1 Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth
Avenue, Pittsburgh, PA 15213, USA
2 Department of Zoology, University of Hawaii at Manoa, 2538 McCarthy Mall,
Honolulu, HI 96822, USA
* Author for correspondence (e-mail: ettensohn{at}andrew.cmu.edu)
Accepted 2 March 2003
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SUMMARY |
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Key words: ß-Catenin, Dishevelled, GSK3ß, Sea urchin embryo, Early patterning
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Introduction |
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Recent studies have demonstrated that ß-catenin has a highly conserved
function in patterning early metazoan embryos. In all deuterostome embryos
that have been closely examined in this regard, including those of amphibians,
fish, chicks, ascidians and sea urchins, ß-catenin becomes localized to
cell nuclei preferentially at one pole of the cleavage-stage embryo
(Schneider et al., 1996;
Larabell et al., 1997
;
Rowning et al., 1997
;
Logan et al., 1999
;
Roeser et al., 1999
;
Imai et al., 2000
). In these
various organisms, nuclear activity of ß-catenin, through its interaction
with LEF/TCF proteins, is required for early axis specification and the
establishment of critical signaling centers in the early embryo
(Heasman et al., 1994
;
Kelly et al., 1995
;
Wylie et al., 1996
;
Pelegri and Maischein, 1998
;
Imai et al., 2000
;
Kelly et al., 2000
). Recent
studies with cnidarian embryos suggest that this mechanism of axis
specification might be very ancient; i.e. its origins may pre-date the
divergence of radially and bilaterally symmetrical metazoans
(Wikramanayake et al.,
2003
).
In the sea urchin, ß-catenin is required for the formation of endoderm
and mesoderm. Overexpression of proteins that interfere with nuclear
localization and/or function of ß-catenin, including cadherins,
GSK3ß and a dominant negative form of TCF/LEF, lead to the development of
`dauerblastula' embryos, which lack mesenchyme cells and gut
(Emily-Fenouil et al., 1998;
Wikramanayake et al., 1998
;
Logan et al., 1999
;
Vonica et al., 2000
).
ß-Catenin appears to have multiple functions in endomesoderm
specification. In the large micromere-primary mesenchyme cell (PMC) lineage,
ß-catenin is an essential activator of a network of early zygotic
transcription factors, including Pmar1, Ets1, Alx1 and T-brain, that regulate
the powerful signaling properties and later morphogenesis of these cells
(Kurokawa et al., 1999
;
Fuchikami et al., 2002
;
Oliveri et al., 2002
,
2003
;
Sweet et al., 2002
;
Ettensohn et al., 2003
). In the
macromeres, ß-catenin is required for the activation of genes involved in
endoderm and non-skeletogenic mesoderm specification, including those that
render the cells responsive to micromere-derived signals
(McClay et al., 2000
;
Davidson et al., 2002
).
ß-Catenin also plays an indirect role in ectoderm specification, through
its influence on vegetally derived signals that pattern the overlying ectoderm
(Wikramanayake et al.,
1998
).
Although the regulation of nuclear localization and function of
ß-catenin is a critical feature of early metazoan development, the
underlying mechanisms are not well understood. It has been proposed that the
differential nuclear accumulation of ß-catenin is a consequence of
regulated proteolytic degradation along the embryo axis. This hypothesis is
based on experiments demonstrating that overexpression of proteins predicted
to interfere with or enhance ß-catenin degradation lead to corresponding
changes in nuclear localization and axis specification (see reviews by
Moon and Kimelman, 1998;
Sokol, 1999
). Although
overexpression studies point strongly to differential degradation, there has
been no direct demonstration that the stability of ß-catenin varies along
an early embryo axis. One study compared the half-life of ß-catenin on
the dorsal and vegetal sides of the cleavage-stage Xenopus embryo
using biochemical methods but reported that ß-catenin was highly and
equally stable (t1/2=3.6-3.8 hours) on both sides of the
embryo (Guger and Gumbiner,
2000
). Mechanisms other than regulated proteolysis have also been
put forward to account for changes in levels of nuclear ß-catenin,
including regulated nuclear import/export and interactions with cytoplasmic
and nuclear anchoring proteins (Henderson
and Fagotto, 2002
). One recent study, for example, has argued that
Wnt signaling increases levels of nuclear ß-catenin not by regulating
GSK3ß-mediated degradation, but by triggering the degradation of a
cytoplasmic anchoring protein (Tolwinski
et al., 2003
).
