Department of Oncology, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Cambridge CB2 2XY, UK
* Author for correspondence (e-mail: ap113{at}hermes.cam.ac.uk)
Accepted 8 October 2002
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SUMMARY |
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Key words: Cell cycle, Cdk inhibitor, Neurone, Differentiation, p27Xic1, Xenopus
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INTRODUCTION |
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Mammals express three members of the Cip/Kip family of cdkis,
p21Cip1, p27Kip1 and p57Kip2
(Sherr and Roberts, 1999).
Although highly expressed in many neural tissues, redundancy and
inaccessibility has complicated analysis of cdki function in nervous system
development in null mouse models (Deng et
al., 1995
; Nakayama et al.,
1996
; Yan et al.,
1997
). As a result, a clear role for cdkis in neural
differentiation has not been demonstrated. One major advantage of the
Xenopus system is that there is only one described cdki,
p27Xic1, which shows structural and functional characteristics of
p21Cip1, p27Kip1 and p57Kip2
(Su et al., 1995
;
Shou and Dunphy, 1996
).
Primary neurone differentiation requires the sequential activation of
proneural basic helix-loop-helix (bHLH) transcription factors such as
neurogenin (X-NGNR-1) (Ma et al.,
1996), Xash3 (Zimmerman et
al., 1993
) and NeuroD (Lee et
al., 1995
), the roles of which have been likened to those of MyoD,
Myf5 and myogenin during muscle differentiation. Although bHLH genes
coordinate cell cycle exit and differentiation in muscle by upregulating cdkis
(Guo et al., 1995
;
Halevy et al., 1995
;
Parker et al., 1995
), no such
relationship has been demonstrated in neural tissue.
We present the first clear in vivo evidence that a cdki has an essential role in the decision to adopt a neural fate. We show that p27Xic1 is highly expressed in cells destined to become primary neurones, and that it is required for primary neurogenesis at a crucial step between X-NGNR-1 and NeuroD. Moreover, p27Xic1 overexpression promotes ectopic neurone formation, while stabilising the proneural gene, X-NGNR-1.
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MATERIALS AND METHODS |
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mRNA injection and morpholino antisense oligonucleotides
Capped RNAs were synthesised in vitro from nuc-ß-gal and
p27Xic1 (Su et al.,
1995); p27Xic1 NT, p27Xic1 CT,
p27Xic1 35-96 and p21Cip1 N50S
(Ohnuma et al., 1999
);
X-NGNR-1 (Ma et al., 1996
);
NeuroD (Lee et al., 1995
);
p21Cip1 (Harper et al.,
1993
); XMyT-1 (Bellefroid et
al., 1996
); Xath3 (Takebayashi
et al., 1997
); Xash3
(Zimmerman et al., 1993
); and
p21Cip1 NT, p21Cip1 CT, and p21Cip1 20-82,
using the SP6 Message Machine kit (Ambion). Embryos are acutely sensitive to
levels of p27Xic1 RNA, so each new RNA batch must be carefully
titrated for effect. The antisense morpholino oligodeoxynucleotide used was:
5'-GCAGGGCGATGTGGAAAGCAGCCAT-3' (Gene Tools LLC).
Whole-mount in situ hybridisation, BrdU detection and antibody
staining
Whole-mount in situ hybridisation was performed as described
(Shimamura et al., 1994).
Linearised Bluescript plasmid from X-NGNR-1 (BamH1/T7), NeuroD (Xba1/T7),
Nßtub (BamH1/T3) or pCS2 XMyT-1 (BamH1/T7) and p27Xic1
(BamH1/T7) was used to generate digoxigenin-11-UTP-labeled antisense RNA
probes. BM Purple was used as a substrate. Double in situ hybridisation was
performed as described (Sive et al.,
2000
) using BCIP (light blue) and NBT/BCIP (purple) as colour
substrates. For 60-70 µm sections, specimens were post-fixed in MEMFA,
embedded in 3% low melting point agarose and sectioned by Leica VT1000M
vibratome, mounted in 100% glycerol and photographed with Nomarski optics.
