UMR 7622, CNRS-University Paris VI. 9, Quai Saint Bernard, 75005 Paris, France
* Author for correspondence (e-mail: michel.gho{at}snv.jussieu.fr)
Accepted 18 February 2005
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SUMMARY |
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Key words: BrdU, Dacapo, G1-phase, In vivo, Microchaete, Tramtrack, Drosophila
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Introduction |
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Control of cell cycle progression depends on an evolutionary conserved
mechanism that regulates the transition between cell-cycle phases. For
example, G1 cyclins associate with the cyclin-dependent kinase Cdk2 (Cdc2c
FlyBase) to promote initiation and progression through the S-phase.
Differential regulation of these Cdk/Cyc complexes gives rise to the different
types of cell cycle. In Drosophila, as in mammals, recent studies
have shown that CycD/Cdk4 acts primarily as a cell growth regulator
(Malumbres et al., 2004;
Meyer et al., 2002a
). By
contrast, Cyclin E (CycE) appears to be the most important cyclin for the G1
to S transition (Knoblich et al.,
1994
; Richardson et al.,
1995
). Thus, maternal CycE is prevalent during the first embryonic
cell cycles that lack a G1-phase. After degradation of the maternal stock of
CycE, zygotic transcription of the cycE gene begins and cells
transcribing cycE continue to proliferate, while those that do not
express CycE stop proliferating. The correlation between CycE expression and
cell proliferation suggests that progression to a postmitotic state depends on
the downregulation of CycE (Richardson et
al., 1995
).
Dacapo (Dap), a protein related to the p21/p27 cyclin-dependent kinase
inhibitor (CKI) family, plays an essential role in the downregulation of CycE
(de Nooij et al., 1996;
Lane et al., 1996
). In
Drosophila dap mutant embryos, most cells in the epidermis and in the
peripheral nervous system fail to exit from the mitotic cell cycle at the
appropriate time, and subsequently undergo an additional mitotic division
(Meyer et al., 2002b
).
The Drosophila cell lineage that generates mechanosensory bristles
has been used as a model system to study cell determination, asymmetric cell
divisions and cell polarity (Jan and Jan,
1998). In the notum, each bristle is composed of four cells: two
outer cells (the socket and the shaft cells) and two inner cells (the neuron
and the sheath cell). These cells originate from a unique precursor cell, pI,
after four rounds of asymmetric divisions that occur during the pupal stages
of development. pI cells are specified in a regular pattern of rows in the
dorsocentral region of the notum around 6-9 hours after pupal formation (APF)
(Usui and Kimura, 1993
) and
begin to divide around 16-17 hours APF to generate two secondary precursor
cells, pIIa and pIIb. Later, pIIb divides before pIIa giving rise to a glial
cell and a tertiary precursor cell, pIIIb. The division of pIIa generates a
socket and a shaft cell. Next, the pIIIb cell divides to produce a neuron and
a sheath cell (Gho et al.,
1999
). Finally, the glial cell undergoes apoptosis
(Fichelson and Gho, 2003
).
Thus, only four cells of the bristle lineage ultimately form each sensory
organ. Upon completion of the lineage, cells enter a postmitotic stage in
which the expression of specific factors controls their differentiation. For
example, the Tramtrack (Ttk) transcription factor is expressed in all cells
except the neuron. Its loss of function transforms sheath cells into neurons,
while its overexpression produces the opposite effect
(Guo et al., 1995
).
We have used the Drosophila mechanosensory bristle as a model
system to study cell cycle progression in a fixed cell lineage. Previous
studies have shown that sensory mother cells are selected during the G2-phase
and arrested in this phase until they resume mitosis and initiate the lineage
(Kimura et al., 1997). Using a
real-time approach at the single cell level, we describe the timing of cell
cycle phases and their regulation in each cell of the bristle lineage. Our
data show that three types of cell cycles are present: the canonical cell
cycle (G1/S/G2/M), a cycle without the G1-phase (S/G2/M) and the endocycle
(S/G). The two former mitotic cell cycles in this lineage depend on the
differential expression of CycE and Dap. In addition, we present evidence that
Ttk, which controls non-neural cell identity acquisition, downregulates CycE
expression and probably participates in the exit of the cell from the cell
cycle and its entrance into a postmitotic state. We propose that atypical cell
cycles, together with asymmetric cell divisions, assure the rapid cadence of
cell divisions observed in this lineage.