In the present study, we have exploited the optical transparency of the sea urchin embryo to measure the half-life of ß-catenin in specific cell lineages in vivo. We report a gradient in ß-catenin stability along the animal-vegetal (A-V) axis during cleavage and show that degradation of ß-catenin in animal blastomeres is dependent on GSK3ß-mediated phosphorylation of the protein. We find that overexpression of a dominant negative form of Dsh blocks nuclear accumulation of ß-catenin in vegetal cells and suppresses mesoderm and endoderm formation. Finally, we report that a GFP-tagged form of Dsh shows a striking, specific localization to the vegetal cortex of the fertilized egg and early vegetal blastomeres, where we propose it is locally activated and subsequently functions to stabilize ß-catenin. Through a detailed mutational analysis of Dsh, we have identified several regions that are required for vegetal targeting, including a short phospholipid-binding motif within the DIX domain of the protein.
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Materials and methods |
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Cloning of LvDsh, LvAxin and LvGSK3ß
A 600-bp fragment of LvDsh was cloned by RT-PCR from mesenchyme
blastula-stage cDNA using degenerate primers. 5'- and 3'-RACE were
used to isolate clones containing the remainder of the coding sequence and the
5' and 3' untranslated regions. A single cDNA clone encoding a
fragment of Axin from Strongylocentrotus purpuratus was identified in
an EST sequencing project (Zhu et al.,
2001). PCR primers designed from this sequence were used to
amplify a fragment of LvAxin from unfertilized egg-stage cDNA. 5'- and
3'-RACE were used to isolate clones containing the remainder of the
sequence. LvGSK3ß was cloned by RT-PCR using the published sequence of
GSK3ß from Paracentrotus lividus and primer sequences described
by Emily-Fenouil et al. (1998
).
Sequences of LvDsh, LvAxin, and LvGSK3ß have been deposited in GenBank
(Accession numbers AY624074, AY624075 and AY624076, respectively).
RNA injection and immunostaining
For mRNA injections, cDNAs were subcloned into pCS2+MT or pCS2+GFP vectors.
Injections of in-vitro transcribed, capped mRNAs were carried out as described
(Sweet et al., 2002).
Immunostaining with anti-ß-catenin antibody was performed following the
protocol of Logan et al.
(1999
), except that embryos
were fixed for 4 hours. Point mutations in LvDsh were introduced using the
Quick-Change Site-Directed Mutagenesis Kit (Stratagene) and deletions were
generated by PCR.
4-D confocal laser scanning microscopy
Four-dimensional microscopy was carried out using a Bio-Rad MRC-600
laser-scanning microscope equipped with 40x and 60x water
immersion objectives. Z-stacks (20-40 images/stack) of 256x256 pixel
images were collected every 2 minutes with a step size of 4 µm, using
Kalman filtering and the Fast1 scan rate. Acquisition of the stacks was
automated using the SOM program (dlapse) accessible through the CoMos software
package (Bio-Rad). A two-dimensional projection (maximum-intensity method) was
generated from each z-stack and transferred to a Macintosh G4 computer. NIH
Image was used to generate time-lapse sequences of the two-dimensional
projections. Representative (non-time-lapse) images of embryos expressing
various GFP-tagged proteins were obtained by confocal microscopy as described
above, except that z-stacks were collected at a slow scan rate with a step
size of 1-2 µm and an image size of 512x512 pixels.