BrdU analysis was performed as described by Hardcastle and Papalopulu
(Hardcastle and Papalopulu,
2000
). Whole-mount antibody staining was performed as described
previously (Sive et al., 2000
)
using anti-phospho-histone H3 (TCS Biologicals).
Western blotting
p27Xic1 was detected using a polyclonal antibody
(Ohnuma et al., 1999). Blots
were stripped (Chemicon International) and probed with anti-ß-tubulin
antibody (1:400) (Santa Cruz Biotechnology).
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RESULTS |
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p27Xic1 is strongly expressed at stage 10.5-11, significantly
before neural differentiation, in three stripes lateral to the midline in the
prospective neural plate (data not shown), reminiscent of expression of genes
involved in primary neurogenesis
(Bellefroid et al., 1996;
Ma et al., 1996
). To examine
this resemblance more closely, we have compared p27Xic1 expression
with that of XMyT-1 (Fig. 1)
(Bellefroid et al., 1996
).
|
XMyT-1, a zinc-finger transcription factor, is one of the earliest markers
of primary neurogenesis. XMyT-1 staining is first detected at stage 11.5 in
cells destined to differentiate into lateral, intermediate and medial primary
neurones (Fig. 1D)
(Bellefroid et al., 1996) and
corresponds to the stripes of stronger p27Xic1 expression
(Fig. 1A). Overlapping
p27Xic1 and XMyT-1 staining can be seen at stage 13 by comparing
single in situ hybridisations (compare Fig.
1B with 1E) and examining double in situ hybridisations
(Fig. 1H). At this stage,
p27Xic1 placodal expression is clear
(Fig. 1B, arrows), but the
medial neural stripe of p27Xic1 staining is obscured by stronger
stain in the underlying myotome. p27Xic1 expression is also
apparent in the epidermis outside the neural plate between stages 10.5 and 15
(Fig. 1G). By stage 15,
p27Xic1 expression in the medial and intermediate stripes is
completely obscured by staining in the underlying myotome. However,
p27Xic1 staining is still clear in the lateral primary neurone
stripes (Fig. 1C) and placodal
regions (Fig. 1C, arrows), and
closely resembles the expression of XMyT-1, X-NGNR-1 and X-Delta-1
(Fig. 1F and data not shown)
(Bellefroid et al., 1996
;
Ma et al., 1996
). Moreover,
p27Xic1 expression is clearly seen in the primary neurone
precursors in the sensorial layer of the neuroectoderm, both overlying and
lateral to the staining in the myotome and notochord
(Fig. 1I)
(Hardcastle and Papalopulu,
2000
). p27Xic1 is not uniform in the neural plate at
stage 15; rather, its scattered distribution is similar to XMyT-1 and is
reminiscent of proneural genes whose expression is restricted by
Notch-mediated lateral inhibition (Fig.
1C).
In cultured myoblasts, the bHLH factor MyoD can transcriptionally
upregulate expression of both muscle structural genes and the cdki,
p21Cip1, thus coordinating cell cycle exit and differentiation
(Guo et al., 1995;
Halevy et al., 1995
;
Parker et al., 1995
).
Analogous bHLH factors such as X-NGNR-1 and Xash3 function during
neurogenesis. However, X-NGNR-1, Xash3 and XMyT-1 alone or in combination are
unable to upregulate p27Xic1 expression either in whole embryos or
in ectodermal explants (data not shown). Nonetheless, p27Xic1 is
probably regulated in vivo by an unknown bHLH transcription factor or
combination of factors, as p27Xic1 transcription increases on
injection of XE12, a bHLH co-factor, and is inhibited in the presence of Id, a
bHLH repressor (data not shown) (Lassar et
al., 1991
; Jen et al.,
1992
).
