Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350, USA
* Author for correspondence (e-mail: hannele{at}u.washington.edu)
Accepted 15 November 2002
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SUMMARY |
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Key words: Oogenesis, Germ plasm, Nuage, Drosophila
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INTRODUCTION |
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One system with high potential for understanding the role of nuage is
Drosophila. In females, Vasa-positive germline granules are
continuously present throughout the life cycle, taking one of two forms, nuage
or pole plasm (Hay et al.,
1988a; Lasko and Ashburner,
1990
). Pole plasm, which contains polar granules, is a determinant
that is both necessary and sufficient to induce formation of the germ lineage
in early embryogenesis (Illmensee and
Mahowald, 1974
; Illmensee and
Mahowald, 1976
) (reviewed by
Mahowald, 2001
). In
Drosophila, nuage is first detectable when primordial germ cells are
formed; it persists through adulthood, where it is present in all germ cell
types of the ovary (Mahowald,
1968
; Mahowald,
1971a
; Mahowald,
1971b
).
In Drosophila, three proteins are known to localize to nuage: Vasa
(Hay et al., 1988a;
Lasko and Ashburner, 1990
),
Aubergine (Harris and Macdonald,
2001
) and Tudor (Bardsley et
al., 1993
). The sequence or mutant phenotype of each gene suggests
a role in post-transcriptional RNA function. Vasa is a DEAD-box RNA helicase
(Hay et al., 1988b
;
Lasko and Ashburner, 1988
;
Liang et al., 1994
) required
for nurse cell-to-oocyte transport of several mRNAs critical to oocyte
patterning (Styhler et al.,
1998
). Vasa is also required for efficient translation of several
key proteins in oogenesis, and itself interacts both physically and
genetically with a Drosophila homolog of yeast Translation Initiation
Factor 2 (dIF2) (Carrera et al.,
2000
). Vasa is thus potentially implicated in translational
control. Aubergine is a member of the RDE1 (for RNAi defective)/AGO1
(Argonaute1) protein family, homologs of which are required in both RNAi and
developmental processes in diverse organisms (reviewed by
Fagard et al., 2000
;
Carmell et al., 2002
).
Aubergine is required, during oogenesis, for efficient translation of Oskar
(Wilson et al., 1996
), which
is pivotal in initiating pole plasm assembly (reviewed by
Rongo and Lehmann, 1996
).
Aubergine is also required for RNAi in late oogenesis
(Kennerdell et al., 2002
).
Tudor, a novel protein (Golumbeski et al.,
1991
), comprises ten copies of an
120 residue motif (the
`Tudor Domain', pfam00567) (Callebaut and
Mornon, 1997
) present in several proteins involved or implicated
in RNA-binding capacities (reviewed by
Ponting, 1997
). The domain has
been suggested to mediate protein-protein interactions
(Selenko et al., 2001
).
Drosophila Tudor is required to mediate transfer of mitochondrial
ribosomal RNAs from mitochondria to the surface of polar granules during pole
cell formation in early embryogenesis
(Amikura et al., 2001
). A role
for Tudor prior to pole plasm assembly, however, has not been described.
Aubergine and vasa are members of a larger group of
female sterile mutants, the spindle (spn) class, which
produces eggshells with variable anteroposterior (AP) and dorsoventral (DV)
axis defects (Schupbach and Wieschaus,
1991; Tearle and
Nüsslein-Volhard, 1987
). In most of the characterized
spn mutants, the etiology of these patterning defects has be traced
to a failure in Gurken (a TGF
homolog) presentation in developing egg
chambers (reviewed by Ray and Schupbach,
1996
; Riechmann and Ephrussi,
2001
). Each spn mutant also has a meiotic progression
defect (Ghabrial et al.,
1998
): by stage 4 in a normal egg chamber, the DNA within the
oocyte nucleus (germinal vesicle) condenses into a compact sphere called a
karyosome (Smith and King,
1968
) after successful meiotic recombination. Mutants in all
spn genes fail to form a karyosome
(Ghabrial et al., 1998
;
Gonzalez-Reyes et al., 1997
;
Styhler et al., 1998
;
Tomancak et al., 1998
). The
characterized spn genes fall into two groups: those whose gene
products are directly required for recombinational DNA repair steps within the
germinal vesicle [e.g. okra (okr), spn-B and
spn-C] and those whose protein sequence or localization suggest
indirect involvement in meiotic progression [e.g. aubergine, vasa and
spn-E] (reviewed by
Gonzalez-Reyes, 1999
). The
spn mutants, as a group, demonstrate that meiotic and patterning
processes intersect during oogenesis.
In this paper, we identify and characterize a null allele of the maelstrom gene, which encodes a novel protein with a human homolog. The mutant displays each of the defects in oocyte development common to the spindle-class. We also demonstrate that Maelstrom localizes to nuage in a Vasa-dependent manner and that maelstrom is required for proper modification of Vasa. Through mutant analysis, we have begun to unravel genetic dependencies of nuage particle assembly.