Measurements of protein half-life in vivo
To calculate the half-life of GFP-tagged proteins, fertilized eggs were
injected with mRNA encoding the protein of interest and allowed to develop for
a period of time sufficient for accumulating levels of the GFP-tagged product
detectable by confocal microscopy. At various times, the translational
inhibitor emetine was added to the cultures (final concentration=100 µM)
and the embryos were cultured continuously in the presence of the inhibitor.
After 30 minutes, when emetine was maximally effective, time-lapse sequences
were collected as described above. Each sequence encompassed 45-60 minutes of
real time. To ensure that the intensity of all fluorescence images was below
saturation, the CoMos software was used to examine histograms of pixel
intensities in the 8-bit images and settings on the MRC-600 were adjusted such
that the brightest pixel in the raw images in each sequence had an intensity
value of <256. From the maximal-intensity projections of each z-stack, NIH
Image was used to measure the mean pixel intensity within selected regions of
each frame. Average pixel intensities were measured in specific cell lineages
by hand-selecting the cells using a free-form tool. These values represented
average pixel intensities over the entire cellular region, i.e. including both
nuclear and cytoplasmic pools of protein. Initial average pixel values (30
minutes after addition of emetine) were set to 1 and the natural log (ln) of
average pixel intensity was plotted versus time using Microsoft Excel. These
plots showed a linear decrease in pixel intensity over time, as expected for
an exponential decay (Fig. 2D).
A trend line was added to each curve and used to determine the R2
value and the equation of the line (y=mx +b). The
slope (m) was used in the equation t1/2=0.693/m
to calculate the half-life (t1/2) of the fluorescence in
each cell lineage.
|
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Results |
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The loss of ß-catenin in animal blastomeres was dependent on
GSK3ß, a serine-threonine kinase that phosphorylates ß-catenin on
several N-terminal residues and targets the protein for ubiquitination and
degradation. We expressed a GFP-tagged form of Xenopus ß-catenin
(Xl-pt-ß-catenin-GFP) in which four N-terminal serine and threonine
residues were converted to alanines. Three of these residues are
phosphorylated by GSK3ß and one by a priming kinase, casein kinase 1
(Yost et al., 1996;
Liu et al., 2002
). This
variant of ß-catenin shows increased stability in biochemical assays and
in vivo (Yost et al., 1996
;
Guger and Gumbiner, 2000
).
Overexpression of Xl-pt-ß-catenin-GFP resulted in persistent nuclear
localization of the protein in all blastomeres throughout cleavage and
blastula stages (Fig. 1F-I).
Similarly, coexpression of Xl-wt-ß-catenin-GFP and a kinase-dead,
dominant negative form of Xenopus GSK3ß (Xl-dn-GSK3ß)
prevented the loss of ß-catenin from animal blastomeres
(Fig. 1J). Overexpression of
Xl-pt-ß-catenin-GFP or Xl-dn-GSK3ß resulted in a vegetalized
phenotype, as previously reported
(Emily-Fenouil et al., 1998
;
Wikramanayake et al., 1998
).
These studies indicated that the restriction of nuclear ß-catenin-GFP to
the vegetal region is dependent on GSK3ß-mediated degradation of the
protein in animal blastomeres.
Measurements of b-catenin half-life in vivo
To measure the half-life of ß-catenin in specific cell lineages, we
injected mRNA encoding Xl-wt-ß-catenin-GFP into fertilized eggs, allowed
the protein to accumulate to levels detectable by confocal microscopy, then
blocked further protein translation with emetine. Control experiments showed
that 100 µM emetine inhibited >90% of new protein synthesis within 20
minutes (Fig. 2E). Levels of
fluorescence were quantified over the subsequent 45-60 minutes and the rate of
fluorescence decay was used to calculate the half-life of the protein in
different blastomeres (Fig. 2).
