1 Section of Molecular, Cell and Developmental Biology, and Institute for
Cellular and Molecular Biology, The University of Texas at Austin, 1
University Station C-0930, Austin, TX 78712-0253, USA
2 Department of Developmental and Molecular Biology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
3 Department of Molecular Genetics, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA
Author for correspondence (e-mail:
d.stein{at}mail.utexas.edu)
Accepted 20 October 2003
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SUMMARY |
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Key words: Myc, dmyc, Proto-oncogene, Nurse cells, Follicle, Egg chamber, Gene amplification, Dacapo, Cyclin E, Polyploidization, Endoreduplication
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Introduction |
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Myc is an essential gene in vertebrates; mice mutant for
Myc die during embryogenesis
(Davis et al., 1993) and
exhibit reduced cell numbers in many developing organs
(Trumpp et al., 2001
). The
function of the Myc oncoprotein in vertebrate cells has been correlated with
the promotion of cell cycle progression; for example, Myc expression
is rapidly upregulated in tissue culture cells exposed to growth factors
(Kelly et al., 1983
), and
overexpression of Myc can force some quiescent cells to enter into S
phase (Eilers et al., 1991
).
In addition to influencing the proliferation of vertebrate cells,
overexpression of Myc can inhibit terminal differentiation
(Coppola and Cole, 1986
), and
in some cases drives cells into apoptosis
(Prendergast, 1999
).
The ability of Myc to participate in these diverse cellular processes is
thought to depend on its function as a sequence-specific transcription factor
of the basic region/helix-loophelix/leucine zipper (bHLH/LZ) class
(Kretzner et al., 1992). Myc
acts as a transcriptional activator in heterodimers with the bHLH/LZ protein
Max, with the two proteins interacting via their LZ domains. It is this
Myc-Max dimer that binds to the E box DNA consensus site CACGTG through bHLH
sequences (Amati and Land,
1994
; Blackwood et al.,
1992
). Early studies of Myc transcriptional regulatory activity
focused on identifying target genes that control cell cycle progression.
However, an accumulating body of evidence now suggests that Myc and its
binding partners regulate the expression of a large number of genes that
regulate diverse functions, including protein synthesis, apoptosis, and DNA
and energy metabolism (Boon et al.,
2001
; Guo et al.,
2000
; Neiman et al.,
2001
; Orian et al.,
2003
; Schuldiner and
Benvenisty, 2001
; Watson et
al., 2002
). Because the transcriptional activation induced by
Myc-Max heterodimers is relatively weak and variable, it has been difficult to
confirm potential target genes. Furthermore, recent data suggests that the
transcriptional activity mediated by Myc may arise from its ability to
influence levels of histone modification and alter chromatin structure
(Amati et al., 2001
;
Eisenman, 2001
). Taken
together, these studies illustrate the complexity of Myc function, and
indicate that much remains to be learned about the normal role of Myc in the
life of the cell.
To take advantage of the powerful genetics available in the
Drosophila system to study the role of Myc proteins in development
and growth, we and others have cloned the single gene encoding a Myc family
protein in Drosophila (dMyc), and have shown that it corresponds to
the diminutive (dm) locus
(Johnston et al., 1999;
Schreiber-Agus et al., 1997
).
Flies homozygous or hemizygous for dm1 are small, with
slender bristles, indicating that dMyc is required for organismal and cellular
growth. In fly imaginal discs, a reduction in dMyc function results in defects
in cell growth but not proliferation; these cells divide at apparently normal
rates but fail to increase in mass
(Johnston et al., 1999
).
At the time dMyc was cloned, the hypomorphic dm1 mutation was the only dm allele available. To elucidate the function of dMyc in Drosophila, we generated a strong dm allele, dm2, that is homozygous lethal. Using this allele, which behaves genetically as a null mutation, we have characterized the role of dMyc in Drosophila oogenesis, a process that encompasses cell proliferation, growth and differentiation. Using mosaic analysis, we show that dMyc is specifically required for cell growth and DNA endoreplication in both the germline and the somatic follicle cells. In contrast to these defects, cell proliferation in both types of cell was unaffected by reduced dMyc activity. Furthermore, although their ability to endoreplicate was severely compromised, dm2 mutant follicle cells appeared to carry out gene amplification normally. Finally, we show that when either the germline or the soma is mutant for dm2, the entire follicle is severely delayed in its progression through oogenesis, which indicates that the growth of the germline and the somatic follicle cells is interdependent.