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Materials and methods |
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Combining time-lapse in-vivo imaging and BrdU incorporation
In-vivo imaging was carried out as described previously on
neuP72> H2B::YFP pupae
(Gho et al., 1999). Imaging
was acquired every 4 minutes on an Olympus BX-41 fluorescence microscope (20
x or 40 x objective) equipped with a CoolSnap camera driven by
Metaview software (Universal Imaging). The temperature was 25°C.
Time-lapse movies were assembled using NIH image software. Sensory clusters
were identified according to their relative positions on the thorax. For each
identified cluster, time from the previous metaphase was recorded to determine
the age of the cells.
At a given time, the notum from the imaged pupae was dissected and incubated with 50 µg/ml BrdU in M3 culture medium complemented with fetal bovine serum (2%), insulin (0.1 U/ml) and ecdyson (0.5 µg/ml), for 10 or 15 minutes at 25°C. Immediately after, the notum was fixed for 20 minutes in 4% paraformaldehyde. To detect bristle lineage cells and/or specific proteins, immunoreaction was realized as described below. The notum was refixed in 4% paraformaldehyde for 15 minutes. To detect incorporated BrdU, the notum was treated twice in 2N HCl for 15 minutes each and subsequently neutralized in 0.1 mol/l Na2B4O7 for 5 minutes. BrdU was detected using mouse monoclonal anti-BrdU (Becton Dickinson 1:50), and Alexa 488- and 568-conjugated secondary antibodies (Molecular Probes, 1:1000). Time of each cell was calculated as the time determined from the in-vivo imaging plus 15 minutes, the period corresponding to the dissection and BrdU incubation. The BrdU index was calculated as the percentage of the number of BrdU-positive clusters relative to the total number of clusters analysed.
To analyse the pattern of CycE expression, in-vivo recordings were obtained as before, but the notum was dissected, immediately fixed and the immunoreaction was performed.
Immunohistology
Dissected nota from pupae at 15-35 hours APF were processed as described in
Gho et al. (Gho et al., 1996).
The following primary antibodies were used: rat anti-
-tubulin (Serotec,
1:500), rabbit anti-ß-gal (Cappel, 1:500), mouse anti-ß-gal
(Promega, 1:1000); mouse anti-Cut (DSHB, 1:500), guinea pig anti-Sens (1:1000)
(Nolo et al., 2000
), rabbit
anti-GFP (Santa-Cruz, 1:500), mouse anti-GFP (Roche, 1:500), rat anti-CycE
(gift from H. Richardson, 1:1000), mouse anti-Dap (gift from H. Hariharan,
1:4), rabbit anti-Dap (gift from C. Lehner, 1:300), rabbit anti-Lamin (gift
from P. Fisher, 1:4000), rat anti-Ttk (gift from F. Schweisguth, 1:300), rat
anti-ELAV (DSHB, 1:10), mouse anti-ELAV (DSHB, 1:100), rat anti-Su(H) (gift
from F. Schweisguth, 1:500). Alexa 488- and 568-conjugated secondary
antibodies anti-mouse, anti-rat, anti-rabbit and anti-guinea pig were
purchased from Molecular Probes and used at 1:1000. Cy5 conjugated antibodies
anti-mouse, -rat or -rabbit were provided from Promega and were used at
1:2000. Images were processed with NIH-Image and Photoshop software.
