Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
* Author for correspondence (e-mail: mlilly{at}helix.nih.gov)
Accepted 17 November 2003
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SUMMARY |
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Key words: Meiosis, Oogenesis, Drosophila, Synaptonemal complex, Oocyte differentiation
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Introduction |
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Drosophila oogenesis starts when a cystoblast, the asymmetric
daughter of the germline stem cell, undergoes four divisions with incomplete
cytokinesis to produce a 16-cell interconnected cyst
(Fig. 1A). Individual cells in
the cyst, referred to as cystocytes, are connected by actin-rich intercellular
bridges called ring canals (Robinson and
Cooley, 1996). All 16 cystocytes complete a long premeiotic S
phase. Subsequently, the two cystocytes with four-ring canals, the
pro-oocytes, develop long synaptonemal complexes (SC) consistent with
pachytene of meiotic prophase I (Mahowald
and Kambysellis, 1980
). Several other cells show similar but
reduced SC structures; however, only the true oocyte maintains its meiotic
state and further condenses its chromatin. The other 15 cystocytes lose their
meiotic characteristics, enter the endocycle and develop as polyploid nurse
cells.
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Several lines of evidence indicate that oocyte differentiation is
contingent on the execution of the proper cell-cycle program of the germline
cyst. As is observed in all animal oocytes, in Drosophila the oocyte
arrests in prophase of meiosis I for the growth phase of oogenesis. During
this developmentally programmed arrest, the p27-like cyclin-dependent kinase
inhibitor Dacapo (Dap) specifically accumulates in the oocyte nucleus
(de Nooij et al., 2000;
Hong et al., 2003
). The
characterization of dap mutants suggests that Dap inhibits
inappropriate DNA replication in oocyte and thus helps maintain the prophase I
meiotic arrest (Hong et al.,
2003
). In the absence of Dap, the oocyte enters the endocycle and
develops as a nurse cell. Thus, inappropriate entry into the endocycle
disrupts oocyte differentiation and can serve as the primary cause of the loss
of the oocyte fate. The characterization of two genes required to repair
double-strand breaks (DSBs) during meiosis demonstrate that meiotic
progression and oocyte differentiation are tightly coupled. The formation of
the both the anteroposterior and dorsoventral axis of the oocyte is dependent
on activity of Gurken (Grk), the TGF
-like ligand for EGF receptor
(Egfr) (reviewed by Van Buskirk and
Schüpbach, 1999
). In okra (okr) and
spindle-B (spnB) mutants the translation of the grk
transcript is inhibited, resulting in pattern defects similar to those
produced by mutants in the grk-Egfr signaling pathway
(Gonzalez-Reyes et al., 1997
;
Ghabrial et al., 1998
).
Surprisingly, okr and spnB encode the Drosophila
homologs of the yeast DSB repair proteins, RAD54 and DMC1. These data indicate
that the pathways that control DSB repair specifically influence the
grk-Egfr signaling pathway during oogenesis
(Ghabrial et al., 1998
).
Why does the oocyte nucleus progress through meiosis while adjacent cystocytes abandon the meiotic cycle in preparation for their development as nurse cells? Although this question is fundamental to a comprehensive understanding of oocyte development, little is known about the pathways that directly promote the differentiation of the oocyte nucleus. We describe the identification and characterization of a novel gene, missing oocyte (mio) that is required for the maintenance of the meiotic cycle during oogenesis. We cloned the mio gene and determined that the Mio protein localizes to the oocyte nucleus at the onset of prophase of meiosis I. In mio mutants, oocyte differentiation and meiotic progression are retarded. Ultimately, mio oocytes exit the meiotic cycle, enter the endocycle and develop as nurse cells. Surprisingly, the mio phenotype is suppressed by inhibiting the formation of the double-stranded breaks that initiate meiotic recombination. Our data strongly suggest that mio mutants define a novel pathway that influences both the nuclear events of meiotic progression and oocyte differentiation.