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Materials and methods |
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Screen for lethal alleles of dm
To isolate lethal mutations in dm, we employed a strategy that
would enable us to specifically select for X-linked lethal mutations that are
rescued by the presence of Dp(1;2)51b, which carries the dm
locus as well as several neighboring loci, including the white (w)
gene. y w males were mutagenized with 25 mM ethyl methansulfonate and
mated to C(1)DX, y w f; Dp(1;2)51b/+ females. 2763 F1 males
of the genotype y w */Y; Dp(1;2)51b/+, (where *
represents a newly mutagenized chromosome), were mated individually to
C(1)DX, y w f; Dp(1;2)51b/+ females. 1823 of these crosses produced
progeny, and these were scored for the presence or absence of
w- males [lacking Dp(1;2)51b]. The presence of
w- males indicated that the X chromosome carried no lethal
mutations, whereas the absence of w- males implied that
the X chromosome carried a lethal mutation that was rescued by the presence of
Dp(1;2)51b. Lines lacking w- males were then
crossed to dm1-carrying females to test for
noncomplementation. Two of the newly-induced lethal mutations proved to be
allelic to dm1. The trans-heterozygous females were small
with slender bristles and were sterile. These alleles were named
dm2 and dm3.
To sequence these alleles, stocks were generated in which the lethal
dm allele was carried in trans to an FM7 balancer carrying a
Krüppel-Gal4, UAS-Green Fluorescent Protein
(Kr-GFP) reporter gene (Casso et
al., 2000). Mutant larvae, identified by their lack of
fluorescence, were collected and DNA was prepared from them. We then used PCR
to amplify the dm coding regions, and the DNA products of these
reactions were sequenced directly.
Generation of germline and follicle cell mitotic clones
Homozygous dm mutant germline and follicle cell clones were
generated by FLP/FRT-mediated site-specific recombination
(Duffy et al., 1998;
Xu and Rubin, 1993
). Mosaic
females carried a dm mutation and the 18D FRT in trans to a
chromosome carrying the 18D FRT and a hGFP reporter gene
(Clarkson and Saint, 1999
).
They carried FLPase on the second chromosome, either under the control of the
heat shock promoter (hs-FLP), or expressed from a UAS-FLP
construct by the somatically-expressed e22c-Gal4 enhancer trap
insertion (Duffy et al.,
1998
). Females carrying hs-FLP were heat shocked for 1
hour at 37°C twice daily for two days. Following heat shock, females were
fed on yeast for 2-10 days, and their ovaries were dissected and examined by
immunohistochemistry. dm mutant cells were identified by the absence
of GFP expression. Staging of egg chambers was carried out according to
Spradling (Spradling,
1993
).
Immunohistochemistry and BrdU labeling
Immunohistochemistry was carried out as described previously
(Niewiadomska et al., 1999),
with the following modifications. For immunostaining, ovaries were fixed for
15 minutes in 5% paraformaldehyde in PBS plus heptane. Prior to staining,
ovaries were blocked by incubation in PBS with 0.1% Tween-20 and 1% BSA. The
following primary antibodies were used: mouse monoclonal anti-Fasciclin III
(1:50 primary dilution) (Patel et al.,
1987
), provided by the Developmental Studies Hybridoma Bank under
the auspices of the NICHD and maintained by the University of Iowa; rabbit
anti-Dacapo (1:500 primary dilution), provided by Christian Lehner
(Lane et al., 1996
); mouse
monoclonal anti-Cyclin E (1:5), provided by Helena Richardson
(Richardson et al., 1995
);
rabbit anti-ORC2 (1:2500), provided by Stephen Bell
(Royzman et al., 1999
);
anti-Broad Complex (1:100), from Greg Guild
(Emery et al., 1994
); rabbit
anti-phosphohistone H3 (1:500) (Upstate Biotechnology); and rabbit anti-dMyc
(1:5000). Antisera against dMyc protein was generated in rabbits following the
injection of a histidine-tagged fusion protein encoding amino acids 80-322 of
dMyc. Rhodamine or Alexa 594-conjugated secondary antibodies were used at a
dilution of 1:500 (Molecular Probes), for the detection of all primary
antibodies.
