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First published online 18 October 2006
doi: 10.1242/dev.02642
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Department of Integrative Biosciences, Oregon Health and Science University, Portland, OR 97239-3097, USA.
* Author for correspondence (e-mail: danilchi{at}ohsu.edu)
Accepted 13 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Axis formation, Left-right asymmetry, Xenopus laevis, Actin, Microtubule, Egg, Cytoskeleton
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Klymkowsky et al., 1991;
Pondel and King, 1988;
Yisraeli et al., 1990). Later,
during the first embryonic cell cycle, the dorsoventral (DV) axis is
superimposed on this oogenetic animal-vegetal polarity via a
microtubule-dependent rotation of the cortex relative to the inner cytoplasm
(Weaver and Kimelman, 2004).
The rotation displaces vegetal pole determinants toward the equator on the
side opposite the sperm entry point
(Weaver and Kimelman, 2004);
the result is the initiation of a dorsoanterior-specific program of gene
expression (Rowning et al.,
1997) via the localized suppression of ß-catenin degradation
(Miller et al., 1999). With
the appearance of the DV axis, anteroposterior and mediolateral dimensions
emerge as well; thus, the direction of vegetal-cortical rotation defines the
embryonic plane of bilateral symmetry. Externally, the embryo remains
bilaterally symmetric, but internally, the various visceral organ systems
develop with consistent left-right (LR) asymmetries. The origins of these LR
asymmetries remain elusive.
Studies in several vertebrate systems indicate that latter steps in the
development of LR asymmetric patterns follow well-conserved genetic pathways
(Kramer and Yost, 2002;
Levin, 2004). Left-sided
nodal expression in the early neurula stage is the earliest conserved
transcriptional event thus far recognized, and in many organisms
nodal induction requires left-directed ciliary flow in the node, the
floorplate or Kupffer's vesicle (Essner et
al., 2005). The case of Xenopus may be somewhat unusual,
in that asymmetries across the incipient embryonic midline develop prior to
the onset of zygotic gene expression, and clearly precede the appearance of
any cilia. For example, an asymmetry in Vg1-signaling, important for normal LR
patterning, is established by the 16-cell stage
(Hyatt and Yost, 1998). The
possibility that the vegetal-cortical microtubule array is biased to generate
such LR differences across the incipient midline during the first cell-cycle
rotation was raised some time ago (Yost,
1991), but no consistent structural asymmetry has ever been
reported. Assuming that the orientation of the microtubule array is directly
related to that of the DV axis, it is difficult to understand how left and
right halves could utilize this presumably symmetrical geometry to achieve
material differences during the pre-cleavage period. However, maternal
products involved with LR symmetrization, such as 14-3-3E
(Bunney et al., 2003) and
H+-V-ATPase (Adams et al.,
2006), become localized asymmetrically during early cleavage,
indicating that a mechanism to localize maternal determinants must operate far
upstream in the LR-specification pathway.
In this report, we address the possibility that the maternal cytoskeleton provides an unambiguous directional cue that serves to redistribute maternal components of the LR pathway unidirectionally along the mediolateral dimension during or shortly after the establishment of bilateral symmetry. Because the mediolateral dimension is not fixed until after fertilization, this directional capacity must initially be distributed uniformly around the animal-vegetal axis. When we used BDM to interfere with cytokinesis in cleaving Xenopus embryos, we noticed that an unusual torsion occurred between separating blastomeres, with the animal pole of each cell rotating several tens of degrees counterclockwise relative to the other, yielding a consistently chiral cleavage pattern. Investigating this effect further, we found that BDM induces shear in a broad belt around the equatorial cortex of the embryo. In parthenogenetically activated eggs this shear resulted in a dramatic, consistently counterclockwise torsion between animal and vegetal hemispheres about the animal-vegetal axis. Contemporaneous with this torsion, a broad continuous array of actin fibers assembled in the equatorial cortex, with contractile fibers paralleling the equatorial plane. Microtubules were not required to modulate this reorganization of the cortex, as torsion and development of the microfilament array continued unabated following nocodazole treatment. Finally, disruption of the actin cortex with BDM during the first cell cycle yielded tadpoles with a high frequency of reversal in cardiac and visceral LR orientation. These results demonstrate the existence of an unambiguously chiral polarization of the maternally derived cortical actin. The geometry of this polarization indicates an oogenetically based mechanism to localize determinants asymmetrically across the incipient embryonic midline, and therefore constitutes one of the farthest-upstream elements yet described in the vertebrate LR-specification pathway.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
some experiments, embryos were devitellinated manually in dishes coated with
2% type V agarose. For parthenogenetic activation, spawned eggs or in
vitro-matured oocytes were maintained at 18°C in full-strength MMR for up
to 3 hours before stimulation via a 3-second pulse of 3 volts, 800 mA DC.
