Department of Oncology, Cambridge University, Hutchison/MRC Research Centre, Addenbrookes Hospital, Hills Road, Cambridge CB2 2XZ, UK
Author for correspondence (e-mail:
ap113{at}cam.ac.uk)
Accepted 5 February 2004
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SUMMARY |
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Key words: Cyclin, Cdk, Cell cycle, Differentiation, Epidermis
![]() |
Introduction |
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Attempts have also been made to study the roles of different cyclins during
development, and most success has been gained from taking a genetic approach
in Drosophila. Overexpression of cyclin E in flies induces both
undifferentiated and differentiating G1 cells to enter S phase in a variety of
tissues (Li et al., 1999;
Neufeld et al., 1998
;
Richardson et al., 1995
).
Ectopic overexpression of cyclin A also triggers entry into S phase, although
this is limited by additional embryonic mechanisms that limit cyclin A
activity, predominantly affecting protein stability
(Sprenger et al., 1997
). Work
on tissue culture cells might imply that most, if not all, cyclins are
important for cell cycle progression in all cells. However, for instance in
Drosophila and Xenopus, expression patterns of various
cyclins differ between tissues in ways that cannot always solely be explained
by the proliferative index of the tissue concerned
(Jones et al., 2000
;
Richardson et al., 1993
;
Vernon and Philpott, 2003a
).
In Xenopus embryos, our experimental system of choice, cell division
is fairly uniform before gastrulation, but after this time proliferation
adopts tissue- and region-specific patterns
(Saka and Smith, 2001
;
Vernon and Philpott, 2003a
).
Methods of cell cycle regulation might differ between these distinct
regions.
A number of G1/S phase cyclins have been identified in Xenopus.
Although cyclin D has not been demonstrated to play an important role in early
Xenopus embryogenesis (T. Hunt, personal communication) (see also
Taieb et al., 1997;
Taieb and Jessus, 1996
), 3
cyclin E (Chevalier et al.,
1996
; Rempel et al.,
1995
) and 2 cyclin A homologues
(Howe et al., 1995
;
Minshull et al., 1990
) have
been cloned. Interestingly, maternal cyclin E and cyclin A1 are degraded after
the midblastula transition (MBT) or at the onset of gastrulation, respectively
(Howe et al., 1995
;
Howe and Newport, 1996
), and
are replaced with zygotic transcripts. Cyclin A1, the pre-MBT form of cyclin
A, complexes predominantly if not exclusively with cdc2, functions in mitosis
and is fully degraded after MBT. It is then replaced by zygotic cyclin A2
(Howe et al., 1995
). Cyclin A2
is found complexed with cdk2 and cdc2 soon after gastrulation but
predominantly complexes with cdk2 from neurula stages onwards
(Howe et al., 1995
;
Strausfeld et al., 1996
).
Thus, Xenopus cyclin A2 behaves more like a homologue of mammalian cyclin A
than Xenopus cyclin A1 does, functioning in S-phase entry
(Strausfeld et al., 1996
).
Both cyclin E1 and cyclin A2 are detectable by in-situ hybridisation at least
through to tailbud stages, although levels and sites of expression appear to
differ somewhat at different developmental stages
(Vernon and Philpott,
2003a
).
Cell division and differentiation are generally thought to be mutually
exclusive (e.g. Edlund and Jessell,
1999; Skapek et al.,
1995
; Zhang et al.,
1999
). While the effects of overexpression of cyclins and cdks
have been studied in Drosophila, and some targeted expression has
been attempted in mammals (Li et al.,
1999
; Miliani de Marval et
al., 2001
; Neufeld et al.,
1998
; Robles et al.,
1996
; Rodriguez-Puebla et al.,
2000
; Yamamoto et al.,
2002
), little work has been performed to investigate the short-
and long-term effects of cyclin/cdk upregulation in multiple tissues in the
vertebrate embryo in vivo. The aim of this study was to determine whether
overexpression of a single cyclin/cdk pair alone was sufficient to promote
proliferation in the early embryo, and to investigate whether this was
incompatible with differentiation. Here, we demonstrate that, while
overexpression of cyclin E results in apoptosis post-MBT, cyclin A2/cdk2
overexpression promotes proliferation in the early tailbud embryo. Moreover,
cyclin A2/cdk2 RNA injection delays differentiation of epidermis and neurons
but has no effect on differentiation of muscle. Thus, cyclin E and cyclin A2
levels can regulate cell cycle and differentiation in a stage- and
tissue-specific manner.
