Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350, USA
* Author for correspondence (e-mail: kimelman{at}u.washington.edu)
SUMMARY
A long-standing question in developmental biology is how amphibians establish a dorsoventral axis. The prevailing view has been that cortical rotation is used to move a dorsalizing activity from the bottom of the egg towards the future dorsal side. We review recent evidence that kinesin-dependent movement of particles containing components of the Wnt intracellular pathway contributes to the formation of the dorsal organizer, and suggest that cortical rotation functions to align and orient microtubules, thereby establishing the direction of particle transport. We propose a new model in which active particle transport and cortical rotation cooperate to generate a robust movement of dorsal determinants towards the future dorsal side of the embryo.
Introduction
One of the major challenges in developmental biology is to understand how polarity is achieved in a developing embryo. For over a century, the amphibian embryo has played a major role in these investigations because its large size and external fertilization have facilitated the analysis of the early steps in the development of the embryonic axes. In the amphibian, the first body axis to be established is the dorsoventral (DV) axis, which ultimately also dictates the orientation of the anteroposterior (AP) axis. A large body of work, mostly from studies using the African clawed frog Xenopus laevis, has described how the initial specification of DV asymmetry depends on physical and biochemical events that occur shortly after fertilization.
Fertilization leads to the movement of maternally deposited dorsalizing
factors from their original location at the bottom of the Xenopus
embryo to a new location near the equator, on the side opposite the point of
sperm entry (Fig. 1A,B). These
factors cause a local stabilization of the Wnt pathway effector
ß-catenin, which is needed to activate zygotic genes of the dorsal
organizer (Fig. 1C,D). As a
result of organizer formation, the side opposite the sperm entry point is
specified as dorsal (reviewed by Moon and
Kimelman, 1998). Therefore, the creation of this DV asymmetry in
ß-catenin levels by the translocation of dorsal factors is a crucial
event in the development of the amphibian embryo. However, we still have a
poor understanding of how it occurs. In particular, there are two important
questions that need to be answered: what is the moving dorsalizing activity,
and how does it get to where it is going?
|
The principal focus of this review is on the second question: how is the
dorsalizing activity transported? The prevailing model has been that a
physical displacement of the egg cortex, an event called cortical rotation, is
responsible for the movement of cortically attached dorsalizing factors
towards the future dorsal side. However, a closer examination of the early
literature, in combination with recent data, points to an alternative
mechanism, in which dorsal determinants travel on subcortical microtubules as
the cargo of kinesin motor proteins. We discuss a potential alternative
purpose for cortical rotation - to align the microtubule tracks on which the
determinants travel. Finally, on the basis of previous models
(Scharf and Gerhart, 1980;
Vincent and Gerhart, 1987
;
Zisckind and Elinson, 1990
;
Kikkawa et al., 1996
;
Rowning et al., 1997
;
Miller et al., 1999b
;
Weaver et al., 2003
), we
propose a new integrated model in which both active transport by motor
proteins and association with the rotating cortex contribute to the net
movement of dorsal determinants towards the dorsal side.
The cell biology of dorsal specification
The unfertilized Xenopus laevis egg is radially symmetrical, with
a darkly pigmented animal hemisphere and a lightly pigmented vegetal
hemisphere. Sperm entry, which can occur anywhere in the animal hemisphere,
causes the outer layer of the egg to loosen from the dense yolky core
cytoplasm. This outer layer is called the cortex, and includes the plasma
membrane of the egg, as well as cytoskeletal elements, the endoplasmic
reticulum (ER) and other components
(Houliston and Elinson,
1991a). The loosening of the cortex from the core creates a
relatively yolk-free area called the shear zone
(Fig. 1E). About midway through
the first cell cycle, the cortex begins to rotate relative to the core in a
process called cortical rotation, which continues until near the end of the
first cell cycle and results in a
30° displacement of the vegetal
cortex away from the sperm entry site and towards the future dorsal region
(Fig. 1F) (reviewed by
Gerhart et al., 1989
;
Houliston and Elinson,
1992
).