Additional control experiments showed that fluorescence decay was dependent on
GSK3ß-mediated phosphorylation of ß-catenin and that photobleaching
of GFP was negligible over the time course of our experiments
(Fig. 2F-G).
These measurements provided direct evidence of a gradient in ß-catenin stability along the A-V axis during early cleavage. The average half-life of ß-catenin-GFP in the mesomere, macromere and micromere territories was 0.24, 0.59 and 1.60 hours, respectively (Fig. 2H). In our emetine experiments, we detected differential degradation of ß-catenin as early as the 8-cell stage (approximately 2 hours postfertilization), the first stage at which cleavage divisions separated animal from vegetal blastomeres. We also made half-life measurements at the 16- and 64-cell stages and consistently observed a gradient in ß-catenin stability at these stages. We found no striking increase or decrease in ß-catenin stability within any single cell lineage between the 8- and 64-cell stages, and therefore data from these various cleavage stages were pooled in the table shown in Fig. 2H.
Potential regulators of ß-catenin degradation: GSK3ß and dishevelled
The differential stability of ß-catenin along the A-V axis suggested
that positive or negative regulators of degradation might be localized (or
activated) in the animal or vegetal regions, respectively. Localized
degradation of GSK3ß has been proposed as a mechanism for regulating
nuclear accumulation of ß-catenin in the early Xenopus embryo
(Dominguez and Green, 2000). We
cloned GSK3ß from L. variegatus (GenBank Accession number
AY624076) and expressed a GFP-tagged form, but half-life measurements showed
that the protein was highly and equally stable in all blastomeres
(Fig. 3). Dorsal translocation
of dishevelled (Dsh) following fertilization has also been proposed to play a
role in Xenopus (Miller et al.,
1999
). We cloned Dsh from L. variegatus (GenBank
Accession number AY624074) and found that LvDsh mRNA was ubiquitously
expressed in eggs and early embryos (data not shown). We expressed a
GFP-tagged form of LvDsh (Lv-wt-Dsh-GFP) and observed a striking, vegetal
cortical localization (VCL) of the protein
(Fig. 4). At the light
microscope level, Lv-wt-Dsh-GFP accumulated in punctate structures associated
with the cortical cytoplasm in the vegetal region
(Fig. 4F). We also observed
punctate localization to the perinuclear region of all cells, including animal
blastomeres. In some embryos injected with high concentrations of mRNA, VCL
could be detected even before first cleavage
(Fig. 4A). Continuous
observation of such embryos showed that the first and second cleavage planes
bisected the domain of Lv-wt-Dsh-GFP localization. The third cleavage
unambiguously identified this region as the vegetal pole. At the 16-cell
stage, the VCL domain was inherited by the micromeres and, to a lesser extent,
the macromeres. The VCL domain became more difficult to detect at later
stages, but could be identified in some embryos as late as the 32-cell
stage.
|
|
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|
Motifs within the DIX domain have been identified that regulate binding to
actin and vesicles in cultured mammalian cells. Point mutations in the
actin-binding motif (K58A) and vesicle (phospholipid)-binding motif
(K68A/E69A) have been shown to abolish the corresponding binding activities
without compromising the structural integrity of the DIX domain
(Capelluto et al., 2002). These
two motifs are well conserved within the DIX domain of LvDsh. A point mutation
in the actin-binding domain of LvDsh (LvDsh.K47A.GFP) that corresponded to the
K58A mutation described by Capelluto et al.
(2002
) did not affect VCL, but
the corresponding double mutation in the phospholipid-binding domain
(LvDsh.K57A/E58A.GFP) completely abolished targeting
(Fig. 5;
Fig. 6E). Two other regions
were identified that were required for targeting: a region between the DIX and
PDZ domains that includes multiple phosphorylation sites
(LvDsh.DIX
PDZ.GFP) and amino acid sequences other than the proline-rich
region that lie between the PDZ and DEP domains (LvDsh.PDZ
DEP.GFP). The
smallest portion of the LvDsh protein sufficient for VCL consisted of
approximately the N-terminal half of the protein
[LvDsh.
