1 Department of Biochemistry and Molecular Biology, University of Chicago, 924
East 57th Street, Chicago, IL 60637, USA
2 Department of Molecular Genetics and Cell Biology, Center for Molecular
Oncology, and Committees on Developmental Biology, Cancer Biology, and
Genetics, University of Chicago, 924 East 57th Street, Chicago, IL 60637,
USA
Author for correspondence (e-mail:
pmueller{at}midway.uchicago.edu)
Accepted 22 December 2003
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SUMMARY |
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Key words: Xenopus, Wee1, Wee2, Cdk, Cell cycle, Morphogenesis, Convergent extension, Somitogenesis
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Introduction |
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Convergent extension, or the simultaneous narrowing and lengthening of a
tissue, is one of the key developmental mechanisms that shapes many features
of embryos (Keller, 2002;
Wallingford et al., 2002
).
Early in Xenopus embryogenesis, gastrula-stage convergent extension
assists in the involution of the axial and paraxial mesoderm over the dorsal
lip (Keller et al., 2000
).
This movement positions these mesodermal tissues underneath the presumptive
neural plate on the dorsal side of the embryo. Later during neurulation,
convergent extension causes the axial mesoderm, the paraxial mesoderm and the
neural plate to delineate from each other, thicken and elongate. As these
tissues elongate, the rest of the embryo elongates with them.
During convergent extension, individual cells within a specified group move
between each other in a single orientation (convergence) and this causes the
tissue encompassing the group of cells to elongate in an orientation
perpendicular to that of the convergence (extension)
(Keller, 2002;
Keller et al., 2000
;
Wallingford et al., 2002
). The
important point is that this is an integrated cell movement that requires the
coordinated action of all cells within the group or tissue. The planar cell
polarity (PCP) pathway is instrumental in coordinating this process in
vertebrates (Keller, 2002
;
Wallingford et al., 2002
). PCP
requires the action of specific Wnts acting through the disheveled protein in
a ß-catenin-independent manner. Numerous studies in Xenopus and
zebrafish have established that mutation or mis-expression of Wnts, Wnt
receptors or downstream effectors disrupts cell polarization, cell elongation
and cell adhesion (Myers et al.,
2002
; Sokol, 1996
;
Tada and Smith, 2000
;
Wallingford and Harland, 2001
;
Wallingford et al., 2000
).
Without these specific cell morphologies, convergent extension in the axial
mesoderm, paraxial mesoderm and neuronal tissue fails resulting in
foreshortened and mis-shapen embryos.
Concomitant with the onset of gastrulation and convergent extension, cells
of the dorsal mesoderm stop dividing in Xenopus
(Cooke, 1979;
Hardcastle and Papalopulu,
2000
; Saka and Smith,
2001
). This absence of proliferation continues into neurulation as
the dorsal mesoderm delineates into axial and paraxial mesoderm. The cells of
the axial mesoderm (notochord) are thought not to divide again, while the
cells of the paraxial mesoderm reenter the cell cycle, but only after a somite
has formed (Saka and Smith,
2001
). Interestingly, as long as a Xenopus embryo has
begun gastrulation, artificially blocking the cell cycle at the whole embryo
level has little effect on the continuation of gastrulation, neurulation or
somitogenesis (Anderson et al.,
1997
; Cooke, 1973
;
Harris and Hartenstein, 1991
).
Although these embryos eventually die, these key morphological events continue
unabated in the absence of cell proliferation.
Numerous mechanisms are used to inhibit progression through the cell cycle
(Morgan, 1997). Ultimately,
these function by regulating the activity of the cyclin dependent kinases
(Cdks). These mechanisms include the binding of Cdk inhibitory factors (CKIs)
and post-translational phosphorylation. In a developmental context, CKIs have
been shown to play important roles in terminal differentiation. For example,
the Xenopus CKI (p27Xic1) is required for differentiation of muscle
and neuronal tissues (Carruthers et al.,
2003
; Vernon et al.,
2003
; Vernon and Philpott,
2003
). Cell cycle progression can also be blocked through
inhibitory phosphorylation of the Cdks. This type of regulation is commonly
used to control entry into mitosis and is dependent on the balance of the
inhibitory Wee kinase activity and the activating Cdc25 phosphatase
activity.
