineage tracing with ß-galactosidase. MAK MO induced Gbx2 in 86% (n=14) of injected embryos and downregulated Otx2 in 70% (n=23) of injected embryos. Upon ß-catenin MO co-injection, the induction of Gbx2 was suppressed, whereas Otx2 levels did not change in the majority (65%, n=40) of injected embryos. We conclude that ß-catenin MO rescued normal brain patterning in MAK-depleted embryos. Thus, the effects of MAK depletion on Otx2 and Gbx2 expression are probably due to canonical pathway upregulation at the midbrain-hindbrain boundary, which is consistent with an inhibitory role for MAK in Wnt/ß-catenin signaling during midbrain patterning.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Liu and Joyner, 2001;
Sokol, 2000). In mouse
embryos, MAK has been implicated in mammary gland development, another process
regulated by Wnt signaling (Gardner et
al., 2000a). In our experiments, Frizzled 3 dramatically altered
MAK intracellular localization by recruiting it to the cell membrane
(Fig. 4A), demonstrating that
MAK can respond to Wnt signaling. The effects of MAK on convergent extension
and on canonical Wnt signaling require its kinase activity, suggesting that
phosphorylation of a putative MAK substrate plays a major role in signaling
outcome. In a search for possible MAK substrates, we found that MAK associates
with Dsh and is able to phosphorylate Dsh in vitro. As Dsh has been implicated
in gastrulation movements (Sokol,
1996) and brain patterning
(Hamblet et al., 2002;
Itoh and Sokol, 1997), it may
be a relevant molecular target of MAK during these developmental
processes.
|
).
Together, these experiments indicate that MAK functions to stimulate
non-canonical Wnt signaling, but negatively regulates the canonical pathway.
Thus, MAK acts in a manner opposite to that of PAR-1, a related SNF1-like
kinase, which activates the canonical pathway but suppresses the noncanonical
pathway by downregulating JNK (Ossipova et
al., 2005; Sun et al.,
2001).
In the isthmic organizer, canonical Wnt signaling may be responsible for
the transcriptional activation of several target genes, including those
encoding Wnt pathway components, e.g. Wnt1
(McMahon and Bradley, 1990),
Axin (Hedgepeth et al., 1999),
LEF1/TCF (Kunz et al., 2004;
Molenaar et al., 1998) and
Frodo (Gillhouse et al.,
2004). Consistent with the hypothesis that MAK modulates Wnt
signaling in the brain, the loss-of-function experiments with embryos injected
with MAK MO resulted in inhibition of the anterior brain marker Otx2
and the isthmus marker En2, but caused upregulation of Gbx2,
the posterior brain marker (Fig.
7). MAK injections did not substantially upregulate Otx2
and En2, but shifted these markers more posteriorly
(Fig. 7J,K). Together with the
lack of effect of MAK on organizer markers
(Fig. 7P), this indicates that
MAK regulates midbrain-hindbrain boundary specification, rather than causes a
more general effect on anteroposterior patterning or Spemann organizer
formation. In these experiments, MAK phenocopies the effect of ß-catenin
MO injection (Wu et al.,
2005), confirming our hypothesis that it is due to MAK-mediated
inhibition of canonical Wnt signaling. The restricted effect of MAK on the
midbrain-hindbrain boundary suggests that the isthmic organizer differs from
other brain regions in the responsiveness to Wnt/ß-catenin signaling,
perhaps by expressing region-specific competence factors. This hypothesis is
supported by our observation that brain-specific expression of xMAK is
restricted to the midbrain-hindbrain boundary.
MAK does not seem to be able to affect dorsal mesoderm markers when
overexpressed in the organizer (Fig.
7P). This finding suggests that MAK is a selective inhibitor of
Wnt signaling in MHB, but not at the earlier developmental stage. One reason
is that MAK is not present in fertilized eggs and does not function in early
axial patterning. It is also possible that the upstream components of Wnt
signaling including MAK are not involved in organizer formation in early
embryogenesis (Harland and Gerhart,
1997; Sokol,
1996), whereas they may be crucial at an earlier stage, e.g.
during oocyte development (Tao et al.,
2005). As secreted Wnt antagonists and Dsh interfering mutants do
not influence organizer development
(Glinka et al., 1998), it has
been hypothesized that the pathway is activated by a ligand-independent
mechanism (Sokol, 1996).
At present, how MAK redirects Wnt signaling from the canonical to the
noncanonical mode is unclear. A simple hypothesis is that it does so by
phosphorylation of Dsh. Thus, the identification of MAK-specific
phosphorylation sites in Dsh will be important in future studies of MAK.
Alternatively, MAK may have other molecular targets that are crucial for
signaling, as other protein kinases, including casein kinase 1 and GSK3,
function in Wnt signaling by phosphorylating multiple substrates
(Lee et al., 2001;
Zeng et al., 2005).
|
ACKNOWLEDGMENTS |
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