(DEP+C).GFP].
Overexpression of a dominant negative form of LvDsh
To test whether Dsh function was required for the vegetal stabilization of
ß-catenin, we overexpressed the DIX domain alone. This region of Dsh is
required for canonical signaling (Kishida
et al., 1999; Rothbacher et
al., 2000
, Penton et al.,
2002
) and overexpression of the DIX domain phenocopies Dsh null
mutations, indicating that it acts as a dominant negative
(Axelrod et al., 1998
). We
found that overexpression of DIX produced an animalized phenotype that was
indistinguishable from the phenotype observed following overexpression of
cadherin (Wikramanayake et al.,
1998
; Logan et al.,
1999
), i.e. lack of endoderm and reduction or complete absence of
mesoderm (Fig. 7A). LvDsh DIX
also blocked the accumulation of ß-catenin in the nuclei of vegetal
blastomeres, as shown by immunostaining with an antibody against endogenous
Lv-ß-catenin (Fig. 7D,E).
At these levels of expression, DIX did not block VCL of Lv-wt-Dsh-GFP
(Fig. 6F), indicating that it
acted by a different mechanism. By contrast to the striking phenotype
resulting from DIX overexpression, overexpression of the PDZ domain of LvDsh
had no effect on embryo morphology (Fig.
7B). Axin, another protein that regulates ß-catenin
degradation, also contains a DIX domain. To determine whether the effects of
DIX domain overexpression were specific to LvDsh, we cloned LvAxin (GenBank
Accession number AY624075) and overexpressed the LvAxin DIX domain. Injection
of LvAxin DIX mRNA at concentrations equal to or higher than LvDsh DIX mRNA
produced no apparent phenotype (Fig.
7C).
|
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Discussion |
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Several observations indicate that the turnover of Xenopus
wild-type ß-catenin-GFP mimics that of the endogenous sea urchin protein.
Most significantly, the pattern of nuclear accumulation of
Xl-wt-ß-catenin-GFP we observed (Fig.
1) closely matches the pattern of nuclear localization of
endogenous, sea urchin ß-catenin detected by immunostaining
(Logan et al., 1999). In
addition, several studies have shown that homologous Wnt pathway proteins from
vertebrates and sea urchins are functionally interchangeable and therefore
likely to be regulated in similar ways. For example, Xenopus and sea
urchin forms of cadherins, GSK3ß and LEF/TCF have been tested in
overexpression studies, and in each case the homologs have similar effects
(Emily-Fenouil et al., 1998
;
Wikramanayake et al., 1998
:
Logan et al., 1999
;
Vonica et al., 2000
). In
cnidarians, much more distant relatives of vertebrates than sea urchins, the
Xenopus wild-type ß-catenin construct used in this study has
been compared to a GFP-tagged form of the endogenous protein and the two have
identical dynamics (Wikramanayake et al.,
2003
).