Recently, we identified Wee2, a developmentally regulated member of the Wee
family of kinases (Leise and Mueller,
2002), in Xenopus. Wee2 is one of three Wee kinase family
members that have been found in all vertebrates examined, the others being
Wee1 and Myt1. Each of these kinases prevents cell cycle progression by
phosphorylating and thus inhibiting the activity of the Cdks. However, these
kinases differ from each other in their specific activities, subcellular
localization, and temporal and spatial patterns of expression during
development (Leise and Mueller,
2002
; Morgan,
1997
). Wee1 is exclusively expressed as a maternal gene product
and its mRNA and protein disappear during gastrulation and neurulation,
respectively. Myt1 has both maternal and zygotic expression. In the developing
embryo, Myt1 is predominantly expressed in neuronal tissues. Finally, Wee2 is
expressed principally in the zygote, reaching high levels of expression by the
end of gastrulation. Significantly, the Wee2 transcript is localized to the
paraxial mesoderm during gastrulation and neurulation, making Wee2 a possible
candidate for causing the low mitotic index in the paraxial mesoderm.
In this study, we have found that Wee2-mediated inhibition of the cell cycle is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopus embryo. Depletion of Wee2 causes an increase in the mitotic index of paraxial mesoderm and prevents convergent extension of this tissue during neurulation. Despite these defects, the early differentiation of the paraxial mesoderm into muscle remains normal. Importantly, the convergent extension defect can be rescued by replacing the depleted, endogenous Wee2 with microinjected Wee2 mRNA. Finally, other mechanisms that upset the balance of inhibitory Cdk phosphorylation and advance the cell cycle cause the same defects as Wee2 depletion. Together, our results suggest that active cell proliferation is incompatible with integrated tissue movements that are used during morphogenesis.
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Materials and methods |
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pCS2-5'UTR-Wee2-GFP was made by subcloning the first 865 bp
(including the 5'UTR) of Wee2 cDNA into pCS2+eGFP Bgl2 (gift of David Turner)
to make an in frame fusion of Wee2 and GFP. pXenGST-Wee2 was made by
subcloning the coding region of Wee2 into pXen1
(MacNicol et al., 1997) (gift
of Angus MacNicol). The coding regions of human CDK2
(Gu et al., 1992
) and CDK2 AF
(Thr14 change to Ala, Tyr15 change to Phe)
(Costanzo et al., 2000
) (gifts
from David Morgan and Jean Gautier, respectively) were amplified by PCR and
subcloned into pCS2+ (Turner and
Weintraub, 1994
) to create pCS2-HCdk2 and pCS2-HCdk2AF. The coding
regions of wild-type (WT) and phosphatase-dead (PD) versions of
Xenopus Cdc25A and Cdc25C (Kim et
al., 1999
) (gifts of James Maller) were amplified by PCR and
subcloned into pCS2+eGFP Bgl2 to create eGFP fusions. Subsequently, the
eGFP-Cdc25 fusions were subcloned into pCARGFP
(Kroll and Amaya, 1996
) (gift
of Kristen Kroll) replacing the GFP of the vector with that from pCS2+eGFP
Bgl2 to create pCARD-eGFP-Cdc25A WT, pCARD-eGFP-Cdc25A PD, pCARD-eGFP-Cdc25C
WT and pCARD-eGFP-Cdc25C PD. All amplified constructs were confirmed by
sequencing. Cdk2 WT, Cdk2 AF, eGFP, Wee2 and 5'UTR-Wee2-GFP capped mRNAs
were prepared from pCS2-HCdk2, pCS2-HCdk2AF, pCS2+eGFP Bgl2, pXenGST-Wee2 and
pCS2-5'UTR-Wee2-GFP as described
(Sive et al., 2000
).
Microinjection of antisense morpholino, mRNA or DNA into Xenopus embryos and dorsal explants
Microinjections were performed as described
(Leise and Mueller, 2002). In
all experiments, non-injected siblings were processed concurrently and exactly
as injected embryos. For all morpholino injections, morpholinos were
resuspended in injection buffer [0.1xMBS
(Sive et al., 2000
) modified
to contain twice as much HEPES] immediately before use at a morpholino
concentration of 2-4 ng/nl. For unilateral morpholino injections, 500 pg of
eGFP mRNA was added to the injection mix, to identify the injected side. For
the morpholino rescue experiment, the indicated amount of GST-Wee2 mRNA was
added to the injection mix. The mRNA and DNA injections were performed as
described (Leise and Mueller,
2002
). Dorsal explants were dissected from stage 12.5/13 embryos
and processed as described (Wilson et al.,
1989
) except that dissections and incubations were in Danilchik's
for Amy (DFA) buffer (Sater et al.,
1993
) and the endoderm was left attached to the explants.