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Materials and methods |
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Molecular characterization of the mio gene
We mapped the mio locus between the proximal breakpoints of
Df(2L)dp79b and
Df(2L)yanJ2. To physically map the extent of this
chromosomal region, we determined the molecular breakpoints of the
deficiencies by quantitative Southern blot analysis. This analysis indicated
mio mapped to a 50 kb genomic region, between 22C3 and 22D2, which
contained 11 predicted open reading frames (ORFs) (FlyBase). From these 11
ORFs, all predicted exons were amplified from genomic DNA samples of
mio1 and mio2
homozygous flies, sequenced and then compared with the sequence from the
Berkeley Drosophila Genome Project (BDGP). To confirm that the single
nucleotide changes found in CG7074 were not due to random
polymorphisms, we sequenced the appropriate parental chromosomes,
y1, w1118, P{y+t7.7
ry+t7.2=Car20y}25F P{ry+t7.2=neoFRT}40A and Canton
S.
The CG7074-coding region was identified by 5' and 3' RACE of mio cDNA using the primers, 5'-GCCGCCGACGATAGGATGTTGCAGGC-3' and 5'-ACTCAGCAGCAGCCCACGAGCAGC-3', respectively, and the SMART RACE cDNA Amplification Kit (Clontech). Full-length mio cDNA was amplified by RT-PCR, cloned into pCR II-TOPO and sequenced. The mio cDNA sequence is available as the mio gene in AE003584 GenBank sequence.
UASp:mio rescue
The full-length mio cDNA was cloned into the pUASp vector
(Rørth, 1998) from the
pCR II-TOPO vector. Transgenic lines were generated in a
w1118 background using standard techniques
(Spradling, 1986
). Three
independent insertions were obtained, all of which mapped to the second
chromosome. Therefore, we crossed the UASp-mio P-element onto the
mio2 chromosome using meiotic recombination. We
expressed UASp-mio in the mio2 mutant
background using the nanos-Gal4:VP16 driver
(Van Doren et al., 1998
).
Production of anti-Mio antibodies
Nucleotides 1865 to 2473 of the mio cDNA (LD45056) were amplified
using primers that added a BamHI site to the 5' end and an
XhoI site to the 3' end, and cloned into pET21a (Novagen) to
generate a His-tag fusion protein. The fusion protein contains amino acids
601-803 of the Mio protein tagged with a 6x His tag at the C terminus. The
fusion proteins was expressed in Escherichia coli BL21 cells
(Novagen) and purified on a nickel column before being used as antigen to
produce rabbit polyclonal anti-Mio antibodies.
Immunostaining and imaging
Immunostaining of ovaries was performed as described previously
(Grieder et al., 2000). The
rabbit
Mio antiserum was used at a concentration of 1:1000. Other
antibodies were used at the following concentrations: rabbit
C(3)G
(Hong et al., 2003
) at 1:1000,
mouse
Orb 6H4 (Lantz et al.,
1994
) at 1:50 (purified IgG, Developmental Studies Hybridoma Bank,
University of Iowa), mouse
Bic-D
(Suter and Steward, 1991
) at
1:5 dilutions. Secondary antibodies conjugated to Alexa 594 or Alexa 488
(Molecular Probes) were used at 1:800 dilution. Double labeling of rabbit
antibodies,
Mio and
C(3)G was performed using
C(3)G
antibody that was directly labeled with Alexa 594 (Alexa Fluor 594 Protein
Labeling Kit, Molecular Probes). Nuclei were visualized with 2 µg/ml
Hoechst 33342 (Molecular Probes). Confocal images of stained ovaries were
captured on a LSM 410 confocal microscope (Zeiss) with a 1.4 NA 63x oil
immersion objective or a 1.4 NA 100x oil immersion objective. Image
analysis was performed using LSM imaging software. Composite figures were
prepared using Photoshop 5.5 (Adobe).