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MATERIALS AND METHODS |
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Southern analysis of maelM391/Df (3L) 79E-F genomic DNA was carried out using standard procedures. We cloned the deletion junction by PCR, using 5' primer M1 (TAC-TAG-TGC-TCA-GCA-ACG-CC), 3' primer M3 (GGC-TTG-TAG-GAG-CTT-GAT-GC) and Vent DNA polymerase (New England Biolabs, Beverely, MA) for amplification. The resulting PCR product was subcloned and sequenced using standard techniques.
Western blotting
Ovaries from 20 females were dissected in Drosophila Ringer's
solution (EBR) (130 mM NaCl, 4.7 mM KCl, 1.9 mM CaC12, 10 mM HEPES, pH 6.9)
and processed as before (Clegg et al.,
1997). Supernatant equivalent to the mass of a single wild-type
ovary was loaded per well of 8% SDS-polyacrylamide gels
(Laemmli, 1970
). Equivalent
loading was verified by Coommassie staining of duplicate lanes. After
electrophoresis, gels were western blotted using standard protocols and probed
with either affinity-purified anti-Maelstrom rabbit polyclonal (1:5,000) or
anti-Vasa rat polyclonal (1:10,000). Immunoreactive bands were visualized with
HRP-conjugated goat anti-rabbit (or anti-rat) secondary antibody (BioRad) at
1:10,000 and enhanced chemiluminescence using an NEN Renaissance kit
(Dupont-NEN, Boston, MA).
Immunocytochemistry
Previously (Clegg et al.,
1997), ovaries were fixed for immunolocalization in 4%
paraformaldehyde for 20 minutes. Here, ovaries were fixed in 6.2% formaldehyde
for 5 minutes, as described previously (Li
et al., 1994
). Briefly, ovaries from groups of ten females were
dissected in EBR and fixed for 5 minutes in 100 µl devitellinizing buffer
under 600 µl n-heptane on a Clay-Adams nutator (Becton-Dickinson, Sparks,
MD). Devitellinizing buffer is composed of 1 vol buffer B, 1 vol reagent grade
formaldehyde (Fisher catalog number F79, which contains 10-15% methanol as a
preservative) and 4 vol H2O. Buffer B is 100 mM
KH2PO4/K2HPO4, pH 6.8, 450 mM KCl,
150 mM NaCl and 20 mM MgCl2. After fixation, ovaries were rinsed
three times in PBS, three times in PBT (PBS plus 0.1% Triton X-100), then
washed in PBT for 2 hours at room temperature. Ovaries were then dissected
into individual ovarioles, washed for an additional 2-16 hours in PBT at
4°C and blocked in PBT-BSA (PBT plus 1% bovine serum albumin) overnight at
4°C. Ovaries were then incubated overnight at 4°C in PBT-BSA plus
primary antibodies, followed by washing in PBT-BSA for 2-3 hours at room
temperature with multiple changes. Secondary antibodies were diluted 1:500 in
PBT-BSA, and incubation was carried out overnight at 4°C. Ovaries were
subsequently washed in multiple changes of PBT-BSA, then PBT, each for 1-2
hours, at room temperature, then stained with DAPI (0.04 µg/ml). After
several quick rinses in PBS over 15 minutes, ovaries were mounted in a
solution of 1xPBS, 70% glycerol and 2% n-propyl gallate (Sigma, St
Louis, MO).
We used the following primary antibodies: anti-ADD87 (an adducin-like
protein) mouse monoclonal 1B1 (1:20)
(Zaccai and Lipshitz, 1996),
anti-alpha-Spectrin rabbit polyclonal (1:500)
(Byers et al., 1987
),
anti-Argonaute1 rabbit polyclonal (1:100)
(Kataoka et al., 2001
),
affinity-purified anti-Argonaute2 rabbit polyclonal (1:100)
(Hammond et al., 2001
),
anti-Bicaudal-D mouse monoclonal (1:20)
(Suter and Steward, 1991
),
anti-C(3)G guinea pig polyclonal (1:500)
(Page and Hawley, 2001
),
affinity-purified anti-Dicer rabbit polyclonal (1:100)
(Bernstein et al., 2001
),
anti-Gurken mouse monoclonal 1D12 (1:20)
(Queenan et al., 1999
),
affinity-purified anti-Maelstrom rabbit polyclonal (1:50-100)
(Clegg et al., 1997
),
anti-nuclear Lamin mouse monoclonals, ADL67 and ADL84 (1:20)
(Stuurman et al., 1995
),
anti-Oskar rabbit polyclonal (1:500) (Paul Lasko), anti-Staufen rabbit
polyclonal (1:500) (Daniel St Johnston) and anti-Vasa rat polyclonal (1:500)
(Paul Lasko). We used Alexa Fluor® (488, 568 or 633)-conjugated goat
anti-rat, anti-mouse, anti-guinea pig or anti-rabbit IgG secondary antibodies
(Molecular Probes, Eugene, OR).
Nuclear transport assay
For the nuclear transport assay, ovaries from groups of ten females were
dissected in EBR and incubated 20 minutes at ambient temperature on a nutator
in 2 ml EBR with or without 50 nM Leptomycin B (Dr Minoru Yoshida, University
of Tokyo). After incubation, ovaries were rinsed briefly in EBR and fixed for
immunolocalization, as indicated above.