Antibody production, endogenous protein isolation, immunoprecipitation, and western analysis
Recombinant, full-length Wee2 protein
(Leise and Mueller, 2002) was
used to immunize rabbits (Covance). The
Wee2 antibody was purified by
affinity chromatography against the first 222 amino acids of Wee2 as described
(Mueller et al., 1995a
). Total
protein was extracted from Xenopus embryos at the indicated stages of
development (Nieuwkoop and Faber,
1994
) in extraction buffer as described
(Hartley et al., 1996
).
Samples (100 µg) were either processed for western analysis directly, or
first subjected to Wee2 immunoprecipitation from stage specific supernatants
(1 mg) using
Wee2 antibodies and then processed for Western analysis as
described (Mueller et al.,
1995a
) using
Wee2,
PSTAIRE (
Cdk1/2) (Santa
Cruz) or
Myt1 (Mueller et al.,
1995b
).
Whole mount in situ hybridization, immunocytochemistry, sectioning, nuclei staining, and paraxial mesoderm volume determination
Whole-mount in situ hybridization was performed as described
(Leise and Mueller, 2002)
using probes specific for MyoD (Dosch et
al., 1997
) (gift of Christof Niehrs), XNot
(von Dassow et al., 1993
)
(gift of David Kimelman), Sox3 (Zygar et
al., 1998
) (gift of Sally Moody), muscle actin (MA)
(Mohun et al., 1988
) and
myosin heavy chain (MHC) (Radice and
Malacinski, 1989
) (gift of Anna Philpott), GFP and Wee2
(Leise and Mueller, 2002
).
Whole-mount immunocytochemistry against phospho-histone H3 (
PH3)
(Upstate Biotechnology) was performed as described
(Leise and Mueller, 2002
),
except that the pre-incubation was performed at 4°C for 1 hour and the
color reaction was performed at 4°C overnight. MyoD (20 µM), XNot (20
µM) and
PH3 (10 µM) stained embryos were subjected to paraffin
sectioning as described (Leise and
Mueller, 2002
; Sive et al.,
2000
). Composite images of
PH3 stained sections were
generated as described (Leise and Mueller,
2002
). SYTOX Green (Molecular Probes) nuclear staining was
performed on mounted MyoD and XNot sections as described
(Newman and Krieg, 2002
).
Nuclei were counted using a Zeiss AxioScope with a 20x objective. The
volume of the paraxial mesoderm was calculated in Photoshop by determining the
number of pixels contained in the MyoD-positive region of every section of
transversely and sagittally cut embryos, and then summing the number of pixels
of all sections.
RNA isolation and RT-PCR
Total RNA was extracted from Xenopus embryos in groups of 10
embryos using the RNAeasy RNA Mini isolation kit (Qiagen) as per the
manufacturer's recommendations. cDNA was prepared using an oligo dT primer as
described (Leise and Mueller,
2002). RT-PCR was performed as described
(Steinbach and Rupp, 1999
)
using the cycling conditions described
(http://www.hhmi.ucla.edu/derobertis/index.html).
The linear range for each primer was empirically determined (25 cycles for MA
and ODC, 30 cycles for MyoD, XNot, Vent1 and MHC), and the lack of DNA
contamination was confirmed by RT reactions. Primers used were MHC
(Vernon and Philpott, 2003
),
MA (Stutz and Spohr, 1986
),
MyoD (Hopwood et al., 1989
),
ODC (Agius et al., 2000
), XNot
(Gont et al., 1993
) and Vent1
(Gawantka et al., 1995
).
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Results |
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To test whether Wee2 might be the direct cause of the low mitotic index
found in the paraxial mesoderm, we used morpholino based antisense technology
to reduce the level of Wee2 protein. Morpholino oligonucleotides inhibit the
translation of specific targets by blocking access of the translational
machinery to the 5'UTR of a mRNA target transcript with high specificity
and low toxicity (Heasman,
2002). We designed two antisense morpholinos (W2MO.1 and W2MO.2)
to the 5'UTR of Wee2. We first tested these in an in vitro translation
assay using a Wee2 5'UTR-GFP fusion construct as the transcript. Unlike
the control morpholino (CMO), both W2MO.1 and W2MO.2 reduced the translation
of this mRNA (Fig. 2A). In this
assay, W2MO.1 was more efficient than W2MO.2. We next asked whether the
morpholinos could reduce the accumulation of Wee2 protein in vivo. Embryos
were either left untreated (sibling) or treated with the various morpholinos
at the two-cell stage by microinjection of both blastomeres. These embryos
were allowed to develop until the controls had reached either stage 13 or
stage 18, and were then processed for western analysis. Both of the Wee2
targeted morpholinos reduced the accumulation of Wee2 protein at stage 13
(data not shown) and at stage 18 (Fig.