Activated eggs produced contractions of the animal cap about 7-10 minutes
post-activation, and `pre-cleavage' surface contraction waves at about 70-80
minutes post-activation, i.e. with the same timing as fertilized sibling
eggs.
Timing of early embryonic events are normalized to the duration of the
90 minute first cell cycle, with 0.0 NT (normalized time) denoting
fertilization and 1.0 NT the onset of first cleavage.
GFP-actin labeling of oocytes
A GFP-actin sequence (Clontech) was inserted into the pCS107 vector, and
transcribed and capped in vitro via mMessage mMachine (Ambion). Stage VI
oocytes, harvested manually from adult Xenopus ovary, were injected
with GFP-actin mRNA and cultured overnight in oocyte culture medium (50%
Leibovitz L-15 containing glutamine, 15 mmol/l HEPES, pH 7.8, 1 µg/ml
insulin, 50 units/ml nystatin, 100 µg/ml gentamycin and 100 units/ml
penicillin/streptomycin). Oocytes expressing GFP-actin were subsequently
matured by overnight exposure to 2 µg/ml progesterone, and then bathed in
oocyte culture medium until activated electrically.
Drug treatments
Dejellied embryos were transferred into culture dishes containing 20 mmol/l
BDM (Sigma) in MMR/3 at specified times following fertilization or
parthenogenetic activation. For some experiments, embryos were transferred to
dishes containing BDM and nocodazole (10 µg/ml), cytochalasin B (10
µg/ml), jasplakinolide (0.4 µg/ml) or latrunculin B (0.1 µg/ml) with
up to 0.25% DMSO as vehicle. These concentrations had previously been
determined to disrupt the contractile ring (not shown). Control experiments
(not shown) indicated no effects of DMSO alone on cleavage, surface
contractility, embryo viability or frequency of LR patterning defects.
Histochemical procedures
Fixation and staining protocols for microfilaments and microtubules were
modified from previously published methods
(Danilchik et al., 2003;
Roeder and Gard, 1994).
Embryos and activated eggs were fixed at various stages in BRB (1 mmol/l
MgCl2, 5 mmol/l EGTA, 80 mmol/l K-PIPES, pH 6.8) containing 3.7%
paraformaldehyde, 0.25% glutaraldehyde and 0.2% Triton X-100 for 2 hours to
overnight at 4°C. Specimens were then rinsed for several hours in three to
four changes of NTBS (155 mmol/l NaCl, 10 mmol/l Tris-Cl, 0.1% NP-40, pH 7.4),
and then devitellinated as needed. Specimens were stained overnight at 4°C
with rhodamine phalloidin or Alexa 546 phalloidin (Molecular Probes, Inc.),
rinsed for several hours with several changes of NTBS, and placed in shallow
depression slides, in NTBS, under coverslips for viewing via confocal
microscopy. For microtubule staining, specimens were incubated with
anti-
ß-tubulin (Biogenesis) followed by Alexa-conjugated secondary
antibodies as previously described
(Danilchik et al., 2003).
Microscopy and time-lapse recording
To observe furrow morphology, still images were taken of cleaving embryos
fixed briefly in Bouin's fixative and rinsed in alkaline 50% ethanol. Stills
and time-lapse sequences (12 seconds/frame) of live embryos and activated eggs
were recorded with an Optronics Microfire (TM) camera mounted on an Olympus
SZH stereoscope. Side views were obtained by recording through a small
mirrored 45° prism (Melles Griot) placed beside upright embryos or
activated eggs. Time-lapse sequences were converted to Quicktime (Apple) with
JPEG compression. Upright confocal imaging of live and fixed specimens was via
a BioRad Radiance 2100 on a Nikon E800 microscope. Confocal time-lapse
sequences were captured at 6 seconds/frame. For analysis, sequences were
converted to TIFF stacks using ImageJ
(http://rsb.info.nih.gov/ij/).
Assay for cardiac left-right orientation
Tadpoles were raised to stage 45-47
(Nieuwkoop and Faber, 1994),
anesthetized with MS-222 (0.01%) and scored visually for direction of heart
looping. As noted by others (Bunney et al.,
2003), reversal of looping of gut and other visceral organs
frequently accompanies that of heart. For this study, however, scoring was
confined to cardiac looping itself.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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trail of pigment behind the twisting animal cortex of
each blastomere. Interestingly, among hundreds of embryos examined, from
dozens of similarly treated spawnings, we never encountered a corresponding
mirror-image (clockwise) motion. This drug response therefore reflects an
underlying chiral organization in the cytoskeleton of the zygote.
