Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution of Washington, 115 W. University Parkway, Baltimore, MD 21210, USA
* Author for correspondence (e-mail: spradling{at}ciwemb.edu)
Accepted 6 January 2003
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SUMMARY |
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Key words: Mitochondria, Oogenesis, Fusome, Balbiani body, Patterning, Germ plasm
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INTRODUCTION |
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Drosophila oogenesis (reviewed by
Spradling, 1993;
Johnstone and Lasko, 2001
)
(Fig. 1A) provides an
attractive system for studying the origin of oocyte organelles. At the
anterior tip of each ovariole, germline stem cells divide asymmetrically to
produce daughters known as cystoblasts. Cystoblasts undergo four rapid,
asymmetric divisions with incomplete cytokinesis to generate interconnected
16-cell groups known as germline cysts. During these cell divisions, the cysts
elaborate a cytoskeletal polarity that ultimately causes one cell to develop
as an oocyte, while the others become nurse cells (reviewed by
Reichmann and Ephrussi, 2001
).
Cyst polarity originates within a special cytoplasmic organelle rich in
membrane skeleton proteins known as the fusome whose branches grow thinner
with each cystocyte division (Lin et al.,
1994
; Lin and Spradling,
1997
; de Cuevas and Spradling,
1998
). As cysts move through germarium region 2, their microtubule
minus ends accumulate on the large fusome segment within one of the two
pro-oocytes (Grieder et al.,
2000
; Huynh et al.,
2001
), resulting in the detection of a new microtubule organizing
center (Theurkauf et al.,
1993
). The movement of cystocyte centrioles along the fusome
toward the future oocyte during this time may cause this microtubule
reorganization (Mahowald and Strassheim,
1970
; Grieder et al.,
2000
; Bolivar et al.,
2001
). The meiotic gradient within developing cysts may also
respond directly to fusome polarity (Huynh et al., 2000). The cytoskeletal
structures that have developed in region 2b cysts causes specific proteins
such as Bicaudal-D (Bic-D), Orb and Cup, and mRNAs from oskar, orb
and Bic-D to accumulate within an initial cell and stimulate it to
differentiate as the oocyte, while the other 15 cells become nurse cells.
|
Recent studies of mitochondria in yeast and cultured animal cells document
three important properties that are relevant to understanding the behavior of
mitochondria during oogenesis. First, mitochondria are highly plastic and can
readily alter their shape from spheres to ellipsoids to complex reticuli
(Bereiter-Hahn and Voth, 1994).
Specific forms depend on the relative number of fusion and fission events
controlled by specific genes (Jensen et
al., 2000
). A candidate Drosophila mitofusin encoded by
the Marf (also known as dmfn) gene is expressed during
oogenesis (Hwa et al., 2002
).
Second, mitochondria are frequently mobile within the cytoplasm, and can move
actively along microtubules in most animal cell types, or actin in plant and
yeast cells (Bereiter-Hahn and Voth,
1994
). Finally, studies of mitochondrial genomes document a high
rate of somatic mutation (Denver et al.,
2000
; Nekhaeva et al.,
2002
), and it has been suggested that maternal mitochondrial
genomes pass through a `mitochondrial bottleneck' in order to maintain their
average fitness (Bergstrom and Pritchett, 1998).
To begin to uncover the molecular mechanisms that support the inheritance of mitochondria and other organelles, we have visualized their number, shape and movement during Drosophila oogenesis. Our observations show that a fraction of the mitochondria, Golgi and other organelles within each 16-cell cyst are delivered into the cytoplasm of the single forming oocyte as a typical Balbiani body under the control of the fusome-dependent system of cyst polarity. The Balbiani-body-derived mitochondrial population of the oocyte remains largely separate from nurse cell mitochondria until nurse cells break down late in oogenesis. Before then, members of this population associate with the polar plasm, leading us to propose that the genomes in Balbiani body-associated mitochondria will be preferentially inherited in grandchildren. Similar mechanisms may act during oogenesis in other species, and these events may underlie the frequent presence of a Balbiani body and the origin of the mitochondrial bottleneck.
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MATERIALS AND METHODS |
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Construction and integration of transgenes
We constructed a mitochondrial marker transgene (`mito-GFP') by fusing the
human COX VIII mitochrondrial targeting signal
(Rizzuto et al., 1995) to the
N terminus of EGFP (Clontech). We synthesized the targeting signal using
overlapping oligos (CGG CTA CGG CTG ACC GTT TTT TGT GGT GTA CTC CGT GCC ATC
ATG TCC GTC CTG ACG CCG CTG CTG and CTT GGC GCG CGG CAC TGG GAG CCG CCG GGC
CGA GCC TGT CAA GCC CCG CAG CAG CAG CGG CGT CAG GAC), and then added a
5' KpnI site (TAT GGT ACC GGC TAC GGC TGA CCG TTT) and a
3' BamHI site (TAT GGA TCC CTT GGC GCG CGG CAC TGG). A 5'
BamHI site (TAT GGA TCC AGT AAA GGA GAA GAA CTT) and 3'
XbaI site (TAT TCT AGA TTT GTA TAG TTC ATC CAT) were added to EGFP.