Clonal analysis and heatshock
Somatic clones were obtained using the FLP/FRT recombination system. The
w; FRT82B ttk1e11 line
(Baonza et al., 2002) was
crossed to the hs-flp, FRT82B ubq-nls::GFP (gift from J.-R. Huynh)
to generate ttk1e11 somatic clones. To induce mitotic
recombination, second instar larvae were heatshocked twice at 38°C for 30
minutes at 1 hour intervals and kept at 25°C for recovery. Heatshocked
overexpression was realized using the hs-Gal4>Ttk69 line. Pupae were
collected at puparium formation and aged to determined time. Heatshock was
performed at 38°C for 30 minutes and pupae were kept for 4 hours at
25°C for recovery.
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Results |
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A similar protocol was applied to determine S-phase timing in the tertiary precursor cell, pIIIb (Fig. 1D-F). Results of this experiment are shown in Fig. 1E and data obtained from 26 pIIb cell divisions are compiled in Fig. 1F. After pIIb mitosis, there was a short period in which no BrdU incorporation was detected in the two progeny cells (glial and pIIIb cells) (Fig. 1E, cluster 1). Fifteen minutes after pIIb telophase, all pIIIb cells were BrdU-positive (Fig. 1E, clusters 2 and 3), indicating that in pIIIb the G1-phase is present, albeit of very short duration. The end of replication, revealed by spots of BrdU incorporation (Fig. 1E, cluster 4), was detected in pIIIb cells around 1 hour after pIIb metaphase. Thus, in pIIIb cells, the S-phase lasts around 45 minutes. As this cell enters mitosis 2 hours after birth, the duration of the G2-phase is around 1 hour, similar to that observed in pIIb cells. During this entire period, we never observed BrdU incorporation in the glial cell, nor in the progeny of pIIa (socket and shaft cells).
Absence of G1-phase in the pIIa and pIIb precursor cells
The above-described experimental protocol does not allow one to detect a
G1-phase shorter than 15 minutes, the time required for dissection and BrdU
incubation. In order to reveal a possible G1-phase in the secondary precursor
cells (pIIa and pIIb), we monitored the beginning of the S-phase by analysing
BrdU incorporation directly during pI mitosis. To distinguish the pI cells and
their descendants, we used the A101 strain that specifically expresses the
lacZ gene in these cells (Usui
and Kimura, 1993). Nota from A101 pupae at 17 hours APF, around
the period of pI divisions, were dissected and incubated for 15 minutes in a
BrdU-containing culture medium. Our data show that BrdU incorporation was
already detectable in telophase cells. Telophase cells were identified by
their small nuclei and the large space between them. To confirm these
observations, triple staining was performed, labelling BrdU incorporation,
nuclear envelope formation (by detection of lamin)
(Newport and Forbes, 1987
) and
bristle lineage cells (by Sens immunoreactivity).
Fig. 2A depicts a case in which
both daughter nuclei are maximally separated; at this moment the nuclear
envelope started to reform, as revealed by lamin immunodetection (note that a
pool of lamin was still localized within the cytoplasm) and BrdU incorporation
was already observed in both nuclei. The midbody, a bundle of microtubules at
the point of cytokinesis, is another landmark of late telophase
(Saxton and McIntosh, 1987
).
Triple staining was performed, labelling BrdU incorporation, midbody (using
-tubulin antibodies) and bristle lineage cells (by ß-gal
detection). Fig. 2B shows a
typical case in which pIIa and pIIb incorporated BrdU at a moment in which a
robust midbody was still present. Moreover, ß-Gal, which is nuclear in
the A101 strain, was still present in the cytoplasm when BrdU incorporation
occurred. These data support the idea that the S-phase starts before the
entire reformation of the nucleus. Taken together, these data suggest that the
S-phase begins in late telophase of the pI division and that the G1-phase is
absent in both secondary precursor cells, pIIb and pIIa.