Cortical rotation coincides with the translocation of the maternal
dorsalizing activity from the vegetal pole towards the prospective dorsal side
of the embryo, in the same direction as cortical rotation
(Kikkawa et al., 1996;
Sakai, 1996
;
Kageura, 1997
). Cytoplasmic
transplant experiments have shown that the dorsalizing activity resides in the
subcortical cytoplasm of the shear zone
(Fig. 1E)
(Holowacz and Elinson, 1993
;
Kageura, 1997
). Both cortical
rotation and the translocation of the dorsal determinants depend on the
assembly of a parallel array of microtubule (MT) bundles that appears in the
vegetal shear zone of the egg about midway through the first cell cycle
(Fig. 2A-C)
(Elinson and Rowning, 1988
)
(reviewed by Houliston and Elinson,
1992
; Chang et al.,
1999
). The MTs that form the parallel array appear to arise from
several sources. Some of them are nucleated by the centriole of the sperm,
which acts as a minus-end MT-organizing center
(Fig. 2D). Other MTs extend
towards the periphery from unknown sources deep in the cytoplasm and bend into
the vegetal shear zone (Fig.
2E). Finally, some array MTs appear to polymerize spontaneously in
the vegetal shear zone (Houliston and
Elinson, 1991b
; Schroeder and
Gard, 1992
).
|
The proper formation of the MT array is crucial for the development of the
dorsal axis. If MT polymerization is blocked during the first cell cycle by
exposure to UV irradiation or to the MT-depolymerizing drug nocodazole, or
with inhibitory antibodies to the MT-associated protein XMAP230, the dorsal
organizer does not form and the resulting embryos lack all dorsoanterior
structures (Scharf and Gerhart,
1983; Elinson and Rowning,
1988
; Cha and Gard,
1999
). In the absence of a functional MT array, cortical rotation
is prevented, and the dorsalizing activity remains stuck in the vegetal pole
of the embryo (Vincent et al.,
1987
; Holowacz and Elinson,
1993
). However, the inherent axis-inducing ability of the
immobilized dorsalizing activity is unaffected: if subcortical cytoplasm is
removed from the vegetal pole of a UV-treated embryo and transplanted into the
marginal region of a host, the host develops secondary axial structures
(Holowacz and Elinson, 1993
).
This result reveals that dorsal specification requires not just the
dorsalizing activity, but also its successful translocation from the vegetal
pole to the marginal zone.
The general view has been that the dorsal determinants associate with the
moving cortex, which carries them up towards the margin during cortical
rotation. The main rationale for this model is that the timing and direction
of the movement of the dorsalizing activity correspond with those of cortical
rotation. However, there is increasing evidence that cortical rotation is not
the primary method used by the embryo to move the dorsalizing activity. In
fact, the existence of an unknown underlying mechanism was first hypothesized
by John Gerhart and colleagues almost 20 years ago, when they observed that
cortical rotation could be uncoupled from the formation of the dorsal axis in
some experimental situations (as discussed later). They therefore proposed
that cortical rotation, rather than acting as the sole mechanism of axis
determination, was instead simply a `cue' that sensitized or oriented the
response of the embryo to some underlying system
(Black and Gerhart, 1985;
Black and Gerhart, 1986
;
Vincent and Gerhart, 1987
).
The nature of this second system was a mystery, although they realized that,
like cortical rotation, it required MTs.