Thus, p27Xic1 is properly spatially and temporally expressed for
involvement in primary neurogenesis. Interestingly, p27Xic1
expression precedes substantial proneural gene expression
(Zimmerman et al., 1993;
Lee et al., 1995
;
Ma et al., 1996
) but coincides
with the time that Rohon-Beard cells (primary sensory neurones) begin to exit
the cell cycle (Lamborghini,
1980
).
p27Xic1 is necessary for neural differentiation
To investigate whether p27Xic1 is necessary for the
differentiation of primary neurones, we used antisense morpholino
oligonucleotides (Mo) to prevent translation of p27Xic1 message
(Heasman et al., 2000).
Injection of p27Xic1 Mo at the two-cell stage prevents the
accumulation of p27Xic1 protein that normally occurs after the MBT
(Shou and Dunphy, 1996
), while
injection of a control morpholino (Con Mo) has no effect
(Fig. 2A).
|
As predicted, ablation of p27Xic1 can increase cell proliferation. Embryos injected with 10 ng p27Xic1 Mo have a 1.27-fold increase in mitotic cells expressing phosphorylated histone H3 (ph3), compared with the uninjected side (n=35, P=0.02, Student's t-test), as well as a small increase in BrdU incorporation (data not shown). The Con Mo had no effect on ph3 or BrdU (data not shown).
Embryos were assayed for primary neurone formation at stage 15 by staining
for X-NGNR-1. X-NGNR-1 is the first proneural gene to be expressed in the
neural plate, and specifies the formation of primary neurones
(Ma et al., 1996). The Con Mo
had no effect on any of the neural markers tested
(Fig. 2B; data not shown).
p27Xic1 Mo injection had very little effect on expression of
X-NGNR-1 at stage 15 (no change in 91% of embryos, n=64),
demonstrating that p27Xic1 is not required for this initial
specification event (Fig. 2C).
However, p27Xic1 Mo, but not Con Mo, reduced expression of XMyT-1,
which is downstream of, and expressed shortly after X-NGNR-1
(Fig. 2D). NeuroD, a direct
downstream target of X-NGNR-1, is also reduced in the absence of
p27Xic1 (Fig. 2E)
(Huang et al., 2000
),
demonstrating a role for p27Xic1 during early differentiation, but
after specification. Interestingly, at stage 18, p27Xic1 Mo
somewhat reduces expression of X-NGNR-1 in 77% of embryos (n=57),
indicating that p27Xic1 may be required for X-NGNR-1 maintenance
(data not shown). Terminal differentiation of primary neurones was examined by
staining for Nßtub, a marker of differentiated neurones. 96%
(n=136) of p27Xic1 Mo-injected embryos showed significant
downregulation or absence of Nßtub on the injected side
(Fig. 2F) that persisted until
tailbud stages (data not shown).
p27Xic1 and a mammalian homologue, p21Cip1, share the
ability to induce Müller glial cells at the expense of neural derivatives
in the Xenopus retina, indicating that they may play similar roles
both in cell cycle regulation and cell fate determination
(Ohnuma et al., 1999). Using
this functional homology, we investigated whether p21Cip1 could
rescue the loss of primary neurones observed upon p27Xic1 Mo
injection. In one typical experiment, after injection of p27Xic1
Mo, 55% of embryos (n=29) had no Nßtub-expressing primary
neurones on the injected side (Fig.