Confocal microscopy
Confocal images were collected using an inverted Leica DMIRBE microscope
equipped with Leica TCS SP/MP confocal and multiphoton attachment using Leica
Confocal Software (LCS) version 2. For each experiment, no less than 150
ovarioles were examined, from which representative figures were prepared.
Variability and penetrance are noted where relevant. Within individual mutant
backgrounds (Fig. 5), Maelstrom
levels proved to be rather variable. In order to compare nuage localization,
we chose ovarioles in which Maelstrom levels were not reduced, relative to
wild type (somatic Maelstrom staining served as an internal control for
individual sections). By contrast, overall Vasa levels in the mutants were
comparable with wild type. However, as the relative distribution of Vasa to
nuage was slightly reduced, we set the gain in confocal sections of mutants
such that peak signal values in mutants were comparable to peak signal values
for the wild type. This results in an apparent relative increase in
cytoplasmic levels of the protein in Fig.
5.
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Whole-mount in situ hybridization
RNA in situ hybridization to ovaries was performed using DIG-labeled DNA
probes as previously described (Clegg et
al., 1997). Light microscopy was performed on a Leitz DMRB with
Nomarski differential interference contrast. Images were acquired with a model
3.2.0 Spot Insight color digital camera (Diagnostics Instrument, Sterling
Heights, Michigan). We used Adobe Photoshop (Adobe Systems, Mountain View, CA)
to crop and assemble all photographic images.
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RESULTS |
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Homologs of Maelstrom can be identified in mosquito (Anopheles gambiae), honey bee (Apis mellifera), mouse and human. A ClustalW alignment (see http://dev.biologists.org/supplemental/) of the Drosophila, mosquito and human homologs shows 7.3% identity and 25.9% similarity shared between the three proteins. Fourth iteration Psi-blast searches with the Drosophila homolog gives overall E-values of e-151 (to the mosquito homolog, agCP12344) and e-150 (to the human homolog, FLJ14904). In addition, a partial potential HMG-box is found in Drosophila Maelstrom (residues 2-50), whereas a canonical HMG-box is found in the human homolog (residues 5-65).
Maelstrom is a spindle-class gene
As hypomorphic alleles of maelstrom showed AP and DV
spindle-class-like defects in the developing oocyte
(Clegg et al., 2001;
Clegg et al., 1997
), we wished
to determine whether the maelstrom null (hereafter referred to as
maelstrom) shared the meiotic progression defect common to the
spindle-class mutants. Specifically, the spn mutants fail to
form a karyosome (Ghabrial et al.,
1998
; Gonzalez-Reyes et al.,
1997
; Styhler et al.,
1998
; Tomancak et al.,
1998
), despite the apparently normal assembly of synaptonemal
complexes within the oocyte nucleus (Huynh
and St Johnston, 2000
). To this end, we examined meiotic
progression in the oocyte nucleus (germinal vesicle) using synaptonemal
complex component, C(3)G, to assess progression to synaptonemal complex
formation; we used oocyte DNA morphology to assess progression to the
karyosome stage (Fig. 2A-E).
C(3)G is normally acquired by oocyte chromosomes in the germarium
(Fig. 2A) and dissociated from
DNA upon karyosome formation (Page and
Hawley, 2001
). In more than 90% of stage 2 or 3 maelstrom
egg chambers, C(3)G signal is present and restricted to the oocyte
(Fig. 2B), where it colocalizes
with DNA in a morphology comparable with wild type
(Fig. 2C). This suggests that
meiosis has proceeded in the mutant to at least zygotene phase of prophase I.
As reported for other spn mutants
(Huynh and St Johnston, 2000
),
maelstrom ovaries show some delay in restriction of synaptonemal
complexes to a single cell (data not shown). When the karyosome forms in
wild-type oocytes (Fig. 2C, WT,
stage 5; Fig. 2D), DNA within
the germinal vesicle loses it association with C(3)G. Despite the dispersion
of C(3)G within the oocyte nucleoplasm by stage 6, maelstrom oocytes
never form karyosomes. Instead, the DNA shows a nuclear morphology distinct
from both stage 1 and karyosome: forming variably distended loops and threads,
often closely apposed to an invariably `deflated' nuclear envelope
(Fig. 2C,
M, stage 5;
Fig. 2E). This DNA morphology,
maintained in maelstrom through at least stage 10B, is similar to
that described for other spn mutants
(Ghabrial et al., 1998
).
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The axial patterning defects displayed by maelstrom hypomorphs
(Clegg et al., 2001;
Clegg et al., 1997
) are fully
penetrant in oocytes of the maelstrom null
(Fig. 2F-K). AP axis
determination in the Drosophila oocyte is a multistep process, the
first known step of which is the establishment of microtubule-mediated
cytoplasmic polarity in the stage 2 oocyte. This asymmetry, which is defective
in spn mutants such as spn-A, spn-B and vasa
(Pare and Suter, 2000
;
Styhler et al., 1998
;
Tomancak et al., 1998
), is a
likely prerequisite for efficient Gurken signaling from the oocyte to the
follicle cells overlying the posterior oocyte. A number of RNAs and proteins
accumulate in the posterior of the wild-type oocyte during stages 2-6 in a
distribution that both requires and reflects the oocyte polarity in this
interval. We assayed polarity in the oocyte indirectly by monitoring the
localization of Bicaudal D (BicD) (Suter
and Steward, 1991
) and multiple RNAs including grk
(shown), osk, bicD and oo18 RNA binding (orb) (not
shown). In normal stage 5/6 oocytes, BicD forms a distinct gradient emanating
from the posterior oocyte cortex (Fig.