BrdU labeling was carried out as described
(Calvi et al., 1998), with the
following modifications. Dissected ovaries were incubated in 1 mg/ml BrdU
(Sigma) in Grace's Medium (Mediatech) for 1 hour at room temperature, followed
by fixation in 5% paraformaldehyde in PBS plus heptane for 10 minutes. Fixed
ovaries were then incubated for 1 hour in PBS with 0.6% Triton X-100, followed
by incubation in DNase buffer [66 mM Tris (pH 7.5), 5 mM MgCl2, 1
mM 2-mercaptoethanol] with 100 U DNase I (Roche). BrdU was detected by a mouse
anti-BrdU antibody (Becton Dickinson) used at a dilution of 1:50, as described
above. Images were captured using a Zeiss Axioplan 2i microscope equipped with
a Zeiss Axiocam digital camera and edited for publication using Adobe
Photoshop.
For the comparison of nuclear sizes in different genetic backgrounds, nuclear area was determined by pixel density of DAPI-stained nuclei using the UTHSCSA Image Tool for Windows Version 1.28 (University of Texas Health Science Center, San Antonio). GFP images of the same nuclei were used to determine genotype.
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Results |
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To determine the lethal period of flies hemizygous for dm2, stocks carrying dm2 in trans to an FM7 balancer expressing a Kr-GFP reporter gene were constructed, which enabled dm2 hemizygous mutant eggs to be identified by their lack of fluorescence. Although dm2 eggs hatched at near normal rates, and many mutant larvae were viable for several days following hatching, they remained small and only about 20% of mutant larvae progressed to pupation. The few pupae that formed were extremely small relative to wild type (Fig. 1C), and none of the mutant pupae eclosed as adult flies.
To investigate whether embryonic development requires maternal loading of
dMyc mRNA, we used the dominant female sterile technique
(Chou and Perrimon, 1996) to
generate germline clones of dm2. However, we found that
oogenesis in these females arrested relatively early and they did not lay eggs
(data not shown). This indicated that dm expression is required in
the germline for the progression of oogenesis. Consequently, we were not able
to determine whether maternally-expressed dMyc is required for embryonic
development.
dMyc is required cell-autonomously in the female germline for growth and endoreplication, but not for cell division
Antibody staining revealed that dMyc protein is abundantly expressed, and
localized to the nuclei of both nurse cells and follicle cells during
oogenesis (Fig. 2A). To
demonstrate the specificity of the antibodies for dMyc, we generated
homozygous-mutant follicle cell clones of dm1, which has
previously been shown to cause a reduction in the expression of dm
RNA in the ovary (Gallant et al.,
1996). Consistent with this,
dm1/dm1 mutant clones exhibited a
strong reduction of staining compared with their wild-type neighbors
(Fig. 2B,C). Antibody staining
was also lost in follicle cells clones homozygous for
dmPG45 (Fig.
2D,E), another lethal allele of dm that has recently been
identified (Bourbon et al.,
2002
), which results from a transposon insertion in the locus.
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The most prominent feature of the dm2 mutant germline clones was the failure of the nurse cell nuclei to undergo their normal dramatic increase in size, suggesting a defect in growth or endoreplication. Following the completion of the mitotic divisions that form the 16-cell germline cyst, the nurse cells undergo a period of rapid cell growth and endoreplication, in which DNA is replicated in the absence of cell division. To assess the ability of the dm2 mutant cells to endoreplicate, we measured bromo-deoxyuridine (BrdU) incorporation in stage 5-8 egg chambers containing germline clones. Because nurse cells become post-mitotic prior to stage 3 of oogenesis, incorporation of BrdU in stage 5-8 egg chambers is a direct measure of endoreplication-associated DNA synthesis. In egg chambers with germline clones of a control chromosome, 28% of the cells were seen to have incorporated BrdU during the one hour labeling period, whereas only 10% of the dm2 mutant cells had (Table 1). This result indicates that dMyc function is required for germline cells to carry out the endoreplication cycles required for normal oogenesis.