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BDM induces ectopic cortical microfilament bundling
To investigate the cytoskeletal basis for the chiral torsion produced by
BDM treatment, we first examined the cortical microfilament distribution in
cleaving embryos at various times following drug exposure
(Fig. 3). Treatment early in
the first cell cycle (<0.4 NT) sometimes produced chaotic and highly
dynamic cortical blebbing, particularly in the animal hemisphere
(Fig. 3A). In many cases, the
blebbing became so vigorous that the surface ripped open. Fluorescent
phalloidin staining revealed a complete reorganization of the normally
amorphous cortical f-actin into long microfilaments arranged in broad,
sometimes branching, swaths of parallel actin fibers around individual blebs
(Fig. 3A'). Exposure to
BDM at the onset of cleavage produced an enhanced offset of the apical stress
folds (arrowheads in Fig. 3B),
indicating the counterclockwise torsion between blastomeres described above.
Thick, ramified microfilament bundles fanned out ectopically as extensions of
the leading tips of the contractile ring
(Fig. 3B', arrows).
Despite this large-scale ectopic remodeling of the cortical actomyosin, the
contractile ring itself developed and advanced more or less normally, and was
evidently capable of sufficient contractility to initiate furrowing in treated
embryos. In summary, BDM treatment appears to provoke or enhance an alignment
of ectopic, contractile cortical actin fibers, but not to interfere directly
with contractile ring assembly or its function.
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). To
learn whether BDM-induced chirality exists in the absence of a mitotic
spindle, we parthenogenetically activated dejellied Xenopus eggs in
the presence of BDM. Eggs activated by electrical or osmotic shock initiate
cdc2/cyclin-dependent cytoplasmic and DNA-replication cycles
(Perez-Mongiovi et al., 1998;
Rankin and Kirschner, 1997),
raise a fertilization envelope and undergo nearly normal, albeit delayed,
cortical surface contraction waves, but ultimately fail to cleave because they
lack a mitotic spindle. BDM induces activated eggs to undergo a rapid,
invariably counterclockwise torsion, and produce a striking spiral pattern of
pigmentation in the animal hemisphere (Fig.
4; see also Movie S2 in the supplementary material). Thus, the
chiral torsion of the egg cortex does not require presence of the sperm
centrosome or mitotic spindle.
BDM-induced torsion does not require microtubules
Although parthenogenetically activated eggs lack a sperm centrosome, they
still generate a microtubule monaster and a vegetal-cortical microtubule
array, and thus microtubule ends could still be interacting with, and
potentially influencing, the cortex during BDM treatment. Because microtubules
are known to modulate the flow of cortical actomyosin of Xenopus
oocytes treated with phorbol ester (Benink
et al., 2000; Canman and
Bement, 1997), we tested whether the BDM-induced torsion could
develop in the continuous presence of nocodazole, a microtubule-depolymerizing
agent. As shown in Fig. 5,
microtubules are not required for this torsional activity: a steady
counterclockwise rotation of the animal caps continued in
BDM/nocodazole-treated eggs for at least 4 hours, through several cell-cycle
equivalents. Thus, the chiral response of the cortex to BDM does not require
recent contact with microtubules. To test whether microtubules present before
activation might have been important for cueing the post-activation BDM
response, we pretreated eggs for several hours with nocodazole. After
activation in the presence of BDM, pretreated eggs underwent essentially the
same chiral torsion as described above (not shown). In summary, the chiral
responsiveness of the cortex of the egg does not depend on the presence of
microtubules at any time before activation or during the first cell cycle, and
therefore seems to be an intrinsic property of cortical actin itself.
Parallel bundles of cortical actin assemble in the equatorial shear zone
To understand the cytoskeletal basis of the animal hemisphere rotation, we
used a mirrored 45° prism to obtain side views of the equatorial region of
living eggs treated with BDM. Analysis of video time-lapse recordings
indicated that the surfaces of both animal and vegetal hemispheres undergo
cortical rearrangements, but a zone of particularly extensive shear develops
between the two hemispheres in a 300 µm-wide belt below the equatorial
pigment boundary. To illustrate the extent and rate of this shear, individual
pigment granules initially spaced approximately 100 µm apart along an
arbitrary animal-vegetal meridian served as surface marks that were tracked
for 20 minutes in a representative side-view time-lapse movie (see Movie S3 in
the supplementary material). As shown in
Fig. 6, the upper part of the
animal hemisphere underwent a relatively small amount of localized deformation
as it rotated atop the vegetal hemisphere. The three adjacent marks above the
equator (marks 1-3) traveled at about the same speed and therefore remained in
relatively close proximity, indicating that the circumpolar animal hemisphere
surface rotated more or less as a coherent unit. By contrast, marks below the
pigmentation boundary (marks 4-6) underwent a much larger amount of relative
motion, indicating that a broad shear zone develops in the equatorial zone.