2F). In maelstrom oocytes, although BicD is present at
levels comparable with wild type (Fig.
2G), a wild-type gradient is not established. Instead, about half
of stage 5/6 maelstrom oocytes show BicD in a diffuse or only vaguely
polarized distribution (not shown). In the remaining oocytes, the marker forms
a randomly localized focus within the ooplasm
(Fig. 2G). Similarly, the
normally polarized distribution of grk and other RNAs is lost in
maelstrom oocytes (Fig.
2H,I). Gurken protein distribution in wild-type oocytes
(Fig. 2J) is comparable with
that of BicD, albeit more punctate in appearance. In maelstrom
oocytes, not only is the gradient lost, but Gurken levels are either highly
reduced (
50%) (Fig. 2K) or
undetectable (
50%) (not shown). The Gurken defect is probably sufficient
to account for the observed polarity defects in mid- to late-stage
maelstrom oocytes, in which variety of polarity markers, including
multiple mRNAs (e.g. osk) and proteins (including Staufen, Oskar,
Vasa), fail to accumulate in the posterior ooplasm. Dorsal appendages are also
invariably vestigial or absent in the maelstrom null (not shown). The
failure in establishing AP polarity in the early oocyte, together with
reduction in Gurken accumulation, the DV phenotypes of the null and hypomorph,
and failure to proceed to karyosome stage collectively puts maelstrom
in the spindle-class of mutants.
The phenotypes of the double-strand break (DSB) repairspecific spn
mutants (e.g. spn-B) can be suppressed by a mutation in
mei-W68 (Ghabrial and Schupbach,
1999). This locus encodes the Drosophila homolog of the
Spo11 protein, which induces double-strand breaks in chromosomes, the
initiating event required for subsequent steps in recombination
(McKim and Hayashi-Hagihara,
1998
). If DSBs do not occur, then genes normally required in the
ensuing recombinational repair steps are not required. Thus, their absence
will not be detected by the elements of the meiotic checkpoint, which responds
to persistent unrepaired DSBs. To resolve the sphere of maelstrom
function, we assessed genetic interaction between mei-W68 and
maelstrom by examining Gurken accumulation in early oocytes of
mei-W68-maelstrom double mutant ovaries. If maelstrom were
required only in recombinational repair, we would expect a suppression of the
Gurken translation defect of the maelstrom null oocyte
(Fig. 2K). We observe that the
Gurken defect of maelstrom oocytes is not, in fact, suppressed by
mei-W68 (Fig. 2L), from which we conclude that maelstrom cannot only be required in a
recombinational repair step.
How meiotic progression status in the oocyte nucleus is transmitted to
effectors of oocyte patterning is a key, and largely unanswered, question. One
candidate effector is Vasa, a target of the pachytene checkpoint, which
displays a mobility shift in spn-B ovaries, in which the checkpoint
is activated (Ghabrial and Schupbach,
1999). Interestingly, Vasa mobility is aberrant in
maelstrom ovaries; two distinct species of Vasa protein are observed:
a minor band with wild-type mobility and a species larger, curiously, than
that reported for spn-B (Fig.
2M). Although the relationship to activated-checkpoint-Vasa is
unclear, our data shows that maelstrom is required for proper Vasa
modification (or processing). It is thus conceivable that any phenotype(s) of
the maelstrom mutant could arise, indirectly, as a result of this
Vasa modification. The apparent mass of Maelstrom, by contrast, was unchanged
in vasa null (vasPH165) background, and
in alleles of each of the spn genes (A-E) and okr (not
shown).
Maelstrom localizes to Nuage
Previously, we reported that Maelstrom protein displayed no distinct
subcellular localization within the germline
(Clegg et al., 1997). We have
since re-examined its localization using a `lighter' fixation-based protocol
(see Materials and Methods). As a result, we find that in addition to
previously observed diffuse nuclear and cytoplasmic germline staining, much of
Maelstrom localizes to highly abundant particles within germline cells
(Fig. 3A; see
http://dev.biologists.org/supplemental/).
The frequency and distribution of Maelstrom particles were reminiscent of that
previously described for nuage, to which Vasa localizes. Double labeling of
Maelstrom and Vasa (Figs 3,
5; see
http://dev.biologists.org/supplemental/)
shows overlap in perinuclear germline granules from stem cells through stage
10 nurse cells. Double labeling of Maelstrom and a nuclear lamin shows that
virtually all distinct Maelstrom particles are closely apposed to the
cytoplasmic face of the nuclear envelope in nurse cells
(Fig. 3B). Because
nanos-GAL4-driven GFP-tagged Aubergine (AubGFP) was reported to
localize to nuage in late stage egg chambers
(Harris and Macdonald, 2001
),
we examined its localization in combination with Vasa and Maelstrom
immunostaining (Fig. 3C). Each
discrete particle in the germarium and early egg chamber labels for Vasa,
Maelstrom and AubGFP, a concordance that is also maintained in stages 7-10.