3A). The remaining 45% of embryos had substantially reduced
primary neurone numbers. Neurones in the lateral stripe were counted in the
embryos with reduced Nßtub expression, and they displayed an average of
22 neurones on the injected side compared with an average of 45 on the
uninjected side (P<0.001). After co-injection with
p21Cip1, 100% of embryos showed some Nßtub-expressing primary
neurones (n=29) with an average number of 44 neurones on the injected
side compared with 45 on the uninjected side
(Fig. 3B) (P<0.001). Thus, depletion of p27Xic1 protein by Mo
injection prevents primary neurone formation. This effect is specific to the
loss of cdki function because neurone formation can be rescued by co-injection
of the mammalian homologue p21Cip1.
|
We wished to determine whether the ability of p21Cip1 to rescue
primary neurogenesis in the absence of p27Xic1 was solely related
to its ability to arrest the cell cycle, or whether p21Cip1 plays
an additional role in determining primary neural cell fate. To address this
question we used N- and C-terminal constructs of p21Cip1 [amino
acids 1-89 (p21Cip1 NT) and 87-164 (p21Cip1 CT)], which
arrest the cell cycle in distinct ways. p21Cip1 NT blocks
cyclin/cdk kinase activity while p21Cip1 CT binds and inactivates
PCNA (Chen et al., 1995). We
assayed the ability of these constructs to downregulate proliferation and
compared this with their ability to rescue p27Xic1 Mo-induced
primary neurone loss. Injection of 20 pg full-length p21Cip1 RNA
substantially downregulates BrdU incorporation (data not shown) and rescues
primary neurones (Fig. 3B).
Although similar amounts of NT and CT construct RNAs (10 and 50 pg,
respectively) greatly reduce or eliminate BrdU incorporation at this stage
(data not shown), only expression of p21Cip1 NT rescues primary
neurogenesis (68% of embryos, n=38) (compare
Fig. 3C with 3D). These data
demonstrate that cell cycle arrest alone is insufficient to promote primary
neurone formation, but rather some further function of the N terminus of
p21Cip1 is required.
Ohnuma et al. (Ohnuma et al.,
1999) have shown that a mutant of p27Xic1 (amino acids
35-96) blocks overall cdk2 kinase activity but cannot induce Müller glial
cells, indicating a distinct, non-cell-cycle requirement for the
p27Xic1 NT region upstream of amino acid 35. To investigate whether
a homologous deletion of p21Cip1 can rescue primary neurones, we
co-injected the mutant p21Cip1 20-82 with p27Xic1 Mo.
Although 50 pg of p21Cip1 20-82 was able to arrest the cell cycle
by inhibiting overall cdk2 kinase (data not shown), it did not rescue primary
neurogenesis (Fig. 3E). Thus, a
separable differentiation function of p21Cip1 is required to induce
primary neurones.
To determine whether inhibition of cdk kinase activity is necessary for
rescue, we injected the p21Cip1 N50S point mutant, previously shown
to have a reduced capacity for cdk2 inhibition
(Ohnuma et al., 1999).
p21Cip1 N50S rescued the loss of primary neurones caused by
injection of p27Xic1 Mo in 66% of embryos (n=45,
Fig. 3F). These data again show
that the ability to promote primary neurogenesis is separable from the ability
to inhibit overall cdk2 kinase activity.
p27Xic1 is required between X-NGNR-1 and NeuroD to promote
neural fate determination
When overexpressed, X-NGNR-1 induces extensive ectopic neurogenesis on the
flank of the embryo (Ma et al.,
1996). Our in situ studies show that p27Xic1 is
expressed in the epidermis outside the neural plate at late gastrula and early
neural plate stages (Fig. 1G)
and is therefore available to cooperate with ectopic X-NGNR-1 to promote
neurogenesis both inside and outside the neural plate. Thus, we investigated
whether p27Xic1 is required for X-NGNR-1-mediated primary neurone
formation in the neural plate and embryonic skin. Embryos were injected with
X-NGNR-1 and either Con Mo or p27Xic1 Mo. While embryos co-injected
with X-NGNR-1 and Con Mo show extensive ectopic neurogenesis in the epidermis
(95%, n=37) (Fig. 4A),
both endogenous and ectopic primary neurogenesis is substantially reduced or
eliminated in the presence of p27Xic1 Mo (100%, n=36)
(Fig. 4B). The downregulation
of Nßtub by p27Xic1 Mo is rescued by co-injection of
p21Cip1 (97%, n=40)
(Fig. 4C), again indicating
that the effect of p27Xic1 Mo on neurogenesis is specific to loss
of cdki activity.