Considerable relative translocation of surface marks occurred across this
zone. For example, marks 3 and 5, initially apart vertically only 210 µm
across the shear zone, became separated laterally by more than 615 µm in
the 20-minute period - a horizontal displacement rate of at least 30
µm/minute. Nearer the vegetal pole, horizontal movement was somewhat
slower: marks 4 and 5 separated laterally from each other at about 16
µm/minute; marks 5 and 6 separated at about 6 µm/minute. Thus, relative
rates of horizontal displacement varied several fold along the animal-vegetal
meridian, with the fastest rates of shear at the equator.
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To observe the behavior of this microfilament array in vivo, we injected
stage VI oocytes with capped mRNA encoding GFP fused to actin. After
incubating overnight, GFP-actin expressing oocytes were matured with
progesterone and then electrically activated as above in the presence of BDM
and nocodazole. These labeled oocytes underwent essentially the same
large-scale counterclockwise torsion as activated eggs, with shearing across a
broad equatorial zone as described above. Large numbers of discrete,
actin-containing microfilament bundles aligned into broad arrays paralleling
the shear (Fig. 8A). Individual
fibers within the array had an apparent thickness of about 0.5 µm and they
ranged in length from about 4 µm to at least 45 µm
(Fig. 8B). Much longer fibers
were within the array, but because the surface of the live egg continually
undulated, individual fibers could not be traced for long distances in any
single confocal section. Analysis of representative transects across the array
indicates relatively uniform spacing (0.90±0.08 µm) of individual
filament bundles (Fig. 8C),
similar to previous measurements of microfilaments in the contractile bands of
Xenopus cleavage furrows (Noguchi
and Mabuchi, 2001). We analyzed motions of individual fibers in
confocal time-lapse movies and measured a nearly uniform sliding of individual
fibers past each other at an average rate of 0.03 µm/minute (see Fig. S1
and Movie S4 in the supplementary material). Importantly, no fibers slid in
the opposite direction. Thus, the rapid counterclockwise rotation of the
animal cap surface seen macroscopically results from the cumulative action of
incremental and unidirectional sliding of adjacent microfilament bundles in
the equatorial belt.
BDM-induced torsion requires f-actin, but not ongoing actin assembly
The contractile band of Xenopus zygotes develops both by
recruitment of cortical actin and new polymerization in situ at the growing
furrow tips (Noguchi and Mabuchi,
2001). As a preliminary test of whether the equatorial
microfilament array in BDM-treated embryos develops via new assembly or
recruitment from preexisting cortical f-actin, we exposed activated eggs
undergoing torsion to various microfilament inhibitors. At doses previously
determined to be effective at halting cytokinesis, latrunculin B (1 µg/ml)
essentially abolished all cortical movements, confirming that the torsion is
indeed dependent on the presence of microfilaments. However, cytochalasin B
(10 µg/ml) and jasplakinolide (0.4 µg/ml), agents that respectively
block and stabilize actin assembly, had no effect on torsion, indicating that
the equatorial actin array develops largely via reorganization of preexisting
cortical microfilaments.
Actin cortex and left-right axis specification
The consistent directionality of the BDM-induced equatorial microfilament
belt indicates the presence of an endogenous circumferential polarization
within the cortex of the egg. Whatever the physical nature of the initial
polarization, its orientation suggests a potential actin-dependent mechanism
for breaking the mirror-image symmetry of the zygote during the first cell
cycle. We hypothesized that this chiral polarization reflects a transport
mechanism used by the zygote or early-cleavage embryo to localize components
of a LR axis-specifying pathway. If this hypothesis is correct, then
disrupting the actin cytoskeleton early in development should result in LR
patterning defects. Supporting this idea, it has already been demonstrated
that exposing Xenopus embryos to sublethal doses of latrunculin B for
several hours during cleavage stages results in randomized cardiac and gut LR
patterning (Qiu et al.,
2005).