While the cortical rotation-dependent model predominated, further clues
about a possible alternative system accumulated. Cytoplasmic transplantation
and removal experiments, which were used to map the location of the
dorsalizing activity at different times, revealed that the dorsalizing
activity is initially concentrated near the vegetal pole
(Fig. 1A). During cortical
rotation, this activity becomes broadly distributed along the dorsal side in a
swath that reaches from the vegetal pole to near the equator
(Fig. 1B) (Yuge et al., 1990;
Fujisue et al., 1993
;
Holowacz and Elinson, 1993
;
Kikkawa et al., 1996
;
Sakai, 1996
;
Kageura, 1997
). This
distribution suggested that the activity can somehow move faster than the
speed of rotation, as the cortex moves only about 30° during the period of
transport. It was therefore very intriguing when unidentified, membrane-bound
organelles in the subcortical shear zone were discovered to translocate very
quickly towards the prospective dorsal side, with velocities two to three
times faster than that of cortical rotation
(Rowning et al., 1997
).
Microinjected carboxylated beads are similarly capable of fast directed
transport in the vegetal shear zone, and can spread more than 60° from
their site of injection in the vegetal pole
(Rowning et al., 1997
). The
velocities and behavior of the organelles and beads strongly suggested that
their transport is mediated by MT motor proteins.
Although the significance of these organelles in the process of dorsal axis
formation remains unknown, the observation that they translocate was the first
evidence that cargo transport by MT motor proteins is likely to occur in the
shear zone. The orientation of the MTs, with their plus-ends directed
dorsalwards, implied the involvement of a plus-end-directed motor protein. All
known plus-end-directed MT motors are members of the large superfamily of
kinesin-related proteins (KRPs) (reviewed by
Vale, 2003) (see also the
Kinesin Home Page at
http://www.proweb.org/~kinesin).
A model was proposed in which the dorsal determinants, rather than being
`passively' carried by virtue of association with the cortex during rotation,
might instead travel as cargo that is associated with kinesin motor proteins
and that moves directly on the MT array
(Kikkawa et al., 1996
;
Rowning et al., 1997
).
In order to determine the validity of this model, it becomes essential to know two things. First, which molecules comprise the dorsalizing activity? And second, can a motor protein be identified that transports them? In the following section, we address the first of these questions, describing work that has been carried out to identify candidate dorsalizing proteins. Although this is still a work in progress, advances made in recent years indicate that these molecules are likely to be ß-catenin regulatory proteins that act in the canonical Wnt signaling pathway.
The Wnt pathway during early frog development
The canonical Wnt signaling pathway is used by numerous organisms to make
cell fate decisions in many developmental and cellular contexts. Its defining
feature is the use of ß-catenin as a downstream effector. In the absence
of a Wnt signal, a host of proteins cooperate to target ß-catenin for
degradation by the ubiquitin-proteasome pathway. Active Wnt signaling causes
the stabilization of ß-catenin, allowing it to accumulate in the nucleus
where it activates Wnt target genes as part of a complex with T-cell
factor/Lymphoid enhancer factor (TCF/LEF) transcription factors.
ß-Catenin regulation by the Wnt pathway has been extensively reviewed
elsewhere (Miller et al.,
1999a; Dominguez and Green,
2001
; Huelsken and Behrens,
2002
).
The localized stabilization of ß-catenin on the future dorsal side of
the early Xenopus embryo (Fig.
1C) is a crucial event in the formation of the dorsal organizer;
depletion of maternal ß-catenin can cause a complete loss of dorsal
structures in the embryo and ectopic expression of ß-catenin ventrally
leads to the formation of a secondary dorsal axis
(Heasman et al., 1994;
Funayama et al., 1995
;
Larabell et al., 1997
). A key
goal, therefore, is to elucidate the biochemical steps that connect the
translocation of the dorsalizing activity to the stabilization of
ß-catenin. Movement of the dorsalizing activity is thought to be
essential for ß-catenin accumulation in the dorsal margin, which does not
occur if vegetal MT polymerization is blocked
(Larabell et al., 1997
). An
obvious hypothesis is that the moving activity is ß-catenin itself, which
accumulates dorsally via active transport. However, as subcortical vegetal
cytoplasm from ß-catenin-depleted Xenopus embryos is still able
to induce a secondary axis when transplanted into a normal host
(Marikawa and Elinson, 1999
),
it appears more likely that the dorsalizing activity consists of an upstream
factor or factors that move to the dorsal region in order to stabilize
ß-catenin there.