Western analysis
Ovaries were dissected in Grace's medium (Gibco) and homogenized in (50 mM
Tris-HCl pH 7.5, 1 mM MgCl2, 1 mM PMSF). After centrifugation to
pellet the debris, supernatants were diluted with an equal volume of
2xSDS loading buffer (Bio Rad), resolved by 7.5% SDS-polyacrylamide
gels, and transferred to Hybond ECL nitrocellulose membrane (Amersham).
Mio serum was used at 1:3000. As a loading control, blots were probed
with anti-
Tubulin antibody at 1:9000 (DM 1A, Sigma). For detection,
blots were incubated with Horseradish Peroxidase-conjugated antibody
(Amersham) at a 1:6000 dilution, and bands were visualized with a
chemiluminescent detection kit (Amersham).
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Results |
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In order to determine if mio influences the specification of the
oocyte identity, we examined the localization of Orb and Bic-D protein in
mio mutant ovaries. In wild-type cysts, the Orb and Bic-D proteins
first accumulate in the cytoplasm of the oocyte, as well as that of several
adjacent cells, in region 2a of the germarium soon after the completion of
premeiotic S phase (Fig. 2A)
(Christerson and McKearin,
1994; Lantz et al.,
1994
). As cysts pass through region 2b Orb and Bic-D staining
becomes restricted to a single centrally localized cell, which will ultimately
become the oocyte. mio cysts clearly specify an oocyte, as indicated
by the specific accumulation of Orb and Bic-D protein within a single cell
(Fig. 2B, and data not shown).
However, the preferential accumulation of Orb in the mutant pro-oocytes is
markedly delayed and is often not visible until late region 2b or region 3
(Fig. 2B). Indeed, the overall
levels of Orb appear to be lower in mio mutants
(Fig. 2A,B).
|
Null mutations in the cyclin-dependent kinase inhibitor dap have a
similar effect on the maintenance of oocyte identity
(Hong et al., 2003). In
dap mutants, an oocyte is initially specified. However, once the
oocyte enters the endocycle, the oocyte identity is gradually lost and egg
chambers develop with 16 polyploid nurse cells. We wanted to examine if
mio influences oocyte differentiation by regulating Dap expression.
In the
10% of the mio2 egg chambers in which
the oocyte does not enter the endocycle, Dap protein accumulates to high
levels in the single oocyte as is observed in wild-type egg chambers. These
data indicate that Dap can be expressed and properly localized to the oocyte
nucleus in the absence of Mio. In the 90% of egg chambers in which the oocyte
becomes polyploid, Dap does not accumulate to high levels in any one cell.
Thus, as is observed with all oocyte-specific markers examined, the specific
accumulation of Dap is lost, or is never properly established, in mio
oocytes that enter the endocycle. We believe this reflects a general problem
in the maintenance of oocyte identity and/or the differential transport
system, as represented by the concomitant loss of Bic-D and Orb accumulations,
rather than a direct role for Mio in the regulation of Dap expression.
However, we cannot rule out the formal possibility that some aspects of the
mio ovarian phenotype are due to the misregulation of Dap.
Meiotic progression is altered in mio mutants
To examine if Mio is required for early meiotic progression, we followed
the distribution of the SC protein C(3)G. In wild-type cysts, the two
pro-oocytes progress to pachytene in region 2a as determined by the completion
of the SC along all bivalents (Mahowald
and Kambysellis, 1980) (Fig.
3A). Cells adjacent to the pro-oocyte also build a less extensive
network of SC. In mio mutants, an increased number of cells enter the
meiotic cycle in region 2a as assayed by C(3)G staining (6.5±2.1,
n=21 versus 3.9±.3, n=14 in wild type) with 76% of
mio cysts having more than five cells in the meiotic cycle
(Fig. 3B). Although too many
cells enter meiosis, mio mutants retain the ability to restrict the
meiotic cycle to a single cell. Thus, as is observed in wild type, by region 3
the majority of mio cysts contain a single C(3)G positive cell
(Fig. 3B).