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Absence of dMyc function in the germline is not associated with changes in expression of the cell cycle regulators Cyclin E or Dacapo
Endoreplication in nurse cells is a regulated cell cycle that is thought to
be controlled by the relative levels of the G1 cyclin, Cyclin E, and by Cyclin
dependent kinase 2 (Cdk2) (Edgar and
Orr-Weaver, 2001). Cycles of Cyclin E protein expression and
breakdown (Lilly and Spradling,
1996
) produce cyclical Cdk2 activity, which is believed to be
necessary for the phosphorylation of components of the pre-replication complex
and the initiation of DNA replication at each endoreplication cycle
(Edgar and Orr-Weaver, 2001
;
Woo and Poon, 2003
). To
determine whether the reduced ability of dm2 mutant nurse
cells to endoreplicate is due to alterations in Cyclin E expression, we used
antibodies against Cyclin E to examine its expression in mosaic germline
cysts. In wild-type cells, because Cyclin E is expressed in a cyclical pattern
(Lilly and Spradling, 1996
),
only a subset of cells is stained by antibodies against Cyclin E at a given
time. In mosaic germline cysts, a subset of the cells of each genotype,
dm2/dm2 and +/+, were positive for
Cyclin E staining (Fig. 4F),
consistent with a cyclical pattern of expression. Thirty-seven out of 131
(28%) dm2 nurse cells, and 74 out of 199 (37%) wild-type
nurse cells, counted in mosaic germline cysts exhibited Cyclin E expression.
The staining observed was of approximately equal intensity in
dm2 versus wild-type nurse cells. Although the basis for
the difference in the proportion of dm2 and wild-type
nurse cells expressing Cyclin E is not clear, this result suggests that Cyclin
E expression cycles relatively normally in dm2 mutant
cells and that a failure in the periodic expression of Cyclin E is not
responsible for the reduced ability of these cells to undergo
endoreplication.
The activity of the Drosophila Cyclin E/Cdk2 complex is inhibited
by Dacapo, the p27cip/kip ortholog
(de Nooij et al., 1996;
Lane et al., 1996
). Like
Cyclin E, Dacapo protein levels oscillate in a periodic manner, but slightly
out of phase with Cyclin E (de Nooij et
al., 2000
). To investigate a possible influence of dMyc on Dacapo
protein expression, we stained mixed germline cysts with antibodies against
Dacapo. Similar to what we observed for Cyclin E, Dacapo protein was detected
in a subset of both dm2 and wild-type nurse cells
(Fig. 4I), consistent with an
oscillating pattern of protein expression. This indicates that the reduced
endoreplication seen in dm2 mutant nurse cells does not
arise through alterations in the expression of Dacapo protein. These results,
together with the ones described above, also indicate that, in these cells,
dMyc is not directly required for the expression of either Dacapo or Cyclin
E.
Taken together, the studies described above demonstrate that dMyc activity is not required in the germline for the mitotic divisions that generate the 16-cell cyst, nor for the specification of the oocyte. By contrast, dMyc plays an essential role during the stages of oogenesis in which growth and DNA endoreplication occur in the nurse cells. Although dm2 mutant nurse cells are capable of undergoing the transition from the mitotic cycle to endoreplication, the number of endocycles that occur in these cells is dramatically reduced relative to wild-type nurse cells.
Follicle cell growth and endoreplication requires dMyc function
Endoreplication is also carried out by the somatically-derived follicle
cells that surround the germline cells during oogenesis
(Calvi et al., 1998). Follicle
cell precursors first associate with the germline cyst shortly after the
fourth mitotic division that generates the 16-cell cyst. The somatic cells
associated with each cyst divide approximately eight times during stages 2-6,
to produce an epithelium of about 1200 follicle cells per egg chamber
(Margolis and Spradling,
1995
). Upon completion of these mitotic divisions, the follicle
cells execute three rounds of endoreplication, which increases their ploidy to
16C (Calvi et al., 1998
).
Similar to our observations of nurse cells, we find that dMyc is required for
efficient DNA endoreplication in follicle cells, but seems not to be essential
for their mitotic proliferation. As described for the generation of germline
clones, we used FLP/FRT-mediated site-specific recombination to produce marked
clones of dm2/dm2 mutant follicle
cells. We recovered clones containing 10-20 mutant cells or more
(Fig. 5A), which indicates that
the loss of dMyc activity does not prevent the cells from carrying out several
mitotic division cycles. In control experiments we generated marked wild-type
clones that contained somewhat larger numbers of cells (data not shown). This
observation may reflect a slightly depressed proliferative ability of the
dm2/dm2 cells, compared with
dm2/+ cells, attributable to their decreased growth rates.