In the canonical Wnt pathway, the regulation of ß-catenin stability is
controlled by a complex of proteins called the ß-catenin destruction
complex. Central to this complex are two large proteins, Axin and Adenomatous
polyposis coli (APC), which are thought to act by shuttling ß-catenin in
and out of the complex (reviewed by Bienz,
1999). In the absence of a Wnt signal, ß-catenin is
phosphorylated within the complex by two kinases, Casein Kinase 1
(CK1
) and GSK3, which marks ß-catenin for ubiquitination and
degradation (Fig. 3A) (reviewed
by Dominguez and Green, 2001
;
Polakis, 2002
). During active
Wnt signaling, this process is inhibited through an only partially understood
mechanism, which causes ß-catenin to be stabilized.
|
Intracellular components of the Wnt pathway are clearly involved in
Xenopus axis specification. One protein of particular interest is the
vertebrate-specific GSK3-binding protein GBP, which is thought to act on the
dorsal side to inhibit GSK3 function (Yost
et al., 1998; Dominguez and
Green, 2000
). GBP binds directly to Dsh, a GSK3 inhibitor that is
activated by a Wnt signal in the canonical pathway
(Yost et al., 1998
;
Li et al., 1999
;
Salic et al., 2000
). In the
Xenopus embryo, Dsh is thought to act in part by recruiting GBP to
the destruction complex (Salic et al.,
2000
). GBP binds GSK3 and removes it from Axin, and also causes
GSK3 degradation (Dominguez and Green,
2000
; Farr et al.,
2000
). GSK3 is thereby prevented from phosphorylating
ß-catenin (Fig. 3B),
causing ß-catenin levels to increase.
How strong is the evidence that GBP and Dsh are components of the
endogenous dorsalizing activity? Like ß-catenin and Wnt, GBP and Dsh are
both capable of inducing the formation of a complete dorsal axis when their
RNAs are microinjected into early Xenopus embryos
(Sokol et al., 1995;
Yost et al., 1998
).
Furthermore, overexpressed GBP mimics the endogenous dorsalizing activity in
that it causes GSK3 degradation in the equatorial region
(Dominguez and Green, 2000
).
Most importantly, GBP has been shown to be required for dorsal axis formation
in Xenopus through the use of antisense oligonucleotides, which can
be injected into the egg to target specific mRNAs for degradation prior to
fertilization (Yost et al.,
1998
). The case is less clear for Dsh, as loss-of-function
experiments reducing Dsh levels in the egg prior to fertilization have not yet
been reported. In embryos, the microinjection of RNA that encodes a
dominant-negative Dsh had no effect on dorsal axis formation; however, the RNA
was injected at the two-cell stage, too late to have an effect if Dsh has a
required role during the translocation events of the first cell cycle
(Sokol, 1996
). A finding that
supports a role for Dsh in this process is that endogenous Dsh protein is
found at higher levels in the dorsal shear zone than in the ventral shear zone
as early as the two-cell stage (Miller et
al., 1999b
). The distribution of endogenous GBP protein in the
embryo is not yet known.
Experiments using various ventralizing molecules to block the activation of
dorsal markers by transplanted vegetal cytoplasm suggest that neither GBP nor
Dsh alone is the key ß-catenin stabilizing factor in the vegetal shear
zone (Marikawa and Elinson,
1999). One possibility is that GBP and Dsh synergize to stabilize
ß-catenin levels, as was recently shown in Xenopus egg extracts
(Salic et al., 2000
), or that
they act in combination with other dorsalizing proteins (see
Weaver et al., 2003
). An
alternative possibility is that GBP, Dsh, or both, are not part of the moving
dorsalizing activity, but are instead somehow activated by it once it reaches
the dorsal equatorial region. Therefore, two key questions need to be
answered: (1) what is the relationship of GBP and Dsh to the moving
dorsalizing activity; and (2) how is this activity transported to the future
dorsal side?