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Mio is required for progression to pachytene in egl mutants
To explore the function of mio further during oogenesis, we made
double-mutant combinations of mio and egl. In egl
mutants, all 16-cyst cells enter the meiotic cycle and form SCs
(Carpenter, 1994;
Page and Hawley, 2001
)
(Fig. 4B). However, this
meiotic state is not stably maintained and ultimately all 16 cystocytes exit
the meiotic cycle and enter the endocycle. We generated females that were
double mutant for mio2 and one of two null
alleles of egl, egl1 or
egl2. Intriguingly, mio, egl females
have an ovarian phenotype significantly stronger than either single mutant.
mio, egl double mutants do not form even the thin SC observed in
egl null mutants. Instead,
C(3)G staining remains diffuse in
all 16-cystocytes with one or two bright clumps of staining
(Fig. 4C). This pattern
suggests that either the nuclei are arrested prior to pachytene, before the
completion of the mature SC, or alternatively mio, egl mutants form a
defective SC. The ability of mutations in mio to enhance the severity
of the eglnull SC phenotype confirms that
mio functions prior to the pachytene stage of meiosis.
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Mio Localizes To Nuclei That Contain Synaptonemal Complexes
In order to determine the precise cellular localization of the Mio protein,
we generated antibodies against a C-terminal region of the protein
(Fig. 5C). Western blot
analysis indicates that the polyclonal antibody recognizes a single band of
98.6 kDa, the predicted molecular weight of the Mio protein
(Fig. 6A). The intensity of the
98.6 kDa band is dramatically increased in ovarian extracts from UASp-mio;
nanos-Gal4:VP16 females, which overexpress the mio mRNA
(Fig. 6A). Ovaries from
mio2 females produce a slightly smaller protein,
92.6 kDa, consistent with the mio2 C-terminal
truncation. Immunostaining of wild-type ovaries indicates that the Mio protein
specifically localizes to oocyte nuclei
(Fig. 6B). Weak Mio staining is
first detected in one or two centrally located cystocytes in region 2a
(Fig. 6C). The staining becomes
dramatically brighter and restricted to a single cell in region 2b. Staining
in the oocyte nucleus after stage 3 is still visible in some
mio1 mutant egg chambers, indicating that the
Mio signal observed in post-germarial stages of oogenesis is at least
partially due to a crossreaction. However, we believe that the following two
observations indicate that the
Mio signal in the germarium is real.
First, no germarial staining is observed in the
mio1 mutant (see Fig. S1A at
http://dev.biologists.org/supplemental),
which is predicted to produce a truncated protein that does not contain the
appropriate antigen (see Fig.
5C). Second, the
Mio signal is dramatically increased in
intensity, but present in the same germarial pattern, when the gene is
overexpressed in the germline from a mio transgene (see Fig. S1B,C at
http://dev.biologists.org/supplemental).
|
The mio phenotype is suppressed by blocking the formation of DSB during meiosis
Mutations in genes required to repair DSB, such as okr and
spnB, affect the formation of the dorsoventral axis of the egg
(Ghabrial et al., 1998). In
okra and spnB mutants this phenotype is suppressed by
blocking the formation of the DSB that initiate meiotic recombination
(Ghabrial and Schüpbach,
1999
). In order to determine if blocking the formation of DSBs
suppresses the mio ovarian phenotypes, we examined mio,
mei-W68 and mio, mei-P22 double-mutant females. Both
mei-W68 (SPO11 homolog) and mei-P22 (novel gene)
are required for DSB formation during meiosis
(McKim and Hayashi-Hagihara,
1998
; Sekelsky et al.,
1999
; Liu et al.,
2002
). Intriguingly, ovaries from mio, mei-W68 and
mio, mei-P22 double-mutants have three to four times more egg
chambers that contain an oocyte than mio single mutants
(Table 3). In addition,
mio, mei-W68 and mio, mei-P22 egg chambers often undergo
vitellogenesis and develop to late stages of oogenesis
(Fig. 7B). By contrast, egg
chambers from mio single mutants rarely develop beyond stage 5
(Fig. 7A). Placing a single
copy of either a mei-W68 or mei-P22 mutation in the
mio background results in a partial suppression of the ovarian
phenotype (Table 3). Thus,
inhibiting the formation of DSBs during meiosis significantly suppresses the
mio 16-nurse cell phenotype and the associated developmental
delay.