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Materials and methods |
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Xenopus cyclin E1, cyclin A2, and cdk2 (the latter two with or without myc-tag or HA-tag) were subcloned into pCS2+ and transcribed using the Ambion Message Machine kit. Typically, 1 ng of each RNA was injected (2 ng in Fig. 2G,H; 1.5 ng in Fig. 6). Where appropriate, 100 pg of Bgal was injected as a lineage tracer.
|
|
In situ hybridisation, BrdU detection, antibody staining and histology
Whole-mount in-situ hybridisations were performed as described
(Shimamura et al., 1994) with
digoxigenin-labelled antisense RNA probes generated from epidermal keratin
(EcoRI/SP6), neural-beta-tubulin (BamHI/T3) and muscle actin (HindIII/T7)
clones. BrdU incorporation and detection was performed essentially as
described in the Roche (Roche, Mannheim, Germany) instructions for the BrdU
Labelling and detection kit 1 (1296 736). Embryos were injected with 1 nmol of
BrdU 1 hour before MEMFA fixation. Anti-BrdU (1:100) was applied to 12 µm
paraffin wax embedded sections overnight at 4°C, washed and detected with
an anti-mouse Ig-rhodamine (1:200) for 1 hour at room temperature.
Whole-mount antibody staining was performed as described
(Sive et al., 2000) using an
anti-phosphohistone H3 antibody (TCS Biologicals, Buckingham, UK) at 1:1000
and detected with an alkaline phosphatase-conjugated secondary antibody using
NBT/BCIP as substrates. Staining for skin differentiation
(Fig. 6) and HA-tagged cyclin
A2 (Fig. 4) was performed on
embryos, fixed in 4% paraformaldehyde and sectioned by cryostat, using
monoclonal antibody culture supernatant
(Jones and Woodland, 1986
)
diluted 1:1 with PBS, or anti-HA at 1:250. For whole-mount staining of nuclei,
embryos were fixed in MEMFA for 1-2 hours, bleached and stained with Hoechst
at 10 µg/ml in PBS. Embryos were de-stained in PBS for at least 12 hours,
then animal caps were cut, mounted under coverslips and viewed directly.
|
![]() |
Results |
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To establish whether overexpression of G1/S phase cyclins was able to increase cdk2 kinase activity in embryonic blastomeres, RNAs encoding Xenopus cyclin A2 or cyclin E were injected into fertilised eggs with or without RNA encoding cdk2. At stage 8, after embryo homogenisation, cdk was immunoprecipitated and tested for its ability to phosphorylate histone H1 (Fig. 1). Injection of RNAs encoding the cyclin subunits alone did not result in a significant increase in cdk2 kinase activity, indicating that either the amount of cdk2 in the extract was limiting or that the newly-synthesised cyclin was unable to complex with or activate the endogenous cdk2 (Fig. 1, lanes 1, 2 and 6). In fact, we observed that overexpression of cdk2 alone led to a modest increase in cdk2 kinase activity (Fig.1, lane 3), indicating that the amount of cdk2 subunit in the early embryo was limiting and that there might have been uncomplexed cyclins present. Overexpression of cyclin E in particular was able to upregulate cdk2 kinase activity when exogenous cdk2 was introduced by RNA injection (Fig.1, lane 4), showing that active complexes can be formed from injected messages. In this system, neither cyclin A2 nor cyclin E overexpression resulted in appreciable activation of endogenous cdc2, as measured by immunoprecipitation assays (data not shown).
|
Thus elevated levels of cyclin E, but not cyclin A2, induce apoptosis in
the early embryo. This effect must be independent of the ability of cyclin E
to globally raise cdk2 kinase activity because cyclin E injection alone, which
does not significantly activate endogenous cdk2 kinase as measured in
immunoprecipitation assays (Fig.