(Owing to the discontinuous nature of the nanos driver, AubGFP is not
highly expressed between approximately stages 3 and 6.) At the ultrastructural
level, most nuage is lost from the oocyte by stage 1, prior to the formation
of the karyosome (Mahowald and Strassheim, 1970). However, occasional
particles of Vasa and Maelstrom can be detected in the ooplasm as late as
stage 4 (Fig. 3E and data not
shown). Although the most conspicuous localization of Maelstrom and Vasa is to
nuage, each protein is also present within the nucleus and cytoplasm of all
germline cells (Fig. 3A-D).
Within the oocyte nucleus, both proteins localize to discrete regions in young
egg chambers: in single confocal sections, Vasa often appears in discrete dot
or dots, exclusive of, but adjacent to an `aura' of concentrated Maelstrom
(Fig. 3E). Maelstrom persists
in the oocyte nucleus as diffuse staining through at least late stage 10B
(Fig. 3F). After onset of pole
plasm assembly (stage 8/9), Vasa accumulates in posterior region of the oocyte
(Fig. 3D, Vasa panel)
(Hay et al., 1988b
;
Lasko and Ashburner, 1990
).
Maelstrom, by contrast, never shows a posterior concentration in the ooplasm
(Fig. 3D, Maelstrom panel).
Although Maelstrom is present in the mature egg and early embryo, its
distribution is again uniform at these stages (not shown). As neither the
Maelstrom nor its RNA (not shown) show preferential posterior accumulation in
the ooplasm, Maelstrom is the first described nuage component that is not also
concentrated in pole plasm.
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Maelstrom shuttles between compartments
Because Maelstrom and Vasa are each present in the nucleus, nuage and
cytoplasm of germline cells, it was of interest to determine whether either
protein could transit between these compartments. We assayed nuclear shuttling
utilizing Leptomycin B (LMB) (Hamamoto et
al., 1983), a specific inhibitor of nuclear transport receptor,
CRM1 (Exportin). CRM1 mediates nuclear export of substrates containing a
leucine-rich nuclear export sequence (NES) in cells as diverse as yeast and
human (Fornerod et al., 1997
;
Fukuda et al., 1997
;
Stade et al., 1997
).
Drosophila CRM1 has been shown to be mechanistically
indistinguishable from its homologs in other systems, including its specific
inactivation by LMB (Fasken et al.,
2000
). LMB treatment of Drosophila ovaries has a marked
effect on Maelstrom protein localization within the germline (compare
Fig. 4C with 4G and
Fig. 4D with 4H), whereas Vasa
protein shows only a slight redistribution (compare
Fig. 4A with 4E and
Fig. 4B with 4F). The effect is
most pronounced in nurse cells and oocytes, where Maelstrom manifests a
nuclear accumulation, with a corresponding depletion in cytoplasm
(Fig. 4H). We surmise that
Maelstrom must transit between cytoplasm and nucleus.
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Maelstrom is dissociated from nuage particles in vasa,
aubergine and spn-E mutants
We do not know if the known nuage proteins act in a common pathway before
their convergence in nuage particles. To begin to answer this question, we
have sought to determine genetic dependencies for nuage particle assembly
(Fig. 5). AubGFP has previously
been shown to depend on vasa function for its nuage localization
(Harris and Macdonald, 2001).
We analyzed Maelstrom and Vasa localization in wild-type, maelstrom, vasa,
aubergine and spn-E backgrounds. Maelstrom protein levels proved
to be quite variable among ovarioles of single mutant backgrounds. So, in
order to compare localization, we examined individual ovarioles in which
Maelstrom levels were not significantly reduced (see Materials and Methods).
Maelstrom and Vasa localization in the wild-type ovariole is shown in
Fig. 5 (parts A1 and
A1'). Virtually no Maelstrom immunoreactive signal is present in the
maelstrom (Fig. 5,
part A2), whereas Vasa is largely maintained in nuage
(Fig. 5, part A2';
Fig. 6D). By contrast, the
perinuclear accumulation of Maelstrom is virtually absent in the vasa
null (Fig. 5, part A3),
suggesting that Maelstrom localization in nuage is Vasa dependent. We examined
Maelstrom's distribution in several vasa point mutants, in the hope
of correlating functional domains in the protein with nuage organizational
function. Of particular interest were two vasa EMS alleles,
vas011 and vas014, each of which
produces a protein devoid of RNA binding and unwinding activities
(Liang et al., 1994
). In both
of these mutants, Vasa and Maelstrom colocalization in nuage is largely
maintained (data not shown). We analyzed Vasa and Maelstrom localization in
several allelic combinations of aubergine, as a null for this gene
has not been described. Maelstrom (Fig.
5, part A4) and Vasa (Fig.