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; Scharf et al.,
1989), we suspected that the cortical rearrangements induced by
early BDM treatment might directly perturb the vegetal-cortical microtubule
array. Comparison of the cortical microtubules in untreated and BDM-treated
zygotes (Fig. 10B,C,
respectively) confirmed this idea: in early-treated embryos, the microtubule
array sometimes became radialized, with microtubule bundles sweeping up past
the equator along multiple meridians. This finding suggests interaction
between the cortical actin and the highly labile microtubule array during its
initial growth in the first cell cycle. The brief, early BDM treatments that randomized cardiac and gut orientation were not of sufficient duration to produce equatorial torsion described above, but clearly were sufficient to redirect normal LR patterning. To observe the actual extent of cortical actin reorganization induced by brief, heart-randomizing treatments, embryos were fixed during the course of 20 minutes exposure to BDM (Fig. 11A1-A3), and then stained with rhodamine-phalloidin for whole-mount confocal microscopy. Cortical contraction first became noticeable about 10 minutes after exposure to BDM, with the appearance of a distinctive horizontal constriction or sulcus at the equatorial pigment boundary (Fig. 11A2,A3, arrows). Interestingly, in all cases observed (n>30 from three separate spawnings), the sulcus first appeared on the side opposite to the sperm entry point (SEP), i.e. prospective dorsal marginal zone. Confocal microscopy revealed that this dorsal sulcus is enriched with microfilaments (Fig. 11B), arranged linearly and running parallel to the equator (Fig. 11C). Thus, in contrast to parthenogenetically activated eggs that produce a uniformly circumferential microfilament array at the equator, the zygotes develop a microfilament array only on the prospective dorsal side.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Any maternal component involved with an
upstream step in LR patterning would have to have an initially uniform
distribution around the animal-vegetal axis, ready to undergo a useful (i.e.
consistently orthogonal to the midsagittal plane) localization only during or
shortly after DV specification. It has been hypothesized
(Yost, 1991) that the
vegetal-cortical microtubule array could accomplish this orthogonal bias, if
the orientation of the microtubule deviated from the midsagittal plane.
However, years of study by numerous investigators have revealed no consistent
structural asymmetries in the vegetal-cortical array
(Houliston and Elinson, 1991;
Larabell et al., 1996;
Marrari et al., 2003;
Vincent et al., 1987) or of
the locomotion of particles along it
(Miller et al., 1999).
Nevertheless, early asymmetries important in LR patterning do develop during
prezygotic development. For example, vegetally localized maternal proteins and
mRNAs enrich asymmetrically across the embryonic midline during early cleavage
stage (Adams et al., 2006;
Bunney et al., 2003). By the
four-cell stage, blastomeres are already asymmetric with respect to serotonin
signaling (Fukumoto et al.,
2005). Interestingly, these localizations are sensitive to
sublethal, chronic doses of the microfilament-depolymerizing drug latrunculin
B.
There are a number of ways in which BDM might affect actin-dependent motion
in the cortex of the early embryo. BDM reportedly functions as a low-affinity
myosin ATPase inhibitor (Cramer and
Mitchison, 1995), but in some situations may also act by
interfering with unspecified non-ATPase functions of nonmuscle myosin II
(Forer and Fabian, 2005). These
potential myosindisrupting effects are difficult to reconcile with the
observed unidirectional sliding of microfilaments past each other through the
cortex. Moreover, eggs exposed to a number of higher-affinity myosin
inhibitors, including blebbistatin and ML7, did not exhibit any effects
similar to that of BDM (M.V.D. and E.E.B., unpublished). Thus, it is worth
considering the other known pharmacological effects of BDM on the
cytoskeleton, including its inhibition of retrograde actin flow in
lamellipodia (Lin et al.,
1996; Zhou et al.,
2002), reflecting direct interference with the assembly kinetics
of actin (Valentijn et al.,
2000). Alternatively, a recent study suggests that BDM blocks
lamellipodial protrusion not by interfering with assembly kinetics per se, but
rather by delocalizing actin-assembly components, principally the Arp2/3
complex, from the plasma membrane (Yarrow
et al., 2003). A delocalization of actin assembly components would
be consistent with the abundant ectopic microfilaments seen throughout the
cortex of BDM-treated eggs, but as shown by our cytochalasin and
jasplakinolide experiments, new assembly is not required to sustain the
torsion once it has begun. Structural and functional similarity between the
ectopic microfilament bands and the contractile ring suggest that BDM may be
activating a rhoA-dependent pathway.