|
Endoreplication in pIIa progeny cells
Previous reports have shown that shaft and socket cells become polyploid by
endoreplication (Hartenstein and Posakony,
1989). To determine the timing of DNA replication in these cells,
nota from pupae between 21 and 36 hours APF were incubated in a BrdU solution
for a period of 1 or 2 hours and processed as previously described. The shaft
and socket cells were identified by their size and their posterior position in
the sensory clusters. In addition, socket cells were recognized by their
specific accumulation of Su(H) (Gho et
al., 1996
). During the period studied, only the shaft and the
socket cells incorporated BrdU. Four patterns of BrdU distribution were
observed in sensory clusters: no BrdU incorporation
(Fig. 3A), only the shaft cell
positive for BrdU (Fig. 3B),
only the socket cell positive for BrdU
(Fig. 3C) and both cells
BrdU-labelled (Fig. 3D). It is
interesting to note that the socket cells underwent endoreplication when they
were already engaged in a specific pattern of cell fate determination as
suggested by the Su(H) immunoreactivity.
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These results reveal some differences in the expression pattern of CycE and Dap (see Fig. 7). Firstly, during pI division, only CycE was present. Then, during pIIb and pIIIb divisions both factors were present. Interestingly, in pIIIb, which exhibits a G1-phase, Dap expression ceased when CycE was still strongly present. During pIIa division, Dap was present while CycE was no longer detected. Finally, we never detected Dap in late pIIa progeny cells.
The delayed entry into S-phase in pIIIb cells arises from Dap-inhibition of CycE/Cdk2 activity
In order to determine whether the differences in Dap and CycE expression
can account for the acquisition of a G1-phase in the bristle lineage, we
tested the possibility that the G1-phase arises in pIIIb due to the inhibition
of the Cdk2/CycE complex by Dap. In an attempt to override the Dap-mediated
inhibition, we analysed the G1-phase in pIIIb in a null dap
background using the dap4 strain
(Hong et al., 2003) in which
some homozygous escapers are viable until the pupal stage. Short duration BrdU
incorporations were performed to analyse the BrdU incorporation pattern in
three-cell clusters. This configuration ensures that the analysis was
performed just after the pIIb division and before the pIIa division.
Fig. 5A shows that BrdU
incorporation took place during pIIb division when the midbody was still
present. In the three-cell clusters analysed, 100% of the pIIIb cells and 93%
of the glial cells were BrdU-labelled (n=56). These data show that,
in dap null mutants, an S-phase can be induced late in telophase in
the two daughter cells arising from the pIIb division: a situation never
observed in control animals.
|
High levels of CycE in the absence of Dap account for the absence of the G1-phase in secondary precursor cells
The Dap-dependent appearance of a G1-phase in the pIIIb cell suggests that
the direct entry into the S-phase after pI division is due to the absence of
this inhibitor. In order to test this hypothesis, we overexpressed Dap and
determined whether or not an S-phase would occur during pI telophase as in the
control situation. Experiments involving BrdU incorporation under conditions
of Dap overexpression showed that at around 17 hours APF there was no DNA
replication during pI telophase (Fig.
5D). This observation was confirmed in 100% of the late pI
telophases analysed, a situation never observed in control conditions (see
Fig. 2B). This absence of
replication was correlated with an equal distribution of Dap immunoreactivity
in both pI daughter cells. However, the S-phase was not abolished, as BrdU
incorporation was observed in pIIa and pIIb cells at later stages (two-cell
clusters without midbody) (not shown). Taken together, these results show that
the balance of the activity between CycE/Cdk2 and Dap regulates the structure
of the cell cycle in the bristle lineage cells. When CycE/Cdk2 overrides the
inhibitory action of Dap, an S-phase is triggered (as is the case for pIIb,
pIIa and in late pIIIb). However, when Dap controls the activity of this
complex, no S-phase occurs and cells enter the G1-phase (as is the case for
pIIIb) or become postmitotic (the terminal cells of the lineage).