5, part A4') localization is shown for a representative,
aubHN2/N11 ovariole. Both aubHN2 and
aubN11 alleles encode truncated proteins
(Harris and Macdonald, 2001
).
In this and other aubergine mutant combinations, the normal
concentration of Maelstrom in nuage is severely depleted in all germline cells
(Fig. 5, part A4). Vasa is
largely maintained in perinuclear localization in this mutant background, but
the normally discrete particles are less obvious; instead, Vasa appears as a
more uniform perinuclear band (Fig.
5A, part 4').
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Spn-E encodes a putative Dex/hD-box RNA helicase, required for
proper localization of several oocyte-destined RNAs and proteins over the
course of oogenesis (Gillespie and Berg,
1995; Pare and Suter,
2000
). While the localization of Spindle-E in the ovary has not
been determined, its involvement in both RNAi and oogenesis
(Aravin et al., 2001
;
Stapleton et al., 2001
;
Kennerdell et al., 2002
), like
Aubergine, prompted its inclusion in our analysis. As with aubergine
mutants, the concentration of Maelstrom in perinuclear particles is lost in
strong spn-E allelic combinations,
spn-E616/hls
125
(Fig. 5A, part 5) and
spn-Ehls3987/hls
125 (not shown). Vasa retains a
perinuclear concentration in spn-E ovaries
(Fig. 5A, part 5'), but
as in aubergine, the normal particulate appearance of nuage is less
pronounced. We extended our localization analysis to include the remaining
members of the better characterized spn-class mutants, spn-A,
spn-B, spn-C, spn-D and okr. Of particular interest was
spn-B, which has been shown to modify Vasa as a consequence of
meiotic checkpoint activation (Ghabrial
and Schupbach, 1999
). The dependency of Maelstrom on Vasa for its
localization could, in principle, be affected if Vasa is aberrant. However, in
multiple allelic combinations of well-characterized spn genes
(spn-B, spn-D and okr) and uncloned spn genes
(spn-A and spn-C), colocalization of Vasa and Maelstrom in
nuage particles was unperturbed at all stages of oogenesis (not shown).
The maelstrom and vasa nulls differ in their RNA
phenotypes
Normally, a variety of RNAs required for proper oocyte function are
transcribed, exported from nurse cell nuclei and transported to the oocyte. As
this process is defective in vasa ovaries
(Styhler et al., 1998), we
wished to assess transport in maelstrom ovarioles, with the aim of
resolving potential functions of individual nuage components. We found that
transport of several of these mRNAs, including oskar
(Fig. 2N,O), orb and
BicD (not shown), is unaltered in maelstrom ovaries.
RNAi/microRNA components are mislocalized in maelstrom egg
chambers
The dissociation of Maelstrom from nuage particles in aubergine
and spn-E backgrounds was intriguing in light of their requirement in
RNAi in Drosophila spermatogenesis and late oogenesis (see
Discussion). Importantly, proteins (or homologs) of RNAi pathway components
also act in micro RNA (miRNA) processing
(Grishok et al., 2001;
Hutvagner et al., 2001
). As
miRNAs have been shown to regulate RNA translation, it is conceivable that
miRNAs are assembled in RNP particles formed in nuage. In this setting, nuage
could represent a step in the generation of specificity in translational
control in the germline. To explore this potential relationship between nuage
and RNAi/miRNA processing pathways, we examined the localization of additional
RNAi components in wild-type and maelstrom ovaries. Argonaute1 and
Argonaute2 are RDE1/AGO1 homologs required for RNAi in Drosophila
(Hammond et al., 2001
;
Williams and Rubin, 2002
).
Dicer is the core RNase of RNAi in Drosophila
(Bernstein et al., 2001
); it is
also required for production of the small RNA effectors of the RNAi and miRNA
pathways in C. elegans (Grishok
et al., 2001
; Hutvagner et
al., 2001
). In vertebrate cell lines, Dicer is primarily
cytoplasmic (Billy et al.,
2001
). In wild-type Drosophila ovarioles, Dicer
(Fig. 6A,A') and AGO1
(Fig. 6G,G') appear
uniform and cytoplasmic in nurse cell cytoplasm; AGO2
(Fig. 6E,E') appears
cytoplasmic but relatively more granular. In maelstrom ovaries, AGO1
distribution is relatively unperturbed
(Fig. 6H,H'). However,
AGO2 and Dicer are both dramatically mislocalized in maelstrom
ovarioles. Beginning around stage 3, Dicer aggregates in discrete, often
perinuclear foci in nurse cells (Fig.
6B,B'). AGO2 is observed in perinuclear regions of nurse
cells (Fig. 6F,F'), which, by contrast, could colocalize with Vasa in nuage
(Fig. 6D,D').