2B). As with the in vitro assay, W2MO.1 reduced translation more
efficiently than W2MO.2. The reduction of the endogenous level of Wee2 protein
was dependent on the dose of Wee2 morpholino
(Fig. 2C). At the highest level
of the more efficient morpholino, W2MO.1, we observed an almost complete
abrogation of Wee2 protein accumulation in late neurulating (stage 18)
embryos. At earlier times in development (stage 13), half as much morpholino
could be used to obtain the same level of Wee2 protein reduction (data not
shown). Importantly, the Wee2-directed morpholinos reduced the level of
endogenous Wee2 protein specifically as they had no effect on a related
protein, Myt1 (Fig. 2B,C) or an
unrelated protein, p90 Rsk (data not shown). In all cases, the highest dose of
control morpholino (CMO) had no effect.
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Knockdown of Wee2 disrupts embryo elongation, somite formation and convergent extension
We next investigated the developmental consequences of depleting Wee2.
Embryos were microinjected with W2MO.1, W2MO.2 or CMO, or left non-injected
(sibling). These were allowed to develop for various periods of time before
being processed for MyoD in situ analysis. Embryos depleted of Wee2 underwent
cleavage and gastrulation normally, suggesting that Wee2 is not required for
these early developmental stages (data not shown). However, as development
continued, numerous defects became apparent. By stage 25 of development,
sibling and CMO-treated embryos had developed an elongated, tadpole-like shape
and each had 15 somites (Fig.
3A, panels 1 and 2). By contrast, the Wee2 morpholino-treated
embryos failed to elongate completely along their anteroposterior axis and
failed to develop defined somites (panels 3 and 4). Although both of the
Wee2-targeted morpholinos gave this phenotype in a high percentage of embryos,
(W2MO.1, 85%, n=35; W2MO.2, 87%, n=32), this phenotype was
never observed in embryos treated with CMO. Using one half or one quarter as
much Wee2 morpholino caused a less severe foreshortening of the embryo, but
the percentage of embryos affected remained as high (data not shown). Embryos
treated with the dose of Wee2 morpholino used in
Fig. 3A (80 ng) died by the
tailbud stage (stage 28), while embryos treated with the same amount of CMO
continued to grow (n=28, 75%). This suggests that the loss of Wee2
may be embryonic lethal. As might be expected from the protein depletion data
(Fig. 2), the short,
somite-deficient phenotype was slightly stronger with W2MO.1 than with W2MO.2
(Fig. 3A, panels 3 and 4).
Because the in vitro and in vivo reduction of Wee2 protein and the in vivo
phenotype were more pronounced with W2MO.1, we used it for the remainder of
this study.
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A priori, the reduced embryo elongation could be caused by a defect in
mesoderm specific gene expression (Kopan
et al., 1994) or by a defect in convergent extension
(Keller, 2002
;
Wallingford et al., 2002
). We
investigated each of these possibilities. First, we compared the levels of
various developmentally regulated mRNAs in CMO- and W2MO.1-treated whole
embryos. We observed no difference in the level of axial (XNot), paraxial
(MyoD) or ventral (Vent1) mesoderm markers
(Dosch et al., 1997
;
Gawantka et al., 1995
;
von Dassow et al., 1993
)
suggesting that the early determination of these tissues remains intact in
Wee2-depleted embryos (Fig.
3C). Furthermore, the early differentiation of paraxial mesoderm
into muscle tissue appears normal in Wee2-depleted embryos, as judged by the
expression of muscle actin (MA) and myosin heavy chain (MHC)
(Mohun et al., 1988
;
Radice and Malacinski, 1989
)
(Fig. 3C). We next directly
examined convergent extension by comparing the ability of dorsal explants from
CMO (n=21) or W2MO.1 (n=18)-treated embryos to extend in
vitro (Wilson et al., 1989
).
When Wee2 protein levels were reduced, the dorsal explants extended to only
65% of the length (on average) of control treated embryos
(Fig. 3D). Conversely,
injection of the control morpholino had no effect on explant extension
compared with sibling controls (data not shown). Together, these results
indicate that the reduced elongation observed in Wee2-depleted embryos is
caused by a defect in some aspect of convergent extension. Furthermore, these
results suggest that despite this convergent extension defect, the
determination of the mesoderm and early differentiation of the paraxial
mesoderm remain normal, at least through late neurulation, as judged by
expression of specific markers.