CycE expression is controlled by Tramtrack p69
CycE protein was always re-expressed before pIIb and pIIIb mitosis but
never before pIIa mitosis. We investigated whether a regulating factor ensured
either the inhibition of cycE expression in pIIa cells and/or the
induction of this expression in pIIb and pIIIb cells. In the embryonic nervous
system, it has been shown that overexpression of the Ttk transcription factor
blocks glial development by repressing expression of cycE
(Badenhorst, 2001). As such we
investigated the possibility that Ttk could prevent cycE expression
in late pIIa cells and its descendants where CycE is not detected. In order to
analyse this hypothesis, we studied the expression of Ttk in the bristle
lineage. The ttk gene encodes two products, Ttk69 and Ttk88, via
alternatively splicing (Read and Manley,
1992
). An analysis of Ttk69 expression in bristle lineage cells
revealed that this protein is expressed in the pIIa cell
(Fig. 6A,B) and in its progeny,
the shaft and the socket cells (Fig.
6C). After the pIIIb division, the sheath cell also becomes Ttk69
immuno-positive (Fig. 6C)
(Lehembre et al., 2000
). Thus,
the expression pattern of Ttk in pIIa and its descendent is consistent with
the possibility that Ttk prevents CycE expression in these cells. Previous
clonal analysis, using the ttkosn allele, which affects
both Ttk69 and Ttk88 proteins, revealed patches of adult cuticule devoid of
outer support cells. In addition, sensory organs were composed only of
neurons, suggesting a transformation of pIIa into pIIb cells
(Guo et al., 1995
). This cell
fate change prevents the analysis of CycE expression in a ttk null
background. Thus, we analysed CycE expression in ttk1e11
somatic clones at 20 hours APF in which the Ttk69 product is specifically
depleted (Lai and Li, 1999
).
Accordingly, in ttk1e11 somatic clones, no Ttk69 was
detected as revealed by the lack of immunoreactivity
(Fig. 6E, arrows). In these
clones, the existence of pIIa progeny in sensory organs was confirmed by the
presence of both Prospero-negative cells
(Fig. 6G", arrowheads)
(Gho et al., 1999
) and
Su(H)-positive socket cells (Fig.
6F, arrows) as well as socket cuticular structures in adult
animals (not shown). These data show that Ttk69 is dispensable for the
acquisition of pIIa fate and allow us to analyse, in a ttk69 mutant
background, the role of Ttk in CycE expression. We observed that inside
ttk1e11 clones, all sensory cells expressed CycE. In
particular, the two pIIa daughter cells, identified by the absence of Prospero
immunoreactivity, strongly accumulated CycE, a situation never observed in
non-clonal tissues (Fig.
6G-G"', arrows). Conversely, overexpression of the active
form of Ttk69 prevented the expression of CycE. Thus, the expression of CycE
in pIIIb, which was always observed in three-cell clusters, was abolished
after overexpression of Ttk69 (Fig.
6D). Taken together these data indicate that Ttk regulates CycE
expression in the bristle lineage. In particular, it prevents the expression
of CycE in pIIa and its descendants. It is important to note that late in
development (up to 24 hours APF), CycE was not detected in sensory organ cells
inside the Ttk clone. This indicates that, in ttk1e11
clones, the CycE upregulation was transitory, suggesting that additional
mechanisms acting late in development can be involved in the control of CycE
expression.
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Discussion |
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Our data show that a similar expression of CycE, peaking during each mitosis, was observed in pI, pIIb and pIIIb cells, which exhibited atypical and canonical cell cycles. Moreover, Dap expression anticipated each mitosis, with the exception of the pI division, in which Dap was not observed. Thus, in the bristle lineage, Dap was expressed only during cell divisions giving rise to at least one postmitotic daughter cell. Therefore, one could hypothesise that Dap expression was controlled by a mechanism that counts cell divisions. In fact, this is not the case, as cell-cycle-arrested pI cells expressed Dap at later stages (Pierre Fichelson and M.G., unpublished). This observation suggests that Dacapo expression is controlled by a transcription factor with activity that changes progressively over time or by a cascade of sequentially activated factors acting independently of cell division.
We observed that neither the CycE nor the Dap proteins were detected in the
shaft and socket cells during the period of endoreplication. This is
unexpected, as it has been shown that one central regulator of Drosophila
endocycles is the complex CycE/Cdk2
(Knoblich et al., 1994).