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Discussion |
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Mio is the earliest nuclear marker for the oocyte that is not a known
component of the SC. In Drosophila, the SC is constructed before the
formation of the DSBs that initiates meiotic recombination
(McKim et al., 1998;
Liu et al., 2002
). The Mio
protein is first expressed in region 2a where it colocalizes to nuclei that
contain SCs. Thus, during oogenesis Mio is expressed specifically in cells
that have entered the meiotic cycle. Although Mio is expressed in both
pro-oocytes in early region 2a, Mio staining always appears asymmetric with
one cell, presumably the true oocyte, having a brighter signal. Intriguingly,
Mio staining is asymmetric before there is noticeable asymmetry in SC
structure, as determined by C(3)G staining. Although Mio does accumulate soon
after the onset of the meiotic cycle, in egl mutants, Mio does not
localize to a single nucleus but remains undetectable. These data strongly
suggest that the construction of the microtubule-based directional transport
system precedes the specific accumulation of Mio in the oocyte. This
localization pattern is consistent with a role for mio downstream of
the initial specification of the oocyte identity.
Mio influences cyst polarity and oocyte differentiation
In mio mutant egg chambers, the oocyte enters the endocycle and
adopts a nurse cell fate. Two observations indicate that Mio acts as early as
region 2a and affects some of the earliest steps in oocyte differentiation. In
wild-type cysts four cells enter meiosis and form SC in region 2a before the
meiotic cycle is restricted to a single cell in region 2b. However, in
mio mutants, too many cells enter meiosis in region 2a and form SC.
This early broadening of the meiotic gradient, with too many cells displaying
oocyte like features, is similar to what is observed in par1 mutants
and is consistent with a delay in the establishment of cyst polarity and/or
the differentiation of the oocyte. In addition, mio mutants are slow
to accumulate oocyte specific markers. In wild-type cysts, oocyte-specific
markers, such as Orb and Bic-D, accumulate in a single centrally localized
cell beginning in region 2a (Spradling,
1993). In mio mutants this accumulation is often not
observed until region 3. Indeed, the initial expression of Orb appears to be
delayed or substantially reduced, with mio cysts throughout the
germarium having lower overall levels of Orb compared with similarly staged
wild-type cysts. These data demonstrate that consistent with its expression at
the onset of prophase of meiosis I, Mio functions early in oogenesis to
promote the polarization of the cyst and acquisition of the oocyte fate.
However, we note that it is not clear if this early meiotic function of Mio is
required for the maintenance of the meiotic cycle or if Mio functions at
multiple times during oogenesis.
How does one reconcile the limited expression pattern of Mio, which specifically accumulates in the nuclei of the two pro-oocytes, with the apparent global disruption in cyst polarity observed in mio mutants? We can think of at least two possible explanations for this apparent paradox. First, Mio may act in the oocyte nucleus to reinforce and maintain the oocyte fate. In this model, subtle alterations in oocyte differentiation may compromise, but not eliminate, the directional transport system and/or other pathways that are required to establish and/or reinforce cyst polarity. This might produce a weak egl-like phenotype and thus allow an inappropriate number of cells to enter meiosis. Alternatively, Mio may be present at undetectable levels in cells beyond the two pro-oocytes. In this model, Mio acts cell autonomously to influence cell cycle regulation and the establishment of cyst polarity in cells throughout the cyst.