We do not yet understand the basis for the consistent directionality of the torsion elicited by BDM. One explanation would be that a cryptic chiral organization already exists on a large scale in the Xenopus egg cortical actin cytoskeleton by the time of fertilization, and that BDM simply reinforces it - e.g. by provoking polarized ectopic bundling onto preexisting, already oriented, microfilaments. Of course, this relegates the organizational problem back to oogenesis. Alternatively, the torsion may depend on chirality at a much smaller scale: the helicity of the microfilaments themselves. In this model, the ectopic bundling of cortical actin initiated by BDM would be chirally organized on a molecular level, incrementally conferring larger-scale chirality to the cortex as a whole.
There are no known cytoskeletal structures or organizations in Xenopus embryos that presage the surprising chiral response of the cortex to BDM. All previously described cortical motions of the early embryo organize around surface singularities, such as the animal pole or the sperm entry point, or symmetrical structures such as the mitotic spindle or vegetal cortical microtubule array. In general, they reflect preexisting maternal organization (the animal-vegetal axis of the oocyte) or later directional cues (the sperm centrosome and mitotic spindle). For example, the animal pole (site of meiotic cleavages) serves as a focus for large-scale, concentric surface contraction waves preceding first cell division. Subsequently, the fertilizing sperm centrosome provides a symmetry-breaking directional cue, culminating in the assembly of the cortical microtubule array perpendicular to the animal-vegetal axis that directs cytoplasmic motions important for DV axis specification. The unusual chiral behavior of the embryo cortex following BDM treatment reflects a topologically different arrangement: one that is organized circumferentially around the animal-vegetal axis. Whatever BDM may be doing at the biochemical level to elicit this response, it is important to recognize that the chiral behavior itself represents an endogenous vectorial cue in the cortical cytoskeleton, one that is potentially used by the pre-cleavage embryo for LR symmetrization. Thus, the initial left- or right-specific accumulation of critical maternal determinants might be accomplished by unidirectional tracking around the equator, and this large-scale handedness could function no matter where the sperm enters and the DV axis develops.
There are at least two ways in which circumferentially polarized cortical
actin (or membrane-tethered microfilament nucleation sites) could function in
symmetry breaking. One involves vesicle transport along microfilaments. Recent
work in Drosophila implicates Myo1A, an unconventional myosin, in LR
asymmetric gut looping (Hozumi et al.,
2006). Although the suggested role for this myosin in
Drosophila is in actin-dependent vesicle transport during a late
stage of gut morphogenesis, a similar function could be served by
circumferentially polarized cortical actin in Xenopus eggs. In this
scenario, vesicles bearing components of a left- or right-determining pathway
would shift unidirectionally around the equator. As we have shown, cortical
actin becomes enriched at the dorsal marginal zone near the end of the first
cell cycle. Thus, vesicles arriving at the marginal zone near the end of the
first cell cycle (e.g. accompanying similar vesicles involved with dorsal
specification) would have an opportunity to interact with microfilaments that,
as we have shown, are capable of organizing horizontally into polarized
arrays.
A second actin-dependent symmetry-breaking scenario in Xenopus
embryogenesis is described in Fig.
12. In this model, actomyosin would produce a shear between deep
and surface components about the animal-vegetal axis. This shear could be
relatively minor, and yet accomplish a significant asymmetry across the
embryonic midline before cleavage. In the cartoon, maternal dorsal-specifying
components (red dots), such as GSK-3 binding protein
(Weaver et al., 2003) or xwnt
11 mRNA (Tao et al., 2005),
are located in the vegetal cortex. Deeper in the cytoplasm, perhaps embedded
in the yolk mass, are maternal elements (blue) involved with LR specification.
These determinants could include, for example, H+-V-ATPase proteins
(Adams et al., 2006) or some
component of Vg1 processing or signaling
(Hyatt and Yost, 1998).
Initially, the unfertilized egg has cylindrical symmetry about its
animal-vegetal axis (Fig.
12A). Fertilization causes a microtubule-dependent shift of dorsal
determinants toward the dorsal marginal zone
(Schroeder et al., 1999;
Yost et al., 1998), while the
deeper determinants shift with the yolk mass toward the sperm entry point; the
relative displacement of deep and shallow components defines the plane of
bilateral symmetry (Fig. 12B).
We suggest that a slight equatorial shift of the shallow components relative
to those deeper in the cytoplasm would be sufficient to produce a significant
desymmetrization across the embryonic midline
(Fig. 12C).
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/22/4517/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adams, D. S., Robinson, K. R., Fukumoto, T., Yuan, S.,
Albertson, R. C., Yelick, P., Kuo, L., McSweeney, M. and Levin, M.
(2006). Early, H+-V-ATPase-dependent proton flux is necessary for
consistent left-right patterning of nonmammalian vertebrates.