Particles and motor proteins in the shear zone
An important breakthrough occurred when Miller and colleagues developed an
assay to monitor the behavior of green fluorescent protein (GFP) fusion
proteins in the vegetal shear zone of live Xenopus eggs during
cortical rotation. Using this assay, both Dsh-GFP and GBP-GFP were found to
form small particles in the vegetal shear zone that move quickly in the
direction of cortical rotation, with roughly the same velocity and saltatory
behavior that was earlier observed for translocating organelles in that region
(Miller et al., 1999b;
Weaver et al., 2003
). The
particle movements depend on the parallel MT array, as evidenced by the fact
that the transport of Dsh-GFP was disrupted by UV irradiation. Conversely,
treatment of embryos with D2O, which causes precocious excessive
polymerization of randomly oriented MTs, causes Dsh-GFP translocation to
become randomized (Miller et al.,
1999b
). Some deletions within conserved regions of Dsh and GBP
prevent them from assembling into particles, suggesting a requirement for
specific protein-protein interactions
(Miller et al., 1999b
;
Weaver et al., 2003
). As
further evidence of the specificity of the behavior, ß-catenin, which is
not a required component of the moving dorsalizing activity, does not form
particles or translocate (Miller et al.,
1999b
). These results show that GFP-tagged GBP and Dsh are able to
translocate to the presumptive dorsal side, supporting the idea that GBP and
Dsh form part of the endogenous moving dorsalizing activity.
But what mediates the transport of GBP and Dsh? A yeast two-hybrid screen
using murine GBP [called Frat1 (frequently rearranged in advanced T-cell
lymphomas)] as bait provided the first insight into this question when it
revealed that GBP binds to a kinesin light chain (KLC)
(Weaver et al., 2003). This
interaction was confirmed by co-immunoprecipitation of tagged mouse and
Xenopus proteins from cultured mammalian cells and Xenopus
embryos, respectively (Weaver et al.,
2003
). KLCs are cargo-binding subunits of conventional kinesin, a
heterotetrameric MT motor. Conventional kinesins, which also contain two
motor-containing heavy chain subunits, move along MTs from the minus- to
plus-ends (reviewed by Verhey and
Rapoport, 2001
), which fits well with the fact that the
dorsalizing activity moves towards the plus-ends of oriented MTs. A
Xenopus KLC, fused to GFP, was shown to exhibit the same behavior as
GBP and Dsh, forming particles that translocate quickly in the direction of
cortical rotation (Fig. 4; see
movies at
http://dev.biologists.org/cgi/content/full/130/22/5425/DC1).
Small deletions in a conserved domain of GBPGFP disrupt both its ability to
form translocating particles and to bind KLC, supporting the idea that GBP
depends on its interaction with KLC in order to move
(Weaver et al., 2003
). As GBP
also binds Dsh, a preliminary model has emerged in which GBP mediates the
transport of dorsalizing particles by binding kinesin.
|
Interestingly, there is evidence that the motor-based transport of dorsal
determinants may also occur in other species. For example, in the zebrafish
embryo, there appears to be one or more transient parallel MT arrays used to
transport dorsal determinants from the vegetal cortex of the large yolk cell
up to the embryonic blastomeres in the animal hemisphere. As in the frog,
microinjected beads can translocate upwards in the periphery of the zebrafish
yolk cell in a manner that suggests active transport by motor proteins
(Jesuthasan and Stahle, 1997)
(reviewed by Pelegri, 2003
).
There is also evidence of a motor-based transport system in one-cell medaka
embryos. In this species, a parallel array of MTs forms in the vegetal region
that are aligned with the DV axis and appears to act as tracks for the
movement of cargo towards the future dorsal side
(Abraham et al., 1995
;
Trimble and Fluck, 1995
).