|
NeuroD, a bHLH factor that promotes terminal neural differentiation, lies
downstream of X-NGNR-1 (Lee et al.,
1995). To determine whether p27Xic1 acts up- or
downstream of NeuroD, we investigated Nßtub expression in response to
NeuroD in the presence of Con Mo or p27Xic1 Mo. As expected, NeuroD
induced extensive ectopic neurogenesis in the presence of Con Mo
(Fig. 4D). Strikingly, however,
although p27Xic1 Mo almost completely blocked Nßtub expression
in 94% (n=70) of X-NGNR-1-injected embryos, NeuroD induced ectopic
Nßtub in 82% (n=120) of embryos even in the presence of
p27Xic1 Mo (Fig.
4E). Therefore, p27Xic1 is required at a crucial early
step in neurogenesis and acts between X-NGNR-1 and NeuroD.
p27Xic1 promotes primary neurogenesis
We have shown that p27Xic1 is necessary for primary neural
differentiation. Next, we wished to determine whether p27Xic1
expression is instructive for primary neurone cell fate determination and
differentiation. Injection of high doses of p27Xic1 message (250 pg
and above) results in rapid cell cycle arrest and embryonic death, triggered
by extensive apoptosis at early gastrula stages
(Hardcastle and Papalopulu,
2000; Finkielstein et al.,
2001
). However, if lower doses are injected, cells divide at a
slower rate but apoptosis is greatly reduced or eliminated. After careful
titration experiments, we chose to inject 30-60 pg p27Xic1 RNA, a
dose that gives modest overexpression compared with endogenous levels
(Fig. 5G). Injecting 30-60 pg
of p27Xic1 into one cell of two-cell stage embryos causes, on
average, 7% embryonic death. The surviving embryos appear healthy, although
slowed cell division is evidenced by a 61% decrease in ph3 expression
(n=10, P<0.001, Student's t-test) and a
reduction of BrdU incorporation (81% of embryos, n=16) on the
injected as compared with the uninjected side
(Fig. 5A).
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p27Xic1 overexpression upregulates the number of primary
neurones on the injected side in the majority of embryos (67%, n=147)
(Fig. 5B,C), but only within
the primary neurone stripes in the neural plate. p27Xic1 does not
lead to an appreciably earlier expression of Nßtub and, therefore, is
unlikely to cause early birth of primary neurones (data not shown).
Interestingly, ectopic p27Xic1 appears to induce neurones in the
superficial layer of the neuroectoderm
(Fig. 5C), which is usually
refractory to primary neurogenesis
(Chalmers et al., 2002).
Expansion of primary neurogenesis in the neural plate is clear even in embryos
where p27Xic1 RNA is overexpressed only in the ectoderm, as
determined by ß-gal tracer staining
(Fig. 5C). Therefore, the
ectopic induction of primary neurones by p27Xic1 is cell autonomous
and not a secondary result of effects on the underlying myotome
(Vernon and Philpott, 2003
).
To exclude the possibility that an increase in primary neurone cell size was
responsible for the apparent increase in Nßtub expression, we used
magnified images to count the number of Nßtub-positive cells in the
lateral stripe on the injected versus the uninjected side. Of the 67% of
embryos that showed an increase in the number of primary neurones, the
injected side had, on average, 1.9 times as many neurones as the uninjected
side (P=0.02, Student's t-test). This effect is dose
dependent; at 30 pg, 39% of embryos had increased Nßtub expression, while
65% of embryos upregulated Nßtub at 60 pg.