![]() |
DISCUSSION |
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---|
Resolving the domains of function of spindle genes
The characterized spn genes currently fall into two general
classes: those that encode proteins that are likely to be directly involved in
meiotic recombinational repair, such as okr, spn-B and spn-C
(Ghabrial et al., 1998;
Ghabrial and Schupbach, 1999
);
and those, such as maelstrom and vasa, whose mutant meiotic
phenotype, protein sequence and/or localization suggest indirect roles. Work
presented in this paper suggests that the spn mutants can be sorted
by an additional criterion: those that are also required for nuage assembly
(vasa, aubergine, maelstrom and spn-E) and those that are
not (spn-A, spn-B, spn-C, spn-D and okra). Taken together,
these data suggest that the Vasa-like group of spn genes are
essential in general `nuage activities' in all cells of the germline. The
activity of the spn-B-class genes, which are involved in
recombination or meiotic checkpoint, could represent one avenue through which
to use or modulate existing nuage functions that are operative within the
germline cyst as a whole. Such nuage-related processes, if inactivated or
defective, could culminate in polarity and translational defects within the
oocyte.
Cell biology of nuage
Within the time frame of the life cycle of the female fly, pole plasm, in
its mature form, is ephemeral (Mahowald,
1971b). Nuage, by contrast, is present in morphologically stable
form from mid-embryogenesis through to late oogenesis in the adult (reviewed
by Mahowald, 1971b
). Nuage is
thus a feature that is specific to established germ cells, perhaps reinstated
by pole plasm components in early embryogenesis. Maelstrom is unique among
previously identified Drosophila nuage components, which are also
concentrated in pole plasm. Maelstrom is thus the first identified
nuage-specific component in Drosophila. Nuage is the most conserved
form of Vasapositive germline granule: it is present in germ cells of diverse
organisms, including mammals, which do not use a pole plasm equivalent
(reviewed by Saffman and Lasko,
1999
). The cell biology of nuage, however, remains largely
unexplored. As nuage components are also present in the nucleus and cytoplasm,
these particles may represent a morphologically distinct form (or kinetic
intermediate) of a nucleocytoplasmic continuum. We have demonstrated that
Maelstrom can shuttle between nuclear and cytoplasmic compartments. The
observation that Vasa showed only a slight redistribution to the nuclear
compartment implies that these proteins are not necessarily always associated.
This begs the question of whether components of nuage converge on and diverge
from perinuclear particles from separate pathways or subcellular compartments.
As more nuclear transport tools become available, we may better resolve how
these particles are formed.
Nuage may function in RNA processing
Within egg chambers, a number of mRNAs crucial for oocyte patterning are
synthesized in nurse cells and transported to the developing oocyte (reviewed
by Riechmann and Ephrussi,
2001). The oocyte represents a discrete compartment in the
continuous cytoplasm of the germline cyst, and precise spatiotemporal control
of nurse cell-derived mRNA translation is crucial for proper development.
Between transcription in nurse cell nuclei and their ultimate translation,
oocyte-destined RNAs are likely to be associated with factors required to
mediate both localization and translational control. Recent work has shown
that heterogeneous nuclear ribonucleoproteins (hnRNP-proteins) associated with
mRNAs during their transit from nucleus to cytoplasm can play key roles in
mRNA localization and translational control (reviewed by
Dreyfuss et al., 2002
).
Similarly, splicing factors have been shown to continue their association with
mRNA into cytoplasm. And although the exchange of nuclear for cytoplasmic
RNA-binding proteins has been demonstrated, the location and regulation of
such exchange processes are poorly understood (reviewed by
Shatkin and Manley, 2000
). A
working hypothesis is that nuage in the Drosophila germline (and
other systems) functions in such an exchange process. In this capacity, nuage
might be a platform for, or represent a kinetic intermediate of, cytoplasmic
ribonucleoprotein (RNP) particle assembly
(Fig. 7). The perinuclear
localization of nuage and the observation that Maelstrom shuttles between
compartments are consistent with this role (Figs
3,
4). Two additional lines of
evidence support the proposition. The first is that nuage ultrastructurally
interfaces with sponge bodies, which are highly abundant, RNA-rich particles
present in the cytoplasm of nurse cells and the oocyte
(Wilsch-Brauninger et al.,
1997
). Mounting evidence points to a role for the sponge body as a
vehicle for transport of RNP complexes between nurse cells and the oocyte
(Cha et al., 2001
;
Nakamura et al., 2001
;
Theurkauf and Hazelrigg, 1998
;
Wilhelm et al., 2000
). Sponge
bodies are also highly enriched for several regulatory proteins, including
Exuperantia (Wilsch-Brauninger et al.,
1997
), DEAD-box RNA helicase Me31B
(Nakamura et al., 2001
) and
cold shock protein YPS (Wilhelm et al.,
2000
), which can be purified as an RNase-sensitive RNP complex
(Nakamura et al., 2001
;
Wilhelm et al., 2000
). This
complex, by purification or localization, is associated with numerous
oocyte-destined RNAs, such as oskar and bicoid
(Nakamura et al., 2001
;
Wilhelm et al., 2000
).
Ultrastructurally, the perinuclear fraction of sponge body particles appear to
embed individual nuage particles
(Wilsch-Brauninger et al.,
1997
), a morphological association that implicates the interface
as one possible segment in what may be a continuous RNP assembly process
(Fig. 7).
|
The second line of evidence is that three of the proteins present in, or
required for, nuage particle assembly (Aubergine, Vasa and Spn-E) are also
implicated in post-transcriptional RNA-related capacities. Aubergine
mutants fail to translate Oskar efficiently, despite proper localization of
oskar mRNA to the oocyte posterior
(Wilson et al., 1996).