The EGFP fragment was subcloned downstream of the targeting sequence in the
pUASp vector (Rørth,
1998
) and transformed into y; w flies
(Grieder et al., 2000
). Males
containing the transgene were crossed to w; NGT40; nosGAL4::VP16
females to drive expression of the transgene. Flies bearing single integrated
copies displayed green ovaries, embryos and larvae, but the signal level in
their germaria was variable. However, strong expression was observed in the
germaria of flies carrying multiple copies of the transgene; hence, they were
used in these experiments.
Immunostaining and fluorescence microscopy
One-day-old females were fed wet yeast overnight prior to analysis. Ovaries
were dissected in room temperature Grace's media (BioWhittaker) and fixed for
20 minutes in 3.7% formaldehyde (Sigma) in Grace's, then rinsed in PBT
(1xPBS, 0.1% Triton-X100, 1 mg/ml BSA). To fix GFP-expressing ovaries,
3.2% EM grade formaldehyde (Ted Pella) was used. Primary antibodies were
incubated overnight at 4°C. Primary antibodies were diluted at follows:
rabbit -Drosophila ATP synthase, ß subunit (1:350, gift
from Dr Rafael Garesse), mouse
-human cytochrome c1 oxidase,
subunit I (COX1, 1:500, Molecular Probes), rabbit
-rat mannosidase 2
(1:300, Dr Kelley Moremen), rabbit
-Drosophila
-Spectrin (1:300) (deCuevas et al.,
1996
), mouse
-Drosophila 1B1 (specific for Hts
protein, 1:100) (Zacci and Lipshitz, 1996), rabbit and rat
-Drosophila Vasa (1:2000 and 1:200, respectively, gift of Dr
Paul Lasko), mouse
-Drosophila Orb (1:100, Dr Paul Schedl,
Developmental Studies Hybridoma Bank), rat
-Drosophila Cup
(1:1000) (Keyes and Spradling,
1997
), mouse and rat anti-
-tubulin (1:350, Sigma; 1:20,
Oxford Biotechnology, respectively), mouse
-phosphotyrosine PY20
(1:1000 ICN Biomedicals), and mouse
-Drosophila lamin ADL67
(1:4 Nico Stuurman). Ovaries were then washed in PBT three times for 20
minutes, then secondary antibody was added in PBT either overnight at 4°C,
or for 4 hours at room temperature. The following secondary antibodies were
used: goat
-rabbit and
-mouse AlexaFluor488, AlexaFluor568,
AlexaFluor546 and AlexaFluor633 (1:200, Molecular Probes); and goat
-rabbit,
-mouse and
-rat Cy3 and Cy5 (1:1000, Jackson
ImmunoResearch) After secondary antibody incubation, ovaries were washed three
times for 10 minutes in PBT, then equilibrated overnight at 4°C in
VectaShield (Vector Laboratories) before mounting. Tubulin antibody labeling
was carried out as described (Grieder et
al., 2000
). Fluorescent in situ hybridization was performed
according to Wilkie et al. (Wilkie et al.,
1999
). Phalloidin staining was carried out according to Frydman
and Spradling (Frydman and Spradling, 2001) using both phalloidin
AlexaFluor546 (1:200, Molecular Probes) and rhodamine phalloidin (1:200,
Jackson ImmunoResearch). For DNA labeling, ovaries were incubated in 20
µg/ml RNAseA for 2 hours during secondary antibody incubation and TOTO3
(1:1500, Molecular Probes) was added for 20 minutes. Confocal analysis was
carried out using Leica TCS NT and Leica TCS SP2 confocal microscopes.
Live ovary imaging
For live ovary imaging, ovaries were dissected from flies expressing
mito-GFP, or wild-type ovaries were incubated with MitoTracker GreenFM
(1:1000, Molecular Probes) for 15 minutes, then briefly rinsed in Grace's. The
ovaries were mounted on a petriPerm 50 hydrophobic plate (Vivascience) in
Grace's and covered with an 18 mm2 cover slip. Using the petriPerm
plates allows the ovarioles to stay alive for 2-3 hours. Ovariole quality was
assessed based on mitochondrial swelling; experiments terminated prior to its
onset. Ooplasmic streaming was defined as vigorous directional movement of
bulk cytoplasm; it contrasted sharply with the relatively slow, random
movement of mitochondria and yolk droplets normally seen. All live imaging and
analysis was carried out using a spinning disk confocal [Leica DM IRE2
inverted microscope, Ultraview confocal system (Perkin Elmer) and MetaMorph
work station (Universal Imaging)].