1, lane 1), is as efficient as cyclin E plus cdk2 at inducing
apoptosis. One possible trigger for apoptosis is the disruption of the normal
nuclear replication and division cycle. To investigate this, we cut animal
caps from cyclin-injected embryos at stage 8.5 and stained them for DNA
content (Fig. 3). Large areas
of the animal pole where cyclin E message had been injected were devoid of any
detectable nuclei (Fig. 3A,
arrow). This lack of nuclei is consistent with elevated levels of cyclin E
specifically blocking DNA replication, as has been demonstrated in egg
extracts (Hua et al., 1997).
This block would result in nuclear loss on subsequent cell division. However,
even in the absence of DNA cell cleavage would continue, as embryos are able
to sense this loss of nuclei only after the MBT when checkpoints kick in
(Dasso and Newport, 1990
) and
when apoptotic pathways are activated in the early gastrula
(Hensey and Gautier, 1997
;
Sible et al., 1997
;
Stack and Newport, 1997
).
Interestingly, overexpression of cyclin E resulted in loss of nuclei with or
without co-injection of cdk2 (Fig.
3A,D). Therefore, the ability of cyclin E to induce nuclear loss
in Xenopus embryos is independent of an effect on overall cdk2 kinase
activity. Injections of RNA encoding cyclin A2 with or without cdk2, cdk2
alone or control Bgal had no effect on nuclear density within the dissected
animal caps, as might be expected since these embryos develop essentially
normally (Fig. 2B,E,C,F and see
below).
|
We initiated experiments to determine the long-term effects of cyclin A2
overexpression on later stages of Xenopus development. As endogenous
A-type cyclins are thought to be unstable
(Funakoshi et al., 1999;
Howe et al., 1995
), we
investigated whether cyclin A2 protein would be maintained at high levels as
late as tailbud stages (stage 23) after injection of cyclin RNA into 2-cell
embryos. Embryos injected with RNA encoding cyclin A2, cdk2, cyclin A2 and
cdk2 together or Bgal as a control were allowed to develop until stage 23.
Cdk2 was then immunoprecipitated and tested for kinase activity against
histone H1 (Fig. 4A). Again,
cyclin A2 alone was not able to significantly activate endogenous cdk2 even
post-MBT (Fig. 4A, lane 3) but
cyclin A2 co-injected with cdk2 RNA led to a substantial enhancement of cdk2
kinase activity (Fig. 4A, lane
4), even as late as stage 23 (tailbud stage).
Levels of cyclin A2 protein were measured by Western blotting
(Fig. 4B). As development
progressed, cyclin A2 protein accumulated in injected embryos
(Fig. 4B, compare lanes 2 and
5). Furthermore, simultaneous overexpression of cdk2 substantially stabilised
overexpressed cyclin A2 protein (Fig.
4B, lane 6). This effect might be analogous to stabilisation of
cyclin E seen on binding to cdk2 in mammalian cells
(Clurman et al., 1996).
However, this stabilisation effect is not seen at stage 9, possibly because
little injected cyclin A2 has accumulated at this time
(Fig. 4B, compare lanes 2, 3
and 7) or due to regulated and abrupt degradation of A-type cyclins at the
onset of gastrulation (Howe et al.,
1995
). To maintain cyclin A2-dependent kinase activity at high
levels in older embryos that we studied in the following experiments, we
co-injected cyclin A2 with cdk2 (cyclin A2/cdk2 RNA).
To investigate the stability of cyclin A2 protein in different tissues, we injected embryos with HA-cyclin A2/cdk2 RNA and allowed them to develop to stage 23. Embryos were sectioned and immunostained for expression of HA-tagged cyclinA2. We saw no overall difference in protein stability between epidermis, notochord, myotome or neural tube in the injected region (Fig. 4C,D). However, there was cell-to-cell variability, as expected from a protein that is degraded in a cell cycle-dependent manner. Cdk2 protein was also equally stable in all these tissue types (data not shown).
Next, to establish whether overexpression of cyclin A2 kinase is sufficient
to force cells through the cell cycle, we injected cyclin A2/cdk2 RNA, along
with Bgal as a lineage tracer, into one cell of a two-cell embryo. Embryos
were allowed to develop to stages 16 and 19 when we investigated proliferation
on the injected versus the uninjected side. We used the mitotic marker
phosphorylated histone H3 to visualise proliferation and measured the number
of cells in mitosis (Saka and Smith,
2001). To quantitate cellular proliferation, regions of equal size
were drawn on the flank of the embryo on the injected versus the uninjected
side and the number of phosphorylated histone H3-expressing cells were
counted. On average, 46% more cells at stage 16 (n=20) and 63% more
cells at stage 19 (n=12) (Fig.