Wee2 is required for neurulation-stage convergent extension of the paraxial mesoderm
To examine the convergent extension defect in more detail, we used markers
specific to the axial mesoderm (XNot), paraxial mesoderm (MyoD, MA, MHC) and
neuronal tissues (Sox3), to determine whether the developmental patterning and
convergent extension of these tissues were affected by Wee2 depletion in the
intact embryo (Dosch et al.,
1997; Mohun et al.,
1988
; Radice and Malacinski,
1989
; von Dassow et al.,
1993
; Zygar et al.,
1998
). Depletion of Wee2 had no effect on when gastrulation was
initiated or completed, as measured by the formation of the dorsal lip and
closure of the blastopore, respectively (data not shown). In addition, by the
end of gastrulation (stage 13), dorsal views show that Wee2-depleted embryos
appeared normal except for a slight mediolateral widening of the axial and
paraxial mesoderm (Fig. 4A,B,
panels 1 and 2 each). Thus, through the end of gastrulation, tissue
rearrangements and convergent extension of the axial and paraxial mesoderm are
relatively unaffected by Wee2 depletion.
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The same is not true for the paraxial mesoderm. Although the gastrula stage
movements were normal, defects in the positioning of the paraxial mesoderm
were apparent by mid-neurulation in Wee2 depleted embryos (77%,
n=18). Dorsal views of stage 15 embryos show that the paraxial
mesoderm failed to complete its anterior and medial migration
(Fig. 4A, compare panels 3 and
4). As neurulation continued (stage 18), the paraxial mesoderm migration
defect became more obvious (70%, n=17). Dorsal and lateral views show
that the paraxial mesoderm failed to complete its migration towards the
midline or extension along the anteroposterior axis
(Fig. 4A, compare panels 5 and
6, and 7 and 8). In addition, anterior views show that the ridge of presomitic
mesoderm failed to form (yellow arrow) and that the neural folds failed to
close (white arrow) (Fig. 4A,
compare panels 9 and 10). These defects in paraxial mesoderm migration were
also observed with the other Wee2 morpholino (W2MO.2, data not shown).
Although depletion of Wee2 disrupted the migration of the paraxial mesoderm,
it had little effect on muscle differentiation, as determined by the
expression of muscle actin (MA) or myosin heavy chain (MHC), early and
mid-stage differentiated muscle markers respectively
(Chanoine and Hardy, 2003). As
with MyoD, these genes are expressed normally, but their spatial distribution
is disrupted (Fig. 3C,
Fig. 4C).
The patterning defect of the paraxial mesoderm suggests that neurula-stage
convergent extension of this tissue might be disrupted
(Keller et al., 2000;
Wallingford and Harland,
2001
). To study this further, we examined a series of anterior to
posterior transverse sections from late neurula-stage embryos that had been
stained with the paraxial mesoderm marker MyoD after morpholino treatment.
Depletion of Wee2 had profound effects on the positioning of the paraxial
mesoderm. Unlike the controls, the paraxial mesoderm failed to migrate toward
the midline in W2MO.1-treated embryos (Fig.
4D, compare panels 1, 3, 5 with 2, 4, 6). Normally, this movement
leads to the dorsoventral thickening of paraxial mesoderm and the formation of
the somitic ridge (arrows in controls). Both of these events are blocked in
Wee2-depleted embryos. In addition, W2MO.1-treated embryos lack the normal
posterior extension of the paraxial mesoderm around the blastopore
(Fig. 4D, compare panels 7 and
8).
Finally, we examined the patterning of the neural plate in late neurula
stage embryos using Sox3 as a pan-neuronal marker
(Fig. 4F). In Wee2-depleted
embryos, the neural plate fails to migrate toward the midline, form normal
neural folds, or extend anteriorly completely (80%, n=10). This
defect in neuronal patterning is unlikely to be a direct effect of Wee2
depletion, as Wee2 is not expressed in neural tissues during neurulation
(Leise and Mueller, 2002). A
likely scenario is that the defect in the convergent extension of the
underlying paraxial mesoderm influences the migration of the overlaying neural
plate (Keller et al., 2000
).
Although neuronal tissues have been shown to undergo convergent extension
independently in explants, it has also been suggested that the complete
migration of the neuronal tissue toward the midline depends on the underlying
mesoderm in vivo. Finally, although the migration of the neural plate is
disrupted in Wee2-depleted embryos, this tissue still differentiates as judged
by the expression of N-tubulin (data not shown)
(Hardcastle and Papalopulu,
2000
). Together, these experiments show that neurula-stage
convergent extension of the paraxial mesoderm is compromised when Wee2 protein
levels are reduced.