Despite these similarities to events in the amphibian embryo, embryos of
teleost fish, such as zebrafish and medaka, do not undergo cortical rotation
(Ho, 1992
). Therefore, it
appears to be possible to achieve the transport of dorsal determinants solely
by translocating the dorsalizing activity directly on MTs. It is not clear how
far the parallels go, but as zebrafish also have maternally deposited GBP and
Dsh, and depend on dorsally stabilized ß-catenin, much of the dorsalizing
machinery may be conserved between fish and frogs
(Sumoy et al., 1999
;
Kimelman and Schier, 2002
) (J.
Waxman and R. T. Moon, personal communication). Interestingly, embryos of more
primitive fish, such as sturgeon, appear to undergo cortical rotation
(Clavert, 1962
), suggesting
that perhaps it is an ancient process that has been lost in more derived fish
species, while the MT-based transport of determinants has been conserved.
Cortical rotation and the alignment of the microtubule array
Cortical rotation is a conserved process that demands energy expenditure by
the embryo during a crucial time in its development
(Vincent et al., 1987). But if
the dorsalizing activity can travel directly on the MT array, what purpose
does the rotation serve? Evidence supports at least two possible functions.
First, cortical rotation might serve to align the polymerizing MTs into
parallel bundles and to orient their plus-ends towards the dorsal side.
Second, as will be discussed later, cortical rotation might directly
contribute to the overall dorsalwards movement of the dorsalizing
activity.
How would rotation align the MTs? Although several scenarios can be
imagined (see Marrari et al.,
2003), the simplest model is that plus-end-directed motor proteins
attached to the cortex move along the MTs and align them as the cortex moves
towards their plus-ends, analogous to a comb moving through hair. The action
of these KRP motors on the subcortical MTs could simultaneously align the MTs
and generate the force required to translocate the cortex relative to the core
cytoplasm. In support of this model, the injection of antibodies that inhibit
KRPs into the vegetal shear zone causes MT flailing, which disrupts the
organization of the MT array and causes a local arrest in cortical rotation
movements (Marrari et al.,
2000
). Interestingly, very recent results have shown that the
inhibition of the minus-end-directed MT motor dynein early in the first cell
cycle of Xenopus development also leads to defects in cortical
rotation, but for different reasons. In contrast to KRPs, dynein is not
required for MT alignment in the shear zone, nor for generating force during
cortical rotation; instead, dynein appears to contribute to the initial
formation of the MT array by pushing MTs from the inner cytoplasm out towards
the cortex (Marrari et al.,
2004
). As KRPs and dyneins make up the only two known classes of
MT motor proteins, these results indicate that members of the KRP superfamily
- the majority of which are plus-end-directed motors - must be responsible for
organizing and aligning the MT array in the shear zone
(Vale, 2003
). Interestingly,
Marrari and colleagues recently observed that the vegetal MT bundles, although
initially thick and wavy in appearance, take on a straighter, more
`fine-combed' appearance towards the end of cortical rotation
(Marrari et al., 2004
). It is
possible that this fine-combing effect is the result of the continual action
of cortically attached motors moving along the length of the MTs.
Does cortical rotation help transport dorsal determinants?