The findings reported here point to differential proteolysis as the
predominant mechanism controlling the polarized nuclear localization of
ß-catenin during cleavage. Nevertheless, other mechanisms might operate
in parallel. ß-Catenin mRNA is uniformly distributed in the egg and early
embryo (Miller and McClay,
1997), and differential localization of maternal ß-catenin
mRNA (or localized transcription of the ß-catenin gene) is therefore
unlikely to be a contributing factor. We cannot rule out differential
translation as a contributing mechanism, however, as our mRNA constructs might
lack important translational regulatory elements, and the rate of translation
of endogenous ß-catenin mRNA in animal and vegetal blastomeres has not
been measured. If differential translation contributes to the polarized
nuclear accumulation of ß-catenin, this effect can be overridden by
experimental manipulations that alter post-translational processing of the
protein. Thus, endogenous ß-catenin can be driven into the nuclei of more
animal blastomeres by treating embryos with LiCl, an inhibitor of GSK3ß
(Logan et al., 1999
). Finally,
another plausible mechanism is regulated nuclear import and/or export,
possibly involving interactions with cytoplasmic or nuclear anchoring proteins
(Henderson and Fagotto, 2002
;
Tolwinski et al., 2003
). Our
observation that wild-type and hyperstable ß-catenin-GFP accumulate in
the nuclei of animal blastomeres (the former transiently and the latter
persistently) argues strongly against the possibility that animal blastomeres
lack factors required for nuclear import or retention. A potential limitation
of overexpression studies, however, is that mechanisms that normally regulate
nuclear import/export of ß-catenin might be overwhelmed. For example,
artificially high levels of ß-catenin might saturate a cytoplasmic
anchoring protein that normally sequesters ß-catenin in the cytoplasm of
animal blastomeres. Although the differential translation and cytoplasmic
sequestration models warrant further study, our findings strongly support the
view that differential degradation is a key mechanism regulating the nuclear
accumulation of ß-catenin during cleavage.
The methods used in the present study do not allow us to determine
precisely when the polarity in ß-catenin degradation first arises. It is
therefore unclear whether differential stability along the A-V axis involves a
local activation of degradation in the animal hemisphere, an inhibition of
degradation in the vegetal region, or a combination of mechanisms. A
difference in ß-catenin stability along the A-V axis is clearly
established by the 8-cell stage, when animal and vegetal blastomeres are first
separated from one another by a horizontal cleavage. The finding that
LvDsh-GFP becomes localized at the vegetal pole even prior to first cleavage,
however, suggests that the molecular machinery underlying differential
degradation is polarized maternally or immediately after fertilization. This
observation also indicates that VCL is not dependent on Wnt signals
transmitted between blastomeres, consistent with other evidence that cell-cell
signaling is not required for vegetal nuclear accumulation of ß-catenin
in the sea urchin embryo (Logan et al.,
1999).
Our finding that endomesoderm specification and nuclear accumulation of
ß-catenin are suppressed by overexpression of the DIX domain of Dsh, a
putative dominant negative form of the protein with respect to canonical Wnt
signaling (Axelrod et al.,
1998), provides the strongest evidence to date that Dsh normally
plays an essential role in early deuterostome embryo polarity. It has been
proposed that Dsh plays a role in axis specification in Xenopus,
based on the finding that Dsh protein becomes concentrated on the dorsal side
of the early embryo (Miller et al.,
1999
) (see below). There are also differences in levels of Dsh
phosphorylation along the dorsal-ventral axis
(Rothbacher et al., 2000
),
although their functional significance (and the role of Dsh phosphorylation in
general) remains unclear (Wharton,
2003
). The function of Dsh in early patterning in Xenopus
has been controversial because dominant negative approaches have not yet
revealed a role for the protein in endogenous axis formation. Dominant
negative forms of Dsh that interfere with Wnt-induced secondary axis formation
do not suppress the formation of the endogenous dorsal axis
(Sokol, 1996
). Others have
noted possible technical reasons for the failure of these constructs to
suppress normal axis formation, however. Levels of expression of dominant
negative constructs might be insufficient for competing with maternal pools of
protein, particularly if the maternal proteins are already complexed with
other molecules (Miller et al.,
1999
; Rothbacher et al.,
2000
).
The molecular mechanism of the dominant negative effect of the DIX domain
is unknown. This is largely because the mechanism by which Dsh stabilizes
ß-catenin has not yet been elucidated. It has been proposed that Dsh acts
by binding to Axin, via the DIX domains of the two proteins. This interaction
might prevent Axin multimerization (Hsu et
al., 1999; Kishida et al.,
1999
; Sakanaka and Williams,
1999
) and/or recruit GBP/FRAT-1, an inhibitor of GSK3ß, to
the degradation complex (Li et al.,
1999
; Ferkey and Kimelman,
2002
; Hino et al.,
2003
). Dsh also forms multimers via the DIX domain, and this might
be important for signaling (Kishida et
al., 1999
; Rothbacher et al.,
2000
). These observations are consistent with a number of
scenarios by which stray DIX domains might disrupt endogenous Dsh-Dsh,
Dsh-Axin and/or Axin-Axin interactions. For example, DIX might compete with
endogenous Dsh for binding to Axin but be unable to recruit GBP/FRAT-1 to the
degradation complex.