A similar situation has been observed in dm mutant clones generated
in other tissues (Johnston et al.,
1999
).
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Cell growth and endoreplication are intimately linked cellular processes
(Edgar and Orr-Weaver, 2001).
Thus, a failure to grow can lead to impaired endoreplication, and vice versa.
In the germline, endoreplication begins shortly after the formation of the
16-cell cyst, making it difficult to ascertain whether the effect of the
dm2 mutation on endoreplication is a direct or indirect
result of reduced growth. In the follicle cell layer, however, we detected a
decrease in the size of dm2 mutant follicle cells relative
to wild-type cells at stage 5, prior to the time at which they begin
endoreplication (Fig. 5G,H).
This finding indicates that the loss of dMyc function directly affects the
ability of the follicle cells to grow, with the effects on endoreplication
potentially being a secondary consequence of this effect.
Chorion gene amplification does not require dMyc function and is independent of endoreplication
Late in oogenesis, the follicle cells are responsible for the secretion of
the eggshell, which is composed of the vitelline membrane, the endochorion and
the exochorion. To facilitate the synthesis of large amounts of chorion
proteins, the follicle cells specifically amplify two clusters of chorion
genes, located on the X and the third chromosomes, as well as two other loci
that have not yet been identified (Calvi et
al., 1998). Although the precise mechanisms that regulate chorion
gene amplification have not been elucidated, Cyclin E activity is known to be
required both to promote amplification of the appropriate gene clusters, and
to prevent the amplification of other genes elsewhere on the chromosomes
(Calvi et al., 1998
).
Chorion gene amplification can be visualized through BrdU incorporation,
and appears as small and discrete foci that stain with antibodies against
BrdU. In stage 11 mosaic egg chambers in which we assessed BrdU incorporation,
we observed that BrdU-staining puncta were present not only in wild-type
follicle cells, but also in dm2/dm2
homozygous mutant cells (Fig.
6D). To obtain supporting evidence that these puncta represented
sites of gene amplification, we stained mosaic follicles with an antibody
directed against a component of the origin recognition complex (ORC2), which
has been shown to be associated with chorion clusters that are undergoing
amplification (Royzman et al.,
1999). Like BrdU, discrete foci of ORC2 staining were observed in
both wild-type and dm2/dm2 mutant
follicle cells (data not shown). These observations indicate that, despite the
inability of dm2 mutant follicle cells to undergo a normal
course of DNA endoreplication, they are nevertheless able to carry out gene
amplification. To our knowledge, dm represents the first identified
mutation that perturbs the follicle cell endocycle without eliminating gene
amplification.
One of the loci thought to be required for chorion gene amplification is
the Broad-Complex (BR-C; broad - Flybase), an early
ecdysone-response gene that acts at many times in the fly life cycle and which
encodes a family of zinc-finger transcription factors. A mutation in the
BR-C locus causes premature arrest of chorion gene amplification,
whereas overexpression of BR-C isoforms leads to the formation of
additional foci of BrdU incorporation in follicle cells that presumably
represent inappropriate sites of gene amplification
(Buszczak et al., 1999).
Consistent with their apparent ability to carry out chorion gene
amplification, we found that dm2/dm2
mutant follicle cells exhibited normal BR-C protein expression (data not
shown).
Loss of dMyc activity in either the germline or the follicle cell layer severely delays the maturation of the entire egg chamber
In addition to the cell-autonomous effects of the loss of dMyc on
dm2 mutant cells, we found that when either the germline
or the follicle cell layer lacked dMyc function the maturation of the entire
follicle was affected. Egg chambers in which all germline cells were
homozygous for dm2 remained small and appeared to be
immature relative to their age (Fig.