However, in salivary glands and in ovary nurse cells, a downregulation of the
CycE activity occurs to allow subsequent DNA replication
(Sauer et al., 1995
). During
embryogenesis, progression through endoreplication requires the CycD/Cdk4
complex (Meyer et al., 2002a
).
Further analysis in our system, particularly concerning the function of the
complex CycD/Cdk4, is necessary to reveal the mechanism underlying the cycles
of endoreplications observed in the shaft and socket cells.
An atypical cell cycle without G1-phases in pIIa and pIIb
Several pieces of evidence indicate that the G1-phase is absent in
secondary precursor cells and that these cells enter the S-phase directly.
Thus, we observed pIIa and pIIb nuclei incorporate BrdU during late pI
telophase, which was distinguishable by (1) the presence of the midbody and
(2) the cytoplasmic location of the nuclear envelope component lamin. That
replication can occur before the end of mitosis has been observed in the
embryonic cell cycle of Xenopus. In these cells, replication starts
at early telophase. At this time, each chromosome becomes surrounded by a
lamin-containing nuclear envelope, the karyomere
(Lemaitre et al., 1998). This
situation was never found during pI mitosis in the bristle lineage. Indeed, we
observed that the nuclear envelope was formed around the entire chromosome set
when we observed BrdU incorporation.
In Drosophila, the master cyclin/Cdk complex involved in the G1-
to S-transition is the CycE/Cdk2 complex
(Knoblich et al., 1994;
Meyer et al., 2002a
).
Sustained expression of CycE has been observed during the rapid early
divisions in Drosophila embryos. It is only after the destruction of
the maternal stock and the zygotic activation of string and
cycE genes that G2- and G1-phases appear in late embryos and larvae,
respectively (Edgar and Lehner,
1996
; Richardson et al.,
1995
). We observed that in the bristle lineage CycE expression
preceded pI division. This expression was required for direct transition to
the S-phase after pI mitosis. Thus, the S-phase was delayed after a reduction
in the CycE activity produced by Dap overexpression. Therefore, sustained
expression of CycE before and after mitosis ensures the direct transition from
M- to S-phases in both the early embryo and the bristle cell lineage.
High levels of CycE are necessary but not sufficient to trigger the
S-phase. For example, pI cells, which are arrested in G2
(Kimura et al., 1997), express
CycE around 2 hours before division. However, these cells do not enter the
S-phase. Similar observations have been made in amnioserosa cells and
non-proliferating pole cells (Richardson
et al., 1993
; Su et al.,
1998
). This refractory property is certainly due to the mechanism
that prevents DNA replication during G2-arrested cells. This mechanism exerts
a control over the licensing factors through mitotic Cdk/cyclin complexes
rather than inactivating the CycE activity
(Sauer et al., 1995
).
A dispensable G1-phase in pIIIb
CycE expression anticipates pIIb division, which confers to its progeny
(the pIIIb and the glial cell) the capacity to precociously enter the S-phase,
as is the case for secondary precursor cells resulting from the pI division.
However, both pIIb descendents express Dap and do not enter the S-phase. Our
data suggest that Dap expression downregulates CycE activity in glial cells,
which rapidly enter a postmitotic state. Dap expression is required to trigger
a postmitotic state, as S-phases can be induced in glial cells in a
dap null mutant homozygous background. Furthermore, glial cell
division was occasionally observed after CycE overexpression in a dap
mutant background (A.A., F.S. and Pierre Fichelson, unpublished). We think
that the ephemeral G1-phase present in pIIIb is due to the fortuitous
inheritance of Dap, which triggers a postmitotic state in its sister cell, the
glia. No change in pIIIb fate was observed after reduction of the G1 duration,
indicating that the G1-phase is dispensable for the identity of this cell.