To determine which region of p27Xic1 is required to promote ectopic primary neurones, we injected p27Xic1 NT (1-96), p27Xic1 CT (97-210) or p27Xic1 35-96. While the NT induced ectopic primary neurones (Fig. 4D) (n=123, average 1.7 fold increase, P<0.001), neither the CT nor 35-96 had any effect (Fig. 5E,F), again indicating that p27Xic1 has a separable N-terminal function required to promote primary neurogenesis that is distinct from its ability to inhibit overall cdk2 kinase activity or block the cell cycle.
In cultured muscle cell systems, the half-life of MyoD protein is extended
by co-expression of p57Kip2
(Reynaud et al., 1999).
Originally, this stabilising property of p57Kip2 was attributed to
its inhibition of cdk-dependent phosphorylation of MyoD
(Reynaud et al., 1999
).
However, a non-phosphorylatable form of MyoD (MyoDAla200) is also
stabilised by co-expression of p57Kip2
(Reynaud et al., 2000
) and an
N-terminal
-helix domain of p57Kip2 is implicated in direct
binding with the basic domain of MyoD and masking of potential degradation
signals (Abu Hatoum et al.,
1998
). Because the processes of myogenesis and neurogenesis are
highly analogous, we investigated the possibility that p27Xic1 was
promoting neurogenesis by stabilising X-NGNR-1 protein. We injected 50 pg of
Myc-tagged X-NGNR-1 alone or in combination with 45 pg p27Xic1, 30
pg NT, 50 pg CT or 50 pg 35-96 and performed western blots on stage 20
embryos. Co-injection of X-NGNR-1 with p27Xic1 and
p27Xic1 NT significantly increased the amount of X-NGNR-1 protein
persisting at this stage, while the CT and 35-96 had no effect
(Fig. 6). However, neither
native nor overexpressed p27Xic1 immunoprecipitates with Myc-tagged
X-NGNR-1 from embryo extracts (data not shown), indicating that
p27Xic1 may not stabilise X-NGNR-1 by direct binding. Therefore,
p27Xic1 both stabilises X-NGNR-1 protein and promotes neurogenesis
in a manner that is distinct from its ability to inhibit cdk2 activity or
arrest the cell cycle, but that may not involve direct binding. By analogy
with MyoD, such mechanisms could involve regulation of DNA binding or nuclear
localisation (Abu Hatoum et al.,
1998
; Floyd et al.,
2001
), but these possibilities remain to be investigated.
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DISCUSSION |
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In the retina, p27Xic1 regulates neural versus glial cell fate
choice in addition to its role in cell cycle inhibition
(Ohnuma et al., 1999). Cdkis
have been previously implicated in the differentiation of neural derivatives
in cultured cell systems such as oligodendrocytes and PC12 cells
(Durand and Raff, 2000
;
Erhardt and Pittman, 1998
).
However, although highly expressed in many neural tissues, redundancy has
complicated analysis of the roles of cdkis during development of the nervous
system in null mouse models (Deng et al.,
1995
; Nakayama et al.,
1996
; Yan et al.,
1997
). Nevertheless, several lines of evidence indicate that
mammalian cdkis may have analogous roles to p27Xic1 during
neurogenesis and gliogenesis. First, in the PC12 neural cell line,
p21Cip1 overexpression enhances differentiation in addition to
arresting the cell cycle (Erhardt and
Pittman, 1998
). Secondly, mice null for p27Kip1 have
disrupted retinal cell differentiation and organisation, processes regulated
by proneural gene function (Nakayama et
al., 1996
). Moreover, loss of p57Kip2 causes
inappropriate S-phase entry in the retina. Importantly, p57Kip2
levels drop on cell cycle exit but it is re-expressed postmitotically in a
subset of amacrine neural precursor, and loss of p57Kip2 leads to a
significant increase in the calbindin amacrine cell subtype
(Dyer and Cepko, 2000
). These
data indicate that p57Kip2 is required for proper amacrine cell
subpopulation distribution in vivo a process also regulated by proneural
genes. Furthermore, Zezula et al. (Zezula et al., 2000) have demonstrated that
p21Cip1-/- oligodendrocytes undergo prompt cell cycle withdrawal,
but fail to differentiate. These results indicate that, in some contexts,
cdkis may play a more general role in neural cell fate determination and
differentiation, in addition to their ability to arrest the cell cycle.