Mio facilitates meiotic progression
The analysis of mio, egl double mutants demonstrate that Mio
influences meiotic progression and/or meiotic chromosome structure early in
prophase of meiosis I, prior to the formation of the mature SC. The phenotype
of mio, egl double mutants is considerable stronger than either
single mutant. In the double mutant the C(3)G pattern never appears even
remotely thread-like, but persists as generalized nuclear staining with one or
more bright dots. This pattern suggests that either the nuclei are arrested
prior to pachytene, before the completion of the mature SC, or alternatively
mio, egl mutants form a defective SC.
Why might the disruption of directional transport to the oocyte uncover an
earlier function for mio? In wild-type cysts, factors required for
meiotic progression are produced in all 16 cystocytes but are quickly
transported to the oocyte where they accumulate to high levels
(Spradling, 1993). In
egl mutants these putative meiotic factors are not enriched in a
single cell, but instead are present in all 16 cystocytes, presumably at a
much lower concentration than is observed in a wild-type oocyte. It is only in
this compromised situation, where factors required for meiotic progression may
be limiting, that Mio becomes an absolute requirement for progression to
pachytene. One possible explanation for these data is that a protein that is
normally concentrated in the oocyte, via the direction transport system, is
partially redundant for Mio function. Additional support for a role for Mio
prior to pachytene comes from the observation that mio single mutants
contain an increased proportion of cysts with fragmented or partially formed
SC in region 2a of the germarium. The increase in the proportion of this
developmental intermediate suggests that mutations in mio cause
subtle alterations in the kinetics of SC formation.
Our data support a model in which Mio acts to facilitate the nuclear events of meiotic progression soon after the completion of premeiotic S phase and the onset of the meiotic developmental program. Considering the presence of WD40 repeats similar to those found in CAF1p48/RbAp48, we speculate that Mio may act to modify chromatin structure in the pro-oocyte nuclei to promote oocyte differentiation and prepare the oocyte nucleus for the upcoming events of meiosis, including meiotic recombination and the meiotic divisions. As discussed below, this model is consistent with a potential role for Mio in DSB repair during meiosis.
Mio may be required to repair the DSBs that initiate meiotic recombination
The mio ovarian phenotype is suppressed by inhibiting the
formation of DSBs during meiosis. In mio single mutants, the oocyte
frequently enters the endocycle and becomes polyploid. However, when placed in
a genetic background in which DSB formation is inhibited, the majority of
mio egg chambers retain an oocyte and develop to late stages of
oogenesis. Intriguingly, mutations in the meiotic checkpoint gene
mei-41 do not suppress mio, indicating that it is the
physical presence of DSB, and not the activation of the meiotic checkpoint,
that contributes to the mio phenotype. However, although clearly
important, the inability to repair DSBs during meiosis is unlikely to be sole
cause of the mio phenotype. Mutations in genes with a direct role in
DSB repair, such as okr and spnB, cause patterning defects
late in oogenesis (Gonzalez-Reyes et al.,
1997; Ghabrial et al.,
1998
) but do not result in the complete abandonment of meiosis and
the polyploidization of the oocyte, as is observed in mio mutants.
Therefore, mio must have additional functions beyond the repair of
DSBs. Considering that Mio localizes to the oocyte nucleus early in meiosis
and functions before the construction of the mature SC, we speculate that
mio acts upstream of the enzymology of DSB repair. Thus, the
inability of mio mutants to repair DSB may be due to a general
alteration in meiotic chromosome structure or alternatively subtle alterations
in the meiotic program.
In the future, the identification of the molecular mechanism by which mio influences the differentiation of the oocyte nucleus as well as the regulation of the meiotic chromosomes will provide insight into the poorly understood pathways that drive early meiotic progression and early oocyte development in metazoans.
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ACKNOWLEDGMENTS |
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Footnotes |
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