Our observations and those of Miller et al.
(Miller et al., 1999) point to
striking similarities in Dsh localization in early sea urchin and
Xenopus embryos. Miller and co-workers reported that in
Xenopus, endogenous Dsh and a GFP-tagged form of the protein
associate with vesicle-like organelles that translocate from the vegetal pole
of the fertilized egg to the future dorsal side during cortical rotation. In
L. variegatus, LvDsh-GFP is associated with granular or vesicular
structures in the vegetal region and this association is dependent on a
vesicle-binding motif within the DIX domain. The major difference appears to
be that Dsh is not redistributed after fertilization in sea urchin eggs, which
lack a cortical rotation. Although the mutational analysis of Miller et al.
(Miller et al., 1999
) was not
as detailed as that presented here, both found that association of Dsh-GFP
with vesicle-like structures was dependent on the DIX domain, but not the
C-terminal region of the protein. One difference between the two studies,
however, is that Miller et al. reported that deletion of the DEP or PDZ
domains eliminated association with vesicles, while we found that deletion of
these domains in LvDsh only partially blocked targeting to the vegetal cortex
and association with vesicles in that region. Dsh has been found associated
with punctate cytoplasmic structures in a variety of cell types
(Axelrod et al., 1998
;
Itoh et al., 2000
), although
it is not known whether these structures are the same as the granular or
vesicular structures observed in eggs and early embryos.
Significantly, in our experiments, we observed no phenotype associated with
overexpression of full length LvDsh, either untagged or GFP-tagged forms, even
at mRNA concentrations sufficiently high to compromise embryo viability.
Others have shown that animal blastomeres can be converted to more vegetal
fates by overexpression of kinase-dead GSK3ß
(Emily-Fenouil et al., 1998), a
mechanism that bypasses Dsh. Moreover, in the present study, we showed that
ß-catenin can be driven into the nuclei of animal blastomeres by
overexpression of kinase-dead GSK3ß or mutation of N-terminal
phosphorylation sites in ß-catenin. These findings show that disruption
of ß-catenin degradation downstream of Dsh leads to nuclear accumulation
of ß-catenin in animal blastomeres and changes in cell fate. Therefore,
our finding that overexpression of wild-type LvDsh does not have the same
effect strongly suggests that the protein is not active in animal cells. We
cannot exclude the possibility that, in our experiments, insufficient levels
of LvDsh were expressed in animal blastomeres to produce effects, although
GFP-tagging confirmed that the protein was expressed persistently in all
cells, including animal blastomeres. An alternative hypothesis, and one that
we currently favor, is that Dsh is activated specifically in the vegetal
region (Fig. 8). This local
activation might involve phosphorylation by a vegetally localized activating
kinase or interactions with other vegetal-specific proteins. In light of such
a model, the targeting of LvDsh-GFP to the vegetal cortex might be interpreted
in one of two ways. First, targeting might be an essential step in Dsh
activation. For example, vegetal targeting might bring Dsh into close
association with a localized, activating kinase, such as Par-1
(Sun et al., 2001
).
Alternatively, VCL might be a consequence of the vegetal activation of Dsh. In
that event, VCL could serve to concentrate the protein to effective levels in
vegetal blastomeres, or it might play no functional role. Further studies will
be required to identify the mechanisms and function of the vegetal cortical
localization of Dsh and the putative, local activation of this protein in the
vegetal region of the embryo.
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ACKNOWLEDGMENTS |
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