7A,B). Follicles that, on the basis of their position in the
ovariole, should be between stages 8-10, instead had the appearance of stage
6-7 follicles. The wild-type follicle cells that surrounded the mutant
germline exhibited unusual patterns of gene expression. For example, Fasciclin
III (Fas III), a cell adhesion marker that is expressed by all follicle cells
very early in oogenesis, is normally downregulated so that only the two polar
cells at each end of the follicle express high levels of Fas III
(López-Schier and St Johnston,
2001). In egg chambers in which all the germline cells were mutant
for dm2, the entire follicle cell layer expressed
inappropriately high levels of Fas III
(Fig. 7B). By contrast,
follicle cell expression of BR-C, which is normally initiated during stage 6
in wild-type follicles, was observed to initiate in egg chambers that contain
dm2 mutant germ cells, despite their failure to grow and
mature normally (Fig. 7D).
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Discussion |
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The phenotype of dm2 mutant cells in the ovary is
consistent with a requirement for dMyc function in both cell growth and the
endocycle, two processes which are interdependent. Conditions that block cell
growth invariably perturb endocycle progression
(Galloni and Edgar, 1999).
Conversely, the inhibition of DNA synthesis during the endocyle, through the
expression of inhibitors of DNA replication or by mutating genes essential for
DNA replication, leads not only to a slowed increase in DNA content but also
to a decrease in overall cell growth (reviewed by
Edgar and Orr-Weaver, 2001
).
We favor the idea that dMyc activity is required initially to enhance growth,
with the effects of dMyc loss-of-function on endoreplication being a secondary
consequence of impaired growth. At oogenic stages prior to the onset of
endoreplication in the follicular epithelium, dm2 mutant
follicle cells are detectably smaller than their wild-type neighbors. This
indicates that reduced dMyc function affects cell growth prior to, and
independently of, its effect on endoreplication. Because of the reciprocal
relationship between growth and endoreplication, the initial growth defect in
dm2 cells may lead to endoreplication defects that then
feed back and contribute to further reductions in cell growth. Ultimately this
growth defect could have the effect of greatly reducing the number of
endoreplication cycles that dm2 mutant cells can complete.
However, we cannot rule out a direct effect of dMyc on endoreplication, in
addition to its influence on growth.
Although endoreplication is severely impaired when dMyc activity is
reduced, dm mutant cells maintain a limited ability to endocycle.
During a one-hour pulse of BrdU, dm2 mutant germline cells
were 3- to 5-fold less likely than wild-type cells to be in the S phase of the
endocycle, as measured by BrdU incorporation. This suggests that relative to
wild-type cells, dm2 mutant cells spend a longer fraction
of the endocycle in G1 phase and a shorter proportion in S phase. A simple
explanation for this observation would be that endoreplicating cells remain in
G1 until they reach a critical metabolic or growth threshold required for the
onset of DNA synthesis, and the rate at which they reach this threshold
depends on the level of dMyc activity. This implies that an important function
of dMyc in the ovary is to promote growth during G1 so that the G1/S
progression can occur. This interpretation would be consistent with the
observation by Johnston et al. (Johnston
et al., 1999) that overexpressing dMyc in wing disc cells
decreases the proportion of the cell cycle spent in G1.
In contrast to its dramatic effects on cell growth and endoreplication, dMyc appears to be largely dispensable for the mitotic proliferation of both germline and somatic cells. Not only did we recover germline and follicle cell clones that resulted from mitotic recombination subsequent to egg chamber formation, we also identified clones that had been produced during the division of the stem cell precursors of these two cell types. This indicates that ovarian cells are capable of dividing many times in the absence of wild-type dMyc activity, which strongly argues against a role for dMyc in mitotic cell cycles in the ovary. Although we favor the notion that mitotic proliferation of these cells can occur in the total absence of dMyc activity, we cannot currently rule out the possibility that the mitotic proliferation that we observe is supported by residual activity associated with the truncated protein produced by the dm2 mutant allele.
In contrast to our observations, investigations carried out in mammalian
tissue culture have suggested a crucial role for Myc in cell cycle
progression. Exposure of quiescent cells to growth factors rapidly induces the
expression of Myc (Kelly et al.,
1983), and forced expression of Myc in various cell types can
induce them to enter S phase (Eilers et
al., 1991
), accelerate their rate of cell division
(Karn et al., 1989
), and alter
their requirements for growth factor stimulation
(Stern et al., 1986
).