Development 133,1657
-1671.
Benink, H. A., Mandato, C. A. and Bement, W. M.
(2000). Analysis of cortical flow models in vivo. Mol.
Biol. Cell 11,2553
-2563.
Bunney, T. D., De Boer, A. H. and Levin, M.
(2003). Fusicoccin signaling reveals 14-3-3 protein function as a
novel step in left-right patterning during amphibian embryogenesis.
Development 130,4847
-4858.
Canman, J. C. and Bement, W. M. (1997). Microtubules suppress actomyosinbased cortical flow in Xenopus oocytes. J. Cell Sci. 110,1907 -1917.[Abstract]
Cramer, L. P. and Mitchison, T. J. (1995).
Myosin is involved in postmitotic cell spreading. J. Cell
Biol. 131,179
-189.
Danilchik, M. V., Bedrick, S. D., Brown, E. E. and Ray, K.
(2003). Furrow microtubules and localized exocytosis in cleaving
Xenopus laevis embryos. J. Cell Sci.
116,273
-283.
Essner, J. J., Amack, J. D., Nyholm, M. K., Harris, E. B. and
Yost, H. J. (2005). Kupffer's vesicle is a ciliated organ of
asymmetry in the zebrafish embryo that initiates left-right development of the
brain, heart and gut. Development
132,1247
-1260.
Forer, A. and Fabian, L. (2005). Does 2,3-butanedione monoxime inhibit nonmuscle myosin? Protoplasma 225,1 -4.[CrossRef][Medline]
Fukumoto, T., Kema, I. P. and Levin, M. (2005). Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos. Curr. Biol. 15,794 -803.[CrossRef][Medline]
Houliston, E. and Elinson, R. P. (1991). Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs. Development 112,107 -117.[Abstract]
Hozumi, S., Maeda, R., Taniguchi, K., Kanai, M., Shirakabe, S., Sasamura, T., Speder, P., Noselli, S., Aigaki, T., Murakami, R. et al. (2006). An unconventional myosin in Drosophila reverses the default handedness in visceral organs. Nature 440,798 -802.[CrossRef][Medline]
Hyatt, B. A. and Yost, H. J. (1998). The left-right coordinator: the role of Vg1 in organizing left-right axis formation. Cell 93,37 -46.[CrossRef][Medline]
Kao, K. R. and Elinson, R. P. (1988). The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos. Dev. Biol. 127, 64-77.[CrossRef][Medline]
Kloc, M. and Etkin, L. D. (1995). Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development 121,287 -297.[Abstract]
Klymkowsky, M. W., Maynell, L. A. and Nislow, C.
(1991). Cytokeratin phosphorylation, cytokeratin filament
severing and the solubilization of the maternal mRNA Vg1. J. Cell
Biol. 114,787
-797.
Kramer, K. L. and Yost, H. J. (2002). Cardiac left-right development: are the early steps conserved? Cold Spring Harb. Symp. Quant. Biol. 67,37 -43.[CrossRef][Medline]
Larabell, C. A., Rowning, B. A., Wells, J., Wu, M. and Gerhart, J. C. (1996). Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation. Development 122,1281 -1289.[Abstract]
Levin, M. (2004). The embryonic origins of
left-right asymmetry. Crit. Rev. Oral Biol. Med.
15,197
-206.
Lin, C. H., Espreafico, E. M., Mooseker, M. S. and Forscher, P. (1996). Myosin drives retrograde F-actin flow in neuronal growth cones. Neuron 16,769 -782.[CrossRef][Medline]
Manes, M. E. and Elinson, R. P. (1980). Ultraviolet light inhibits gray crescent formation in the frog egg. Wilhelm Roux Arch. Dev. Biol. 189, 73-77.
Marrari, Y., Clarke, E. J., Rouviere, C. and Houliston, E. (2003). Analysis of microtubule movement on isolated Xenopus egg cortices provides evidence that the cortical rotation involves dynein as well as Kinesin Related Proteins and is regulated by local microtubule polymerisation. Dev. Biol. 257, 55-70.[CrossRef][Medline]
Miller, J. R., Rowning, B. A., Larabell, C. A., Yang-Snyder, J.
A., Bates, R. L. and Moon, R. T. (1999). Establishment of the
dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of
dishevelled that is dependent on cortical rotation. J. Cell
Biol. 146,427
-437.
Nieuwkoop, P. D. and Faber, J. (1994). Normal Table of Xenopus laevis (Daudin). New York: Garland Publishing.