However, in contrast to the role of p27Xic1 during primary neurone
differentiation, Ohnuma et al. (Ohnuma et
al., 2002
) show that cdk kinase inhibition is essential to the
ability of p27Xic1 to synergise with proneural genes and promote
early neural cell types in the retina.
The data presented here indicate that cell cycle exit is essential but
insufficient for primary neurone differentiation. The NT and CT of
p27Xic1 both arrest the cell cycle (data not shown), but only the
NT induces ectopic primary neurones (Fig.
5). Indeed, previous studies have shown that premature cell cycle
arrest does not affect primary neurogenesis
(Harris and Hartenstein, 1991;
Kroll et al., 1998
). Instead,
our results suggest that a differentiation function of p27Xic1,
which is complementary to, but distinct from, its ability to arrest the cell
cycle is required for primary neurone formation. An N-terminal mutant of
p27Xic1 that retains its ability to inhibit cdk2 kinase activity
but is missing amino acids 31 to 35 is unable to induce extra primary neurones
(Fig. 5F). Additionally, only
the full NT of p21Cip1 can rescue the p27Xic1 Mo-induced
loss of primary neurones (Fig.
3). Moreover, the p21Cip1 N50S mutant, which has an
impaired ability to inhibit cdk2 but contains an otherwise intact N-terminal
region, is able to rescue the p27Xic1 Mo phenotype
(Fig. 3F).
Overexpression of p27Xic1 results in the formation of ectopic
neurones, but only within the neural plate
(Fig. 5). p27Xic1
does not lead to an appreciable earlier expression of Nßtub and therefore
is unlikely to cause early birth (data not shown). p27Xic1 doses
higher than 75 pg did not enhance, and sometimes inhibited, primary neurone
formation (data not shown), indicating acute sensitivity to p27Xic1
levels. This sensitivity may account for the difference between results
presented here and those of Hardcastle and Papalopulu
(Hardcastle and Papalopulu,
2000) who failed to detect ectopic neurones inside or outside of
the neural plate on injecting 250pg of p27Xic1 message, a dose that
induced 70% embryonic death. High doses of p27Xic1 may arrest
neural plate cells before instructive proneural genes have accumulated or may
result in selective death of the overexpressing cells. Indeed, work in
keratinocytes has indicated that sustained overexpression of
p21Cip1 at high levels may, in fact, inhibit differentiation
(Di Cunto et al., 1998
).
However, our data clearly indicate that modest p27Xic1
overexpression promotes primary neurone formation within expanded proneural
domains.
Full-length p27Xic1 and p27Xic1 NT stabilise X-NGNR-1 protein (Fig. 6) and promote primary neurogenesis (Fig. 5), although the molecular mechanism of this stabilisation is unknown. Initial co-immunoprecipitation studies indicate that p27Xic1 does not bind directly to overexpressed X-NGNR-1 (data not shown). Future investigations into the effects of p27Xic1 on X-NGNR-1 DNA binding, nuclear localisation and degradation may reveal alternative mechanisms for the observed upregulation of primary neurogenesis upon p27Xic1 overexpression.
In summary, the model presented in Fig. 7 proposes that p27Xic1 acts in parallel with X-NGNR-1 at an early stage of neurogenesis, and requires functions independent of its ability to arrest the cell cycle or inhibit overall cdk2 kinase activity. p27Xic1 may function in part by regulating X-NGNR-1 stability, and this is also independent of its ability to inhibit cdk2. By combining distinct cell cycle and differentiation functions, a single molecule, p27Xic1, may provide a powerful way to coordinate the processes of division and differentiation during primary neurogenesis.
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ACKNOWLEDGMENTS |
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