Correspondingly, reduction of Myc expression is correlated with exit
from the mitotic cycle and cell differentiation
(Heikkila et al., 1987
;
Hurlin et al., 1995
). In the
developing Drosophila wing disc, overexpression of dMyc dramatically
shortened the length of G1, but a concomitant increase in the length of S
phase and G2 resulted in no change in the length of the cell cycle overall
(Johnston et al., 1999
). Thus,
in Drosophila, the primary function of dMyc may be to promote cell
growth rather than cell proliferation.
Overexpression of dMyc has been proposed to accelerate the G1/S transition
in wing disc cells by activating Cyclin E through a post-transcriptional
mechanism (Johnston et al.,
1999; Prober and Edgar,
2000
). Because Cyclin E has been identified as an important
regulator of the endocycle in nurse cells
(Duronio and O'Farrell, 1995
;
Knoblich et al., 1994
;
Lilly and Spradling, 1996
), we
examined the protein expression of both Cyclin E and Dacapo, the
Drosophila p27 Cip/Kip homolog that specifically inhibits Cyclin
E/Cdk2 activity (de Nooij et al.,
1996
; Lane et al.,
1996
). Both Cyclin E and Dacapo protein expression continued to
cycle in dm2 mutant nurse cells. These observations
suggest that the effect of the dm2 mutation on the
endocycle does not result from an influence on the pattern of Cyclin E
expression. Consistent with this conclusion, Cyclin E protein levels also
continue to oscillate in larval fat body and salivary gland cells that
overexpress dMyc (S. Pierce and R. Eisenman, personal communication).
Perturbations in Cyclin E expression can also lead to the differentiation of
multiple oocytes in a single cyst (Lilly
and Spradling, 1996
). Our finding that only one oocyte
differentiated in dm2 mutant cysts provides further
evidence that Cyclin E regulation was relatively normal.
In mammalian cells, expression of the Dacapo homologs p21CIP1 and p27KIP1
are repressed by Myc (reviewed by Gartel
and Shchors, 2003). In the Drosophila ovary, Dacapo
present in the oocyte nucleus prevents it from undergoing DNA endoreplication,
helping to maintain it in prophase I of meiosis
(Hong et al., 2003
). These
findings suggested a possible mechanism whereby dMyc-mediated inhibition of
dacapo gene expression in nurse cells might facilitate their
endoreplication. However, the fact that Dacapo protein did not show an obvious
increase in expression in dm2 mutant germ cells suggests
that in contrast to what is seen in mammals, dacapo expression is not
directly influenced by dMyc in these cells, nor is dMyc-mediated repression of
dacapo expression an important mechanism regulating nurse cell
endoreplication.
In Drosophila, cell growth is known to be regulated by two
distinct but interacting signalling pathways: one mediated through the insulin
receptor (InR) and phosphatidylinositol-3-OH kinase (PI3K), and the other
through the nutrition-sensing protein kinase TOR (target of rapamycin)
(reviewed by Johnston and Gallant,
2002; Oldham and Hafen,
2003
). The dm mutant phenotype closely resembles that of
mutants affecting the dTOR effector protein ribosomal S6 kinase
(Perrimon et al., 1996
;
Montagne et al., 1999
). In
mammals, S6 kinases have been shown to promote the translation of ribosomal
proteins and translation factors
(Jefferies et al., 1997
). In a
recent study that examined the binding to DNA of the Drosophila Myc
network proteins Myc, Max and Mad/Mnt, a number of genes involved in ribosome
biogenesis and proteins synthesis were identified as dMyc targets
(Orian et al., 2003
). Taken
together, these findings suggest that dMyc, like dS6K, may exert its effect on
growth through the enhancement of protein translation.
One protein through which dMyc might exert its effects on translation is
the product of the pitchoune (pit) gene, a putative DEAD-box RNA
helicase (Zaffran et al.,
1998) whose human homolog, MrDB, has been shown to be a
transcriptional target of Myc-Max heterodimers
(Grandori et al., 1996
).
pit mutants exhibit a constellation of phenotypes similar to that
observed for dm2 mutants, and constitutive expression of
dMyc induces expression of pit in embryos
(Zaffran et al., 1998
) and
third instar larvae (Orian et al.,
2003
). The identity of Pitchoune protein as a DEAD-box RNA
helicase and its subcellular localization in the nucleolus suggests that it
may be involved in rRNA processing or ribosome biogenesis
(Zaffran et al., 1998
).