Thus, the G1-phase in pIIIb seems to be a secondary effect of the glial cell
commitment to a postmitotic state. Taken together, our results suggest that,
in non-terminal cells, the expression of CycE late in the G2-phase leads to
the next cell cycle essentially devoid of G1-phase.
Tramtrack-69 downregulates Cyclin E activity
The ttk gene encodes two alternatively spliced zinc-finger
transcription factors, Ttk69 and Ttk88
(Read and Manley, 1992).
Previous analyses have shown that Ttk products act downstream of Notch and are
involved in the control of non-neural cell fates
(Guo et al., 1995
). The
persistent expression of Ttk69 in the socket, shaft and late sheath cells is
coherent with this function. Surprisingly, our clonal analysis using the
ttk1e11 allele, which affects specifically Ttk69
(Lai and Li, 1999
), showed
that socket cells and neurons are found in sensory organs inside ttk
clones. These results suggested that Ttk69 is dispensable for acquisition of
socket identity. Clonal analysis using ttk alleles affecting
specifically Ttk88 will be required to determine whether this form is
necessary or only sufficient to determine a socket fate.
In the embryonic central nervous system, it has been proposed that Ttk69,
apart from its role as a determinant, controls cell proliferation. This
control seems to involve the repression of cycE and string
gene expression (Badenhorst,
2001; Baonza et al.,
2002
). We took advantage of the fact that the pIIa fate was not
abolished in ttk1e11 to analyse the role of Ttk in
cycE expression. We have shown that, in a ttk1e11
mutant background where Ttk69 is lost, pIIa daughter cells ectopically
expressed CycE. Conversely, overexpression of Ttk69 resulted in a loss of
cycE expression in pIIb cells. These results show that the Ttk
expression in pIIa and its daughter cells prevents expression of CycE. The
effect of Ttk69 on CycE was transitory, and CycE accumulation in pIIa progeny
cells was not observed in ttk1e11 clones at 24 hours APF.
Although CycE ectopic expression was transitory, we have observed
supplementary cell divisions in sensory organs inside
ttk1e11 clones. Accordingly, 80% of clonal sensory organs
were composed of five to six cells, compared with only four in control
clusters (data not shown). The transitory ectopic expression of CycE suggests
that Ttk69 is responsible only in part for downregulation of cycE in
pIIa progeny. Thus, additional mechanisms must also be involved in the control
of CycE expression. In particular, one possibility is that Ttk88 can
compensate for the lack of Ttk69. Unfortunately, the role of Ttk in
determining cell fate identity prevents us from analysing CycE expression in a
completely null ttk background. In any case, our results suggest
that, in the bristle lineage, apart from its role in establishing cell fate
identity, Ttk is involved in the cell cycle exit, and/or maintenance of a
postmitotic state, via the downregulation of CycE.
Atypical cell cycles and asymmetric cell divisions assure a rapid cadence of cell divisions in the bristle lineage
The period between each division in the bristle lineage is around 2.5
hours. This high rate of cell division suggests that in this lineage cell size
is not a limiting factor for cell cycle progression. At each division,
Notch-mediated cell decisions are biased by the presence in one daughter cell
of Numb and Neuralized that assure the activation of the Notch pathway in the
other daughter cell (Jan and Jan,
1998; Le Borgne and
Schweisguth, 2003
). Thus, the asymmetric nature of cell divisions
in the bristle lineage could shortening the amount of time required for the
activation of the Notch pathway in one daughter cell. In addition, previous
analysis of the Notch-mediated cell decisions during vulva formation of
Caenorhabditis elegans (Ambros,
1999
) suggests that the Notch response can be implemented during
the S-/G2-phase. Therefore, it is expected that the reduction, or even the
elimination, of the G1-phase does not have an impact on the Notch-mediated
cell fate decisions. Taken together, these data suggest that the asymmetry in
cell divisions and the absence of the G1-phase can be seen as mechanisms that
have been introduced into stochastic cell lineages during evolution, thus
allowing a very rapid succession of cell divisions.
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ACKNOWLEDGMENTS |
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REFERENCES |
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