5A,C) were undergoing mitosis on the skin of the injected side of
the embryo compared with the uninjected side. By contrast, Bgal-injected
control embryos showed no increase in number of phosphorylated histone
H3-expressing cells on the injected side (data not shown). Therefore,
overexpression of cyclin A2/cdk2 is able to increase cellular proliferation
after MBT.
|
To investigate whether cyclin A2/cdk2 overexpression would similarly
promote proliferation in other early embryonic tissues, numbers of
BrdU-labelled cells were counted in each half of the neural tube and myotome.
There was no significant difference in the number of cells incorporating BrdU
on the injected versus the uninjected side in these tissues
(Fig. 5E,F,G): on average 15%
BrdU-positive cells on the injected side versus 16% on the uninjected side for
the muscle section and on average 54% BrdU-positive cells on both injected and
uninjected sides for the neural tube. Nor was the myotomal tissue area visibly
changed. Similarly, cyclin A2/cdk2 overexpression did not lead to
proliferation in the notochord (Fig.
5E), which is postmitotic by this stage
(Saka and Smith, 2001). These
data indicate that in nerve and muscle at this stage any increased
proliferative signal given by cyclin A2/cdk2 overexpression is overridden by
intrinsic or extrinsic factors that control the rate of cell division.
However, this is clearly not the case in the skin, in which cyclin A2/cdk2
overexpression alone can promote cell cycling.
While cyclin A2/cdk2 overexpression promotes ectopic cell division, we wanted to know whether these cells divide more rapidly, i.e. at a smaller size. To measure approximate cell size, we measured the average internuclear distance along the surface of either normal or thickened skin to produce a measurement of average cell diameter. In a typical experiment, internuclear distance was 26.3 µm±1.27 on the injected side versus 24.7 µm±1.22 on the uninjected side (n=11), demonstrating that there is not a significant difference in cell size in cyclin A2/cdk2-overexpressing regions and the control non-expressing regions. These data indicate that cyclin A2/cdk2 upregulation is not sufficient to allow cells to proliferate at a smaller than normal size.
Cells in the developing embryo are subject to many signals, extrinsic and intrinsic, which instruct cells to divide or to differentiate. While it has become increasingly apparent that cells must exit the cell cycle before terminal differentiation can occur, the ways in which cell cycle regulators and differentiation factors are coordinated is obscure. We wished to determine whether overexpression of cyclin A2/cdk2 alone was enough to overcome endogenous signals within the embryo promoting differentiation of the skin. First, we allowed injected embryos to grow up to tailbud stages and viewed them externally. We observed that overexpression of cyclin A2/cdk2, but not cyclin A2 alone or cdk2 alone, resulted in regions of hyperpigmentation in the injected region of the embryo, indicating that normal skin formation or architecture had been disrupted. This hyperpigmentation was often accompanied by raised regions of epidermis (`lumps', Fig. 6G, arrow). The penetrance of these phenotypes varied somewhat from batch to batch of embryos, but a typical experiment is shown in Table 1. However, we noted that even where hyperpigmentation and lumps were visible, skin integrity was always maintained, indicating that epidermal differentiation was occurring.
|
Thus, while overexpression of cyclinA2/cdk2 does not prevent epidermal differentiation, we wished to determine whether it was sufficient to delay differentiation in embryonic tissues. Hence, we injected cyclinA2/cdk2 RNA and allowed embryos to develop to neural plate and tailbud stages. Embryos at increasing developmental stages were stained by in-situ hybridisation for expression of: the skin marker epidermal keratin; the somatic muscle marker muscle actin; and neural beta tubulin, a marker of primary neurons, which are the first neurons to differentiate within the neural plate (Fig. 7). After cyclin A2/cdk2 overexpression, epidermal keratin expression was significantly delayed on the injected side of the embryo until stage 18 (Fig. 7A-C) while primary neurons failed to differentiate until tailbud stages (Fig. 7D-F). By contrast, expression of muscle actin (or the terminal muscle marker heavy chain myosin, data not shown) was not delayed by overexpression of cyclin A2/cdk2 (Fig. 7G-I). Thus, simple upregulation of a single cyclin/cdk pair is sufficient to overcome endogenous signals to differentiate, but only for a limited time and only in specific tissues.