Aubergine is concentrated in two distinct regions within germline cysts: in
nuage and also in pole plasm (Harris and
Macdonald, 2001
). Thus, aubergine function may be
required for proper Oskar translation before oskar mRNA is even
transported to the oocyte posterior. Vasa is a DEAD-box RNA helicase, a class
of proteins thought to act as RNA chaperones (reviewed by
Tanner and Linder, 2001
).
Vasa-null egg chambers display a systemic failure in mRNA targeting.
This could be attributable, in part, to the loss of interface of nuage with
sponge bodies, as vasa null ovaries are devoid of nuage at the
ultrastructural level (Liang et al.,
1994
). spn-E encodes a Dex/hD class (putative) RNA
helicase. spn-E ovaries contain enlarged sponge bodies
(Pare and Suter, 2000
) and
display defects in the localization of several normally oocyte-destined
molecules, including bicoid RNA, as well as BicD(GFP) and Dynein
Heavy Chain (Gillespie and Berg,
1995
; Pare and Suter,
2000
) (D. E. Gillespie, PhD thesis, University of Washington,
Seattle WA, 1996). Each of these molecules is largely retained in mutant nurse
cells, accumulating in the vicinity of ring canals. The specificity of the
localization defect suggests an underlying defect in transport through ring
canals, which is a discrete, microtubule-independent step in nurse
cell-to-oocyte transport (Theurkauf and
Hazelrigg, 1998
). This defect together with the nuage assembly
defect of spn-E are consistent with the hypothesis that nuage may
function in formation of RNPs required for correct mRNA localization in the
Drosophila germline.
Nuage and the miRNA pathway
A tantalizing possibility for nuage is that it may function at some point
in miRNA and/or RNP particle assembly processes
(Fig. 7). This hypothesis is
supported by the mislocalization of Dicer and AGO2 to perinuclear regions of
germline cells in the maelstrom mutant
(Fig. 6B,B',F,F').
Such discrete redistributions of proteins could reflect an accretion of
intermediate in a normally maelstrom-mediated step. A connection is
further insinuated by the requirement of both aubergine and
spn-E in the assembly of nuage particles. In mutants of both genes,
Maelstrom is dissociated from perinuclear Vasa particles in all germline cells
(Fig. 5). The dissociation is
interesting because both aubergine and spn-E have a common
requirement in double-stranded RNA-mediated gene silencing in both
Drosophila spermatogenesis and late oogenesis
(Aravin et al., 2001;
Kennerdell et al., 2002
;
Stapleton et al., 2001
). In
the testis, spn-E is required for silencing of retrotransposons (e.g.
copia) and both spn-E and aubergine are required
for silencing of genomic tandem repeats (e.g. Stellate). Mutants in
either gene relieve RNAi-mediated suppression of respective target genes. The
fact that each is also required for proper mRNA localization or translation
raises the possibility that these proteins could function as common components
in the allied miRNA pathway. RNAi and miRNA are mechanistically related: each
pathway processes a dsRNA substrate, using a common processing factor, Dicer,
to generate the respective small RNA effectors of each pathway. In C.
elegans, 24 RDE1/AGO1 homologs have been identified
(Grishok et al., 2001
). The
studied homologs are required in either RNAi or miRNA processing, but in not
both pathways (Grishok et al.,
2001
; Hutvagner et al.,
2001
). The Drosophila genome encodes only five RDE1/AGO1
homologs: Piwi, Aubergine, AGO1, AGO2 and AGO3
(Williams and Rubin, 2002
),
which may necessitate dual usage in both miRNA and RNAi pathways.
Recent reports suggest that hundreds of miRNAs exist in metazoans
(Grosshans and Slack, 2002;
Lagos-Quintana et al., 2002
).
These miRNAs are thought to be modulators of target mRNA translation, although
additional functions have been hypothesized (reviewed by
Ambros, 2001
). Indeed, miRNAs
might represent a common means of post-transcriptional regulation of gene
expression in both vertebrates and invertebrates. It is known for many cell
types, including neurons and oocytes, that translation and localization of
mRNA is controlled by RNA-binding proteins. However, the specificity of this
process is poorly understood. In some cases, a sequence-specific RNA binding
protein is found to be involved
(Crittenden et al., 2002
;
Wharton et al., 1998
); in
other cases a combinatorial action of many hnRNPs is proposed
(Dreyfuss et al., 2002
). The
abundance of miRNAs raises the possibility that these small RNAs could
generate the missing specificity: miRNAs bound to target mRNAs are predicted
to form a loop structure that could be recognized by multiple RNA-binding
proteins, allowing for assembly of a full RNP particle. In the context of
oogenesis, miRNAs could provide an added level of control by conferring
specificity through nucleation or regulated assembly of translational (and
possibly localization) control factors on RNAs (see
Fig. 7). The data presented in
this paper suggest that nuage function may be involved in the miRNA or RNAi
pathways. Future experiments should be aimed at determining the role of nuage
components in miRNA precursor maturation or in assembly of mature miRNAs with
their target mRNAs.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
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