Exogenous Wee2 can rescue the Wee2 depletion phenotype
An important control in morpholino-mediated depletions is the rescue of the
induced phenotype. To accomplish this, we microinjected both blastomeres of
two-cell embryos with various mixtures of constant amounts of W2MO.1 and
increasing amounts of Wee2 mRNA that lacks the morpholino target site.
Subsequently, these embryos and non-injected siblings were raised to stage 19
and processed for MyoD or Sox3 in situ analysis
(Fig. 5). As expected,
microinjection of W2MO.1 with buffer alone recapitulated the defects observed
previously (n=23, 89%). There was a lack of paraxial mesoderm
convergent extension as marked by the lateral extension of MyoD and a lack of
neural plate movement as marked by the lateral extension of Sox3 and the wide
neural plate devoid of defined neural folds (compare panels 1-4 with 13-16).
Embryos co-injected with as little as 20 pg of Wee2 mRNA showed a partial
rescue of these defects (panels 5-8; n=53, 87%), while embryos
co-injected with 40 pg of Wee2 mRNA appeared almost normal (panels 9-12;
n=59, 64%). Attempts to completely rescue the phenotype failed
because all embryos microinjected with just twofold more Wee2 mRNA (80 pg)
died during gastrulation (data not shown). Furthermore, even the embryos that
displayed partial rescue with lower amount of mRNA (panels 5-12), died shortly
after controls completed neurulation. These early embryonic deaths were not
totally unexpected because Wee2 is a potent cell cycle inhibitor and because
expression of Wee2 throughout the embryo may be deleterious
(Leise and Mueller, 2002).
However, the partial rescue observed with moderate levels of Wee2 mRNA
indicates that Wee2 is necessary for convergent extension of paraxial
mesoderm.
|
Embryos injected with 230 pg of Cdk2WT per blastomere developed normally (Fig. 6A, panels 1 and 3, 92%, n=26). By contrast, embryos injected with 230 pg of Cdk2AF displayed reduced mediolateral migration and reduced anteroposterior extension (convergent extension) of the paraxial mesoderm and failed to form proper neural folds (arrow) (panels 2 and 4, 80%, n=25). The defect in paraxial mesoderm convergent extension can be clearly observed in an anterior to posterior series of transverse sections from late neural stage embryos prepared for MyoD in situ analysis (Fig. 6B). Microinjection of Cdk2AF (right panels), but not Cdk2WT (left panels), blocks mediolateral convergence to the midline (compare panels 1, 3, 5 with 2, 4, 6), posterior extension around the blastopore (compare panels 7 and 8), and somitic ridge formation (arrows in Cdk2WT). Microinjection of AF or WT Cdk2 had little effect on the axial mesoderm, except that the notochord was not internalized in the Cdk2AF-injected embryos (85% n=20) (Fig. 6A, compare panels 5 and 6). Interestingly, expression of Cdk2AF led to a 40% increase in the number of paraxial mesoderm cells, but no significant change in the number of axial mesoderm cells (Fig. 6C). Although this is consistent with the tissue specificity of the convergent extension defect, it raises the possibility that a phosphorylation-independent mechanism is used to control Cdk2 activity and cell cycle arrest in the axial mesoderm.
|
As an alternative approach to promote premature cell cycle progression, we
used a developmentally regulated promoter to mis-express Cdc25 specifically in
differentiating muscle. The Cdc25 phosphatases are the antagonists of the Wee
kinases (Morgan, 1997). Two
Cdc25 phosphatases have been identified in Xenopus, Cdc25A and
Cdc25C, and both have been shown to promote entry into mitosis in developing
Xenopus embryos (Kim et al.,
1999
). We placed phosphatase-active (WT) and -dead (PD) versions
of Cdc25A- and Cdc25C-GFP fusion constructs under the control of the cardiac
actin promoter (Kroll and Amaya,
1996
). This promoter has been shown to express exogenous genes in
the paraxial mesoderm as this tissue differentiates into muscle
(Mohun et al., 1989
). Thus,
transcripts begin to appear during mid-gastrulation, but high levels of
expression in the paraxial mesoderm are not detected until neurulation. When
injected as plasmid DNA, cells will express the construct in a largely tissue
specific, but mosaic pattern, depending on whether the cell has passively
inherited the DNA (Sive et al.,
2000
). In the first set of experiments, we microinjected 100 pg of
the Cdc25A- or Cdc25C-GFP fusion plasmids into one blastomere of a two-cell
embryo. These unilaterally treated embryos were allowed to develop until stage
19 before being processed for GFP in situ analysis to detect the position of
Cdc25 expressing cells (Fig.