|
![]() |
Discussion |
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In mammalian cell cycles, data that come largely from experiments in
cultured cell systems suggest that accumulation of the cyclin, rather than the
amount of cdk subunit, is rate-limiting for generation of cdk2-dependent H1
kinase activity and subsequent cell cycle progression
(Connell-Crowley et al., 1998;
Ohtsubo and Roberts, 1993
;
Ohtsubo et al., 1995
;
Resnitzky et al., 1994
;
Resnitzky et al., 1995
;
Rosenberg et al., 1995
).
Strikingly in the pre-MBT embryo, this assumption seems not to hold true:
overexpression of either cyclin E1 or cyclin A2 alone did not produce a
significant increase in cdk2 kinase activity in embryo extracts
(Fig. 1)
(Strausfeld et al., 1996
).
While free cdk2 that is unable to complex with overexpressed cyclin E1 or A2
may exist in the embryo, overexpression of cdk2 alone by RNA injection also
enhanced cdk2-dependent H1 kinase activity. This finding indicates that there
may in fact be a pool of excess cyclins in pre-MBT embryos. Cyclin/cdk
overexpression is unlikely to be titrating out cdk inhibitors (cdkis) at this
stage, as the only described cdki in Xenopus, Xic1, is not expressed
at significant levels prior to MBT (Shou
and Dunphy, 1996
; Su et al.,
1995
).
Microinjection of cyclin E RNA into Xenopus embryos resulted only
in a mild slowing of cell cleavage pre-MBT. However, on closer inspection of
dissected animal pole tissue, we determined that nuclei were largely absent
from injected areas, even though cleavage had occurred. This observation is
consistent with elevated levels of cyclin E blocking DNA replication, as has
been demonstrated in egg extracts (Hua et
al., 1997), which would result in nuclear loss on subsequent cell
division. The fact that cleavage proceeds in the absence of nuclear DNA when
cyclin E is overexpressed (Fig.
2) demonstrates the absence of active checkpoints in the embryo
pre-MBT that monitor DNA replication and/or genome stability and chromosome
segregation (Dasso and Newport,
1990
). However, some slowing of cleavage does occur in the most
highly overexpressing regions at this early stage
(Fig. 2a,d, arrows), indicating
either that nuclear events can be sensed to some extent or that cyclin E
overexpression has a direct effect on cleavage events, possibly via an effect
on centrosome duplication (Hinchcliffe et
al., 1999
) or on cdc2 activity. Apoptosis in these cyclin
E-injected embryos, which apparently occurs in response to the absence of
nuclear DNA, occurs at gastrula stages, when the apoptosis machinery becomes
activated (Hensey and Gautier,
1997
; Sible et al.,
1997
; Stack and Newport,
1997
). Interestingly, overexpression of cyclin E in mammalian
cells does not block DNA replication, nor does it result in obvious apoptosis
(Ohtsubo and Roberts, 1993
;
Ohtsubo et al., 1995
;
Resnitzky et al., 1994
), but
instead it promotes genetic instability
(Spruck et al., 1999
). Cyclin
E is often highly expressed in tumours
(Donnellan and Chetty, 1999
),
and the genetic instability caused could lead to loss of genes involved in
apoptotic pathways in the outgrowing cells.
Importantly, we note that loss of nuclear DNA occurs after injection of
cyclin E message alone, with or without additional cdk2. As overexpression of
cyclin E alone does not lead to an overall increase in the level of
immunoprecipitable cdk2 kinase activity
(Fig. 1), nor will it activate
cdc2 (data not shown), it is possible that increased kinase activity is not
required to produce cyclin E-induced loss of nuclei. Alternatively, cyclin E
overexpression might raise kinase activity only locally, which is not detected
by overall cdk2 immunoprecipitation from lysed embryos, and this local
activation might be sufficient to induce nuclear loss. Overexpression of
cyclin A2 with or without cdk2 has no appreciable effect on either cell or
nuclear division prior to MBT. This observation demonstrates that
overexpression of cyclin E has specific effects that are not recapitulated by
overexpression of cyclin A2, indicating distinct targets for these two
molecules. This difference is surprising, given that endogenous cyclin A2 can
compensate for the requirement of cyclin E for DNA replication in egg extracts
(Jackson et al., 1995).