On the basis of the results described above, it is possible to imagine a
scenario in which cortical rotation serves only to align MTs, which are then
used as tracks by kinesin motors that directly transport the dorsalizing
activity to the equatorial region. But does cortical rotation also have a more
direct role in moving dorsal determinants, as the traditional view of its
purpose holds? As already described, cortical rotation can be experimentally
uncoupled from axis formation by D2O treatment, which indicates
that dorsal determinant transport can occur robustly in the absence of
rotation. Yet, some evidence suggests that cortical rotation contributes to
the overall movement of dorsal determinants towards the equator. In UV-treated
embryos, where motor-based cargo transport is unlikely to occur because of a
lack of polymerized MTs, dorsal axis formation can be rescued by
centrifugation or by tilting the embryo, which mimics cortical rotation by
forcing a displacement of the core cytoplasm relative to the immobilized
cortex (Scharf and Gerhart,
1980; Black and Gerhart,
1986
; Zisckind and Elinson,
1990
). This rescue could occur if some dorsal determinants were
associated with the vegetal cortex, causing their relative displacement
towards the equatorial region of the core during centrifugation. Furthermore,
although the most potent dorsalizing activity is concentrated tightly in a
region at the vegetal pole, weak dorsalizing activity can be found in
subcortical cytoplasm as far as 60° from the vegetal pole prior to
cortical rotation (Kikkawa et al.,
1996
). It is therefore possible that a 30° rotation would be
sufficient to transport enough dorsal determinants to the equator to induce
axis formation. Thus, although there is good evidence for the particle model
of transport, the traditional model in which cortical rotation moves dorsal
determinants must still be taken into consideration. One way to reconcile the
evidence that supports each of these models is to propose that dorsal
determinant transport relies on a combination of both transport mechanisms,
which together are more robust than either one acting alone. In support of
this idea, translocating GBP-GFP particles in live eggs frequently appear to
jump on and off of the MT array, interspersing periods of fast transport with
slower periods moving at the speed of the cortex (see movie at
http://dev.biologists.org/cgi/content/full/130/22/5425/DC1)
(Weaver et al., 2003
). Thus,
cortical rotation might help keep determinants moving in the right
direction.
An improved model for axis determination
To bring together the results discussed in this review, we suggest an updated model for how the dorsal determinants cause ß-catenin stabilization on the dorsal side of the Xenopus embryo. During oogenesis, particles are assembled at the vegetal pole, which contain kinesin, GBP, Dsh and, most probably, other components (Fig. 5D). When the sperm fertilizes the egg, it activates embryogenesis and introduces a centriole, which acts as an initial minus-end MT-organizing center (Fig. 5A). A combination of MT polymerization and cortical rotation acts to orient the MTs throughout the vegetal shear zone, such that midway through the first cell cycle most of the cortical MTs are oriented in the same direction (Fig. 5B). The MTs serve as tracks on which the particles can move. We suggest that the particles move by alternating periods of fast transport on the MTs with slower transport, when they are associated with the cortex (Fig. 5E). Cooperation between these two methods of transport may be important to ensure that the bulk of the dorsalizing particles undergo a net dorsalwards movement. Once the particles have moved to their new location, the dorsalizing proteins disassemble from the particles to inhibit the phosphorylation of ß-catenin by GSK3, thus allowing ß-catenin to escape degradation and accumulate locally (Fig. 5C,F). ß-catenin later activates the transcription of dorsal organizer genes, ultimately resulting in the formation of the DV and AP axes.
|
Evidence is accumulating to support the idea that the early dorsal
organizer in frogs is activated by the kinesin-dependent transport of
particles containing regulators of the Wnt intracellular pathway. Although
many questions remain to be answered (Box
1), this view ties together the `classical' embryological studies
of Gerhart and colleagues with more recent molecular studies. Although some
aspects of the proposed particle transport process are specific to amphibian
development, particularly the phenomenon of cortical rotation, this mechanism
fits well with the emerging view that a wide range of biological systems use
MT motor-dependent transport to move signaling proteins and other
developmentally important molecules asymmetrically within embryos, oocytes and
cells (Cohen, 2002;
Schnapp, 2003
;
Betley et al., 2004
;
Gunawardena and Goldstein,
2004
).
Box 1. Key unanswered questions regarding the transport of dorsal determinants in
Xenopus embryos
|
ACKNOWLEDGMENTS
We thank Wilson Clements and Tom Reh for comments on the manuscript, and David Gard for supplying pictures and for helpful suggestions. We also thank Josh Waxman and Randall Moon for sharing unpublished results, and Carolyn Larabell for help with Fig. 4.
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