7A). Although cells that express the phosphatase-dead versions of
either Cdc25 converge to the midline normally (odd numbered panels), many
cells that express active versions of these phosphatases fail to do so (even
numbered panels). In addition, cells expressing the active, but not dead,
phosphatases fail to migrate anteriorly (compare panels 1 and 2, and 3 and 4).
These defects appear to have global effects on the embryo in that the somitic
ridge is improperly formed (arrow) on the injected side of most embryos
expressing active Cdc25 (compare panels 5 and 6, and 7 and 8) and that the
embryos curve towards the injected side in about half the cases (panel 4).
These global defects were not observed with the phosphatase-dead
constructs.
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Discussion |
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Cell divisions are rapid and synchronous during early development in
Xenopus. However, the rate of these divisions slows and becomes
spatially restricted with the onset of gastrulation
(Masui and Wang, 1998). In
particular, the cells of the paraxial mesoderm stop dividing as this tissue
undergoes the concerted cell movement of convergent extension. Eventually, the
cells of the paraxial mesoderm re-enter the cell cycle as they form
significant parts of the muscle, skeletal and dermal elements of the embryo.
Wee2, one of three Cdk inhibitory kinases found in vertebrates, is expressed
in the paraxial mesoderm from mid-gastrulation onwards. Like other Wee
kinases, Wee2 inhibits Cdk activity by phosphorylating a conserved tyrosine
residue in the active site of the Cdk, and therefore prevents entry into
mitosis (Leise and Mueller,
2002
). In this work, we have shown that Wee2 plays a causative
role in the low mitotic index of the paraxial mesoderm as depletion of the
Wee2 protein increases the proliferation of this tissue during neurulation. As
might be expected, tissues that do not express Wee2 show no change in their
normal mitotic index with the depletion of Wee2.
The absence of cell proliferation in the paraxial mesoderm is important for normal development. Embryos bilaterally depleted of Wee2 fail to elongate completely, while embryos unilaterally depleted of Wee2 curve towards the depleted side. These elongation and curvature defects are the result of aberrant convergent extension of the paraxial mesoderm during neurulation. Because of the lack of convergent extension, the somitic ridge fails to form and the neural plate fails to move toward the midline. The occurrence of these defects is not limited to the morpholino-mediated depletion of Wee2, but can be phenocopied by other manipulations that cause premature advancement in the cell cycle. These include the mis-expression of wild-type forms of Cdc25 and non-phosphorylatable forms of Cdk2. Finally, Wee2-depleted embryos fail to undergo somitogenesis. However, because somitogenesis is a late event during neurulation, our results do not allow us to resolve whether the absence of somitogenesis is a direct effect of Wee2 depletion or the result of prior inhibition of convergent extension and positioning of the paraxial mesoderm.
In contrast to the deficiencies in convergent extension of the paraxial
mesoderm, other morphogenetic movements appear to be intact in Wee2-depleted
embryos. Gastrula stage cell movements such as involution of the mesoderm and
closure of the blastopore are initiated and completed on schedule. In
addition, the axial mesoderm (notochord) appears to extend and develop
normally during neurulation. Finally, as expected, we do not see the extreme
phenotype that is observed when the PCP pathway is globally disrupted in the
intact embryo. As the PCP pathway controls convergent extension in three
tissues, the axial mesoderm, the paraxial mesoderm and the neural tissue,
global disruption of this pathway results in embryos that are not only
foreshortened but also that have incomplete closure of the neural folds and
extreme dorsal flexure (Sokol,
1996; Tada and Smith,
2000
; Wallingford and Harland,
2001
; Wallingford et al.,
2000
). In Wee2-depleted embryos, only the paraxial mesoderm was
affected.
Other developmental events also appear to be intact in Wee2-depleted
embryos. For example, the continued expression of mesoderm markers MyoD, XNot
and Vent1 suggests that the fate of the paraxial, axial and ventral mesoderm
cells remain unchanged. Furthermore, the expression of muscle actin (MA) and
myosin heavy chain (MHC) suggests that early myogenic differentiation is
unperturbed (Chanoine and Hardy,
2003). Finally, neuronal induction and differentiation appear
relatively normal as judged by the expression of the pan-neuronal maker Sox3
and the neuronal differentiation marker N-tubulin (data not shown)
(Hardcastle and Papalopulu,
2000
). Thus, the need for Wee2-mediated control of the cell cycle
during the neurulation stage of development is limited to processes that
permit orchestrated cell movement and patterning of the paraxial mesoderm.