While the effect of cyclin E overexpression on nuclear maintenance in vivo is interesting and deserves further study, the focus of this work was to determine the effects of cyclin/cdk overexpression on development after the MBT. As overexpression of cyclin E induces apoptosis at gastrulation, we chose to concentrate on the effects of cyclin A2/cdk2 overexpression on later developmental processes.
Studies of phosphohistone H3 distribution in Xenopus demonstrated
that cell proliferation is widespread in many areas of the neurula and tailbud
stage embryo (Saka and Smith,
2001). We find that cyclin E1 and cyclin A2 messages have
overlapping distributions and are most highly expressed in the anterior neural
tube (Vernon and Philpott,
2003a
). While this distribution might be expected, since the
neural tube is one of the most actively proliferating tissues at this time
(Saka and Smith, 2001
), it is
surprising that expression is strikingly lower in other tissues that are still
dividing at these stages, such as the embryonic epidermis
(Saka and Smith, 2001
;
Vernon and Philpott, 2003a
).
Overexpression of cyclin A2/cdk2 alone is enough to promote enhanced
proliferation in the embryo (Fig.
5). However, a dramatic increase in the proportion of
proliferating cells occurs only in the epidermis. Strikingly, cyclin A2/cdk2
overexpression apparently cannot promote proliferation in the notochord or
myotome, where cells normally exit the cell cycle at early to mid-neural plate
stages (Fig. 5E,F)
(Saka and Smith, 2001
;
Vernon and Philpott, 2003b
),
even though the proteins are stable in these tissues
(Fig. 4C,D). Moreover,
proliferation is not enhanced in the neural tube
(Fig. 5G), where cyclin
messages are normally abundant (Vernon and
Philpott, 2003a
). This indicates that in tailbud-stage embryos, of
the tissues studied, cyclin A2/cdk2 overexpression is limiting only for
proliferation in the skin, where G1/S phase cyclin message levels are usually
low but where many cells are still proliferating
(Vernon and Philpott, 2003a
).
This might reflect the fact that there are high levels of the cdk inhibitor
Xic1 in the myotome, nervous system and notochord but only low levels in the
epidermis (Hardcastle and Papalopulu,
2000
; Ohnuma et al.,
1999
; Vernon et al.,
2003
; Vernon and Philpott,
2003b
), and indicate that different tissues emphasise different
methods of cell cycle regulation during embryogenesis.
Studies in Drosophila have shown that overexpression of cyclin E
can result in cells dividing at a smaller size than usual, but the final area
occupied by overexpressing cells is normal as extra cells are lost by
apoptosis (Li et al., 1999).
Interestingly, in some circumstances, while cyclin E overexpression
accelerates passage through G1 phase, cell cycle length is not changed as
there is a corresponding lengthening of S phase
(Neufeld et al., 1998
). By
contrast, overexpression of E2F, a transcription factor required to upregulate
a variety of S-phase progression factors as well as the M-phase String
protein, accelerates the cell cycle. However, this accelerated proliferation
correlates with decreased cell size, thus tissue volume is not affected
(Neufeld et al., 1998
). To
determine whether cyclin A2/cdk2 overexpression caused cells to divide at a
smaller than usual size, we measured the internuclear distance between cells
overexpressing cyclin A2/cdk2 compared with uninjected cells in skin sections
and found no significant difference. This is indicative of cell division
occurring at normal cell size. Indeed, in cyclin A2/cdk2 overexpressing
embryos, tissue expressing epidermal markers is more than the usual two cell
layers thick (Fig. 6), again
indicating that cells do not divide at a smaller size, but instead continue
proliferating when they would normally have exited the cell cycle and so must
occupy a greater than usual volume (Zuber
et al., 1999
). Therefore, cyclin A2/cdk2 overexpression appears to
be insufficient to accelerate the overall cell cycle rate at the expense of
cell growth in a vertebrate embryo, but instead it prolongs the period of
proliferation.
In most systems, proliferation and differentiation are mutually exclusive.