Noguchi, T. and Mabuchi, I. (2001). Reorganization of actin cytoskeleton at the growing end of the cleavage furrow of Xenopus egg during cytokinesis. J. Cell Sci. 114,401 -412.[Abstract]
Perez-Mongiovi, D., Chang, P. and Houliston, E. (1998). A propagated wave of MPF activation accompanies surface contraction waves at first mitosis in Xenopus. J. Cell Sci. 111,385 -393.[Abstract]
Pondel, M. D. and King, M. L. (1988). Localized
maternal mRNA related to transforming growth factor beta mRNA is concentrated
in a cytokeratin-enriched fraction from Xenopus oocytes. Proc.
Natl. Acad. Sci. USA 85,7612
-7616.
Qiu, D., Cheng, S. M., Wozniak, L., McSweeney, M., Perrone, E. and Levin, M. (2005). Localization and loss-of-function implicates ciliary proteins in early, cytoplasmic roles in left-right asymmetry. Dev. Dyn. 234,176 -189.[CrossRef][Medline]
Rankin, S. and Kirschner, M. W. (1997). The surface contraction waves of Xenopus eggs reflect the metachronous cell-cycle state of the cytoplasm. Curr. Biol. 7, 451-454.[CrossRef][Medline]
Roeder, A. D. and Gard, D. L. (1994). Confocal microscopy of F-actin distribution in Xenopus oocytes. Zygote 2,111 -124.[Medline]
Rowning, B. A., Wells, J., Wu, M., Gerhart, J. C., Moon, R. T.
and Larabell, C. A. (1997). Microtubule-mediated transport of
organelles and localization of beta-catenin to the future dorsal side of
Xenopus eggs. Proc. Natl. Acad. Sci. USA
94,1224
-1229.
Scharf, S. R., Rowning, B., Wu, M. and Gerhart, J. C. (1989). Hyperdorsoanterior embryos from Xenopus eggs treated with D2O. Dev. Biol. 134,175 -188.[CrossRef][Medline]
Schroeder, K. E., Condic, M. L., Eisenberg, L. M. and Yost, H. J. (1999). Spatially regulated translation in embryos: asymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus. Dev. Biol. 214,288 -297.[CrossRef][Medline]
Shibazaki, Y., Shimizu, M. and Kuroda, R. (2004). Body handedness is directed by genetically determined cytoskeletal dynamics in the early embryo. Curr. Biol. 14,1462 -1467.[CrossRef][Medline]
Tao, Q., Yokota, C., Puck, H., Kofron, M., Birsoy, B., Yan, D., Asashima, M., Wylie, C. C., Lin, X. and Heasman, J. (2005). Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120,857 -871.[CrossRef][Medline]
Valentijn, J. A., Valentijn, K., Pastore, L. M. and Jamieson, J.
D. (2000). Actin coating of secretory granules during
regulated exocytosis correlates with the release of rab3D. Proc.
Natl. Acad. Sci. USA 97,1091
-1095.
Vincent, J. P., Scharf, S. R. and Gerhart, J. C. (1987). Subcortical rotation in Xenopus eggs: a preliminary study of its mechanochemical basis. Cell Motil. Cytoskeleton 8, 143-154.[CrossRef][Medline]
Weaver, C. and Kimelman, D. (2004). Move it or
lose it: axis specification in Xenopus. Development
131,3491
-3499.
Weaver, C., Farr, G. H., 3rd, Pan, W., Rowning, B. A., Wang, J.,
Mao, J., Wu, D., Li, L., Larabell, C. A. and Kimelman, D.
(2003). GBP binds kinesin light chain and translocates during
cortical rotation in Xenopus eggs. Development
130,5425
-5436.
Yarrow, J. C., Lechler, T., Li, R. and Mitchison, T. J. (2003). Rapid delocalization of actin leading edge components with BDM treatment. BMC Cell Biol. 4, 5.[CrossRef][Medline]
Yisraeli, J. K., Sokol, S. and Melton, D. A. (1990). A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development 108,289 -298.[Abstract]
Yost, C., Farr, G. H., 3rd, Pierce, S. B., Ferkey, D. M., Chen, M. M. and Kimelman, D. (1998). GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis. Cell 93,1031 -1041.[CrossRef][Medline]
Yost, H. J. (1991). Development of the left-right axis in amphibians. Ciba Found. Symp. 162, 165-176; discussion 176-181.[Medline]
Zhou, F. Q., Waterman-Storer, C. M. and Cohan, C. S.
(2002). Focal loss of actin bundles causes microtubule
redistribution and growth cone turning. J. Cell Biol.
157,839
-849.
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