During Drosophila gastrulation, a similar cell cycle arrest is
observed in the ventral mesoderm as it undergoes invagination and ventral
furrow formation (Foe, 1989).
As with the paraxial mesoderm of Xenopus, this block is transient;
once the ventral mesoderm cells of Drosophila have completed
invagination, they re-enter the cell cycle. In wild-type flies, the ventral
mesoderm cells are arrested due to the expression of Tribbles, a gene product
that promotes the proteolysis of Cdc25
(Großhans and Wieschaus,
2000
; Mata et al.,
2000
; Seher and Leptin,
2000
). As Cdc25 is the antagonist of Wee2, degradation of Cdc25
has the same effect as expression of Wee2, namely the maintenance of Cdks in
their inhibited, phosphorylated state. In fact, forced cell cycle advancement
induced by the mis-expression of Cdc25 or by the loss of Tribbles blocks the
rapid invagination of the ventral mesoderm in flies
(Edgar and O'Farrell, 1990
;
Großhans and Wieschaus,
2000
; Mata et al.,
2000
; Seher and Leptin,
2000
). Thus, at least two types of integrated cell movements,
convergent extension of the paraxial mesoderm in Xenopus and
invagination of the ventral mesoderm cells in Drosophila, use
inhibitory phosphorylation of the Cdks to prevent cell division during these
movements.
Although cell cycle arrest is important in some cases of integrated cell
movement, it is not an absolute or universal requirement for cell migration.
For example, although the ventral mesoderm of Tribbles mutants undergoes
inappropriate cell division, this tissue eventually migrates into the embryo
(Mata et al., 2000;
Seher and Leptin, 2000
).
However, this movement is slow, as well as disorganized, and causes a majority
of the Tribbles mutants to die during embryogenesis. Thus, Tribbles ensures
that the cell movement is rapid and coordinated. In a second example, cell
cycle arrest may not be essential for movement of the presumptive neuronal
tissue of Xenopus. Like the mesoderm, the neural ectoderm undergoes
convergence and extension during neurulation. However, unlike the mesoderm, at
least part of this tissue is proliferating during this process
(Hartenstein, 1989
). This
might partly explain why the force generated by convergent extension of the
neuronal tissue is less than that of the underlying mesoderm
(Keller et al., 2000
).
Together, these examples suggest that integrated cell movements may not so
much need cell cycle arrest, as function more efficiently and rapidly when
cell division is prevented. Still, in the cases of convergent extension of the
paraxial mesoderm or ventral furrow formation, cell cycle arrest is required
for normal development.
As Wee2 is expressed in the paraxial mesoderm from mid-gastrulation
onwards, it is not surprising that morpholino-mediated depletion of Wee2 only
disrupts events after the completion of gastrulation. However, this raises the
question of what causes the cell cycle arrest of the paraxial mesoderm earlier
during gastrulation and the cell cycle arrest of the axial mesoderm throughout
gastrulation and neurulation. One possible candidate is the maternally
expressed Wee1, a kinase related to Wee2
(Mueller et al., 1995a).
Although Wee1 mRNA disappears during gastrulation, Wee1 protein is present
through neurulation (Murakami and Vande
Woude, 1998
) (data not shown). This stable pool of Wee1 protein
might block the cell cycle in these tissues. Alternatively, maternally
expressed Myt1 (another Cdk inhibitory kinase) may contribute to this arrest
(Mueller et al., 1995b
).
Finally, mechanisms independent of inhibitory Cdk phosphorylation may be
involved. A likely candidate is p27Xic1, the only Cdk binding inhibitor found
to date in Xenopus. However, in this context, it is interesting that
whereas depletion of p27Xic1 profoundly perturbed differentiation of muscle
and neuronal tissues, it did not appear to effect convergent extension
(Carruthers et al., 2003
;
Vernon et al., 2003
;
Vernon and Philpott, 2003
).
Thus, although our results implicate Wee2 as the major mediator of cell cycle
arrest in paraxial mesoderm during neurulation, they do not rule out
contributions by other cell cycle inhibitors.
In conclusion, we have shown that Wee2 coordinates cell cycle regulation with critical morphogenetic movements. Wee2 is required both to prevent cell division and to allow the complete convergent extension and somitogenesis of the paraxial mesoderm during neurulation. Our results suggest that convergent extension and cell division are incompatible in the paraxial mesoderm of Xenopus. By arresting the cell cycle, Wee2 prevents the morphological catastrophe that results from tissues trying to carry out these processes simultaneously.
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ACKNOWLEDGMENTS |
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Footnotes |
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