We wished to determine whether overexpression of cyclin A2/cdk2 delayed or
prevented differentiation in the early embryo. Cyclin A2/cdk2 overexpression
was able to significantly delay the appearance of epidermal keratin
(Fig. 7A-C). However,
differentiation did eventually occur (Fig.
6A; Fig. 7C),
demonstrating that promotion of cell cycling in this way is sufficient to
delay, but not to overcome, signals promoting epidermal differentiation. Mice
have been generated in which D-type cyclins or cdk4 are specifically
upregulated in epithelia, especially in the skin, and this results in
hyperproliferation of the epidermis
(Miliani de Marval et al.,
2001; Robles et al.,
1996
; Rodriguez-Puebla et al.,
2000
; Yamamoto et al.,
2002
). Interestingly, under these circumstances differentiation
appears to be largely normal. We note that D-type cyclins, which act in G1
phase of the cell cycle and earlier than cyclin E or cyclin A, are not
detected in the epidermis of Xenopus embryos
(Vernon and Philpott,
2003a
).
The mammalian skin condition psoriasis results from hyperproliferation and
thickening of the epidermis coupled to perturbed differentiation. Although
epidermis structure differs significantly between species, we see a strikingly
similar phenotype on overexpressing cyclin A/cdk2 in Xenopus embryos.
Interestingly, psoriasis in humans is also accompanied by overexpression of
cyclin A (Miracco et al.,
2000), and our results suggest that this upregulation may help
drive the condition. Moreover, a number of cancers, including those derived
from skin, show elevated levels of A-type cyclins
(Balasubramanian et al., 1998
;
Kim et al., 2002
). Our results
indicate that skin may be particularly susceptible to an increase in cyclin
A-dependent kinase level. This overexpressing population of cells maintained
in the cell cycle inappropriately might then be targets of further genetic
`hits', ultimately resulting in tumour formation. Indeed, a study looking at
UV-induced skin cancers in mice showed that cyclin A overexpression occurs
early on in the process of tumour formation
(Kim et al., 2002
).
At neural plate and tailbud stages, most of the cells destined to become
neurons are still dividing and consequently express high levels of cyclin
A2/cdk2 (Hartenstein, 1989;
Saka and Smith, 2001
;
Vernon and Philpott, 2003a
).
However, a small subset of cells found in three stripes lateral to the midline
exit the cell cycle early and differentiate into primary neurons
(Hartenstein, 1989
;
Lamborghini, 1980
).
Overexpression of cyclin A2/cdk2 also delays differentiation of these cells
(Fig. 7D-F). One can envisage
several ways in which elevated cyclin A2/cdk2 could delay primary neuron
differentiation. First, cyclin A2/cdk2 can promote cellular proliferation,
preventing cell cycle exit, which is incompatible with differentiation.
Second, we have recently shown that the cdki Xic1 is absolutely required for
differentiation of primary neurons, and this activity extends beyond its
ability to regulate the cell cycle (Vernon
et al., 2003
). While the exact mechanism of Xic1 action has yet to
be elucidated, cyclin A2/cdk2 overexpression might inhibit primary
neurogenesis by sequestering the required Xic1 protein. Interestingly,
however, cyclin A2/cdk2 overexpression does not delay muscle differentiation,
a process also shown to be Xic1-dependent, again showing that different
tissues may rely on distinct molecular methods to regulate the balance between
proliferation and differentiation. Third, the levels of the myogenic factor
MyoD are regulated by ubiquitin-mediated proteolysis (e.g.
Reynaud et al., 2000
;
Tintignac et al., 2000
), which
is, in turn, controlled by cell cycle kinases and inhibitors. It is possible
that proneural gene products are similarly regulated. We are currently
investigating this possibility.
Our results demonstrate that overexpression of a single cyclin/cdk pair in a vertebrate can have substantial effects on the balance between division and differentiation in the early embryo, in a tissue-specific manner. Studies in experimentally tractable systems, such as those described here in Xenopus embryos, may prove very informative for understanding the role of cyclin/cdk upregulation in diseases of hyperproliferation, such as psoriasis and cancer, in which it might affect both cell proliferation and differentiation in a tissue-dependent manner.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Northwestern University, 2145 Sheridan Road, Evanston, IL
60208, USA
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