Perturbation of Delta/Notch signaling between the germline and the follicle
cells has been observed to disrupt follicle cell endoreplication
(Deng et al., 2001;
López-Schier and St Johnston,
2001
). Follicle cells homozygous for loss-of-function alleles of
Notch exhibit a delay in exiting the mitotic division cycle, which
leads to overproliferation of the follicular epithelium and the formation of
abnormally small mutant cells with small nuclei. This phenotype has been
interpreted as an inability of the Notch mutant cells to undergo
their normal program of differentiation
(López-Schier and St Johnston,
2001
). By contrast, our observations suggest that dm
mutant follicle cells exhibit reduced levels of postmitotic DNA synthesis not
because they cannot make the transition from mitosis to the endocycle, but
because they are unable to grow sufficiently well to support the endocycle. In
addition, the execution of chorion gene amplification by
dm2 follicle cells suggests that they adopt at least some
of the characteristics of mature follicle cells. This observation may reflect
the possibility that dMyc regulates distinct effectors of endocycle DNA
replication that do not participate in gene amplification. Alternatively, the
metabolic and synthetic needs of cells undergoing gene amplification may be
much lower than those of endocycling cells, and may not require the concerted
action of the dMyc-activated gene network.
In addition to the effects of loss of dMyc activity on the growth of
homozygous mutant germline or follicle cells, we also observed a profound
effect on the growth and development of the entire egg chamber when either the
complete germline or the entire follicular epithelium was mutant for
dm2. In both cases, the follicles were delayed in their
development and rarely progressed to vitellogenic stages, in which yolk uptake
can be detected in the oocyte. These results suggest that the growth of the
somatic and germline components of the ovary are tightly coordinated. In
addition to their failure to grow, the genotypically wild-type follicle cells
surrounding dm2 mutant germline clones exhibited some
signs of immaturity, such as perdurance of uniform FasIII expression.
Surprisingly, expression of the BR-C by these cells appeared to be determined
by their age, based upon their position within the ovariole, rather than on
the maturity of the egg chamber, as judged by its size. This raises the
interesting possibility that follicle cells can assess their age by a
mechanism that is independent of the growth state of the egg chamber in which
they are contained. Alternatively, the ability of the follicular epithelium to
respond to ecdysone signaling may depend in part on influences external to an
individual follicle. It has been well documented that signaling between the
soma and the germline is required for the establishment of the dorsoventral
and anteroposterior axes of both the follicle and the future embryo
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995
). However,
the signals that communicate the growth status of one tissue to the other are
less well understood. Our finding that the loss of dMyc function in either the
germline or in the soma is sufficient to prevent the growth of the
complementary soma or germline, respectively, may provide a useful tool for
investigating how this information is transferred between the two tissues
In contrast to clones comprising entire germline cysts, the loss of dMyc activity from a subset of nurse cells led to an apparently autonomous deficiency in growth that did not affect the wild-type nurse cells present in the same cyst. The recovery of such mixed-phenotype mosaic cysts also permitted us to conclude that the reduced growth and impaired endoreplication detected in dm2 mutant nurse cells did not result from the failure of a general cyst-wide developmental transition that requires the function of dMyc. Indeed, the ability to generate such mixed phenotype cysts was quite surprising, as the nurse cells are interconnected by cytoplasmic bridges and are typically considered to share a common cytoplasm. The restriction of hGFP to the nurse cells in which it was synthesized, and the phenotypic differences between the dm2/dm2 and +/+ nurse cells in the same cyst, convincingly demonstrate that there are restrictions that limit the intercellular movement of at least some gene products between nurse cells. The use of the hGFP marker will make it possible to investigate whether the products of other genes expressed by the nurse cells are similarly confined to their cells of origin.
In summary, our analysis of the function of dMyc in the Drosophila ovary is consistent with the conclusions of other recent work that indicate that Myc family proteins profoundly influence the ability of cells to grow. By combining the information obtained from genomics-based molecular studies with the genetic analysis of putative target genes, it should be possible to elucidate the role of dMyc in different tissues, and to identify those genes that act as its downstream effectors. The dm ovarian phenotype will provide a useful framework in which to investigate the function of the dMyc network in growth and endoreplication.
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ACKNOWLEDGMENTS |
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Footnotes |
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