Electron microscopy
Whole ovaries were dissected in Grace's media, then fixed for 1 hour at
4°C in 1% gluteraldehyde, 1% OsO4, 0.1 M cacodylate buffer, 2
mM Ca (pH 7.5). The ovaries were then washed three times for 5 minutes in
cacodylate buffer and embedded in agarose at 55°C. They were then rinsed
for 5 minutes in 0.05 M maleate (pH 6.5), and incubated for 1.5 hours in 0.5%
uranyl acetate, 0.05 M maleate. After rinsing in H2O, they were put
through an ethanol dehydration series (35% twice for 5 minutes, 50% for 10
minutes, 75% for 10 minutes, 95% for 10 minutes and 100% three times for 10
minutes) then incubated twice for 10 minutes in propylene oxide and for 1 hour
in 1:1 propylene oxide:resin [Epon 812:Quetol 651 (2:1)], 1% silicone 200, 2%
BDMA. After three changes of resin (1 hour each), the ovaries were polymerized
at 50°C overnight then at 70°C overnight. Images were captured with a
Phillips Tecnai 12 microscope and recorded with a GATAN multiscan CCD camera
using Digital Micrograph software.
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RESULTS |
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Mitochondria change shape and distribute equally during asymmetric
stem cell and cystocyte divisions.
Germline stem cells (GSCs) and their progeny divide unequally during cyst
production (Fig. 2A)
(de Cuevas and Spradling,
1998). To learn if mitochondria were distributed unequally in
forming cysts, we followed the behavior of mitochondria, fusomes and actin
using specific antibodies. GSCs in G2 (Fig.
2B) contain between 100-200 unbranched, non-reticulate
mitochondria that are about 0.35 µm in diameter and range in length up to 3
µm. Most stem cell mitochondria are preferentially located near the stem
cell fusome (Fig. 2B, blue
sphere) in the same position as most cellular microtubules
(Fig. 2C). Were they to remain
near the fusome during mitosis, mitochondria would be distributed very
unevenly between the daughter cells, and there would be a strong bias for
cystoblasts to acquire the mitochondria not bound to the fusome. However, we
found that in stem cells, mitochondria fragment and associate with the outer
edge of the mitotic spindle just prior to mitosis
(Fig. 2D). Equal-sized daughter
cells are produced that contain approximately the same number of mitochondria
(Table 1). Mitochondria in the
daughter stem cell elongate and re-associate with the fusome
(Fig. 2E, left cell). By
contrast, those in the cystoblast remain more spherical and distribute evenly
throughout the cell cytoplasm (Fig.
2E, right cell). During the remaining cystocyte divisions, the
mitochondria remain round and fusome-independent
(Fig. 2F), but continue to
associate with the spindle during mitosis. Thus, despite cytoplasmic
asymmetries, mitochondria segregate equally into each cell of the 16-cell
cyst.
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Mitochondria move along the fusome toward the oocyte in developing
16-cell cysts
Completed cysts continue to undergo programmed changes in their fusome and
microtubule cytoskeletons that are central to oocyte development during the
2-4 days they spend traversing germarium region 2 (see
Fig. 1A). To determine whether
mitochondria are affected by these cytoskeletal changes, we next studied the
subcellular location and movement of mitochondria within region 2 cysts
(Fig. 3). These experiments
revealed that many mitochondria in developing 16-cell cysts associate with the
fusome, like centrioles. These mitochondria begin to localize along the
branched arms of the fusome in region 2a cysts
(Fig. 3A). As cyst development
proceeds, they move towards the center of the fusome while remaining tightly
associated (Fig. 3B,C). In so
doing, they follow the developmental behavior of microtubule minus ends and
migrating centrioles (Grieder et al.,
2000; Bolivar et al.,
2001
). However, the mitochondria are less centrally concentrated
on the fusome in region 2b than microtubule minus ends, possibly because there
is insufficient space to further concentrate such a large number of
organelles. A small percentage of other mitochondria, indistinguishable in
size and shape from those undergoing translocation, remain free in all the
cystocytes.
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Mitochondria are delivered along the fusome into the oocyte to form a
Balbiani body
The translocating centrioles and microtubule minus ends enter the anterior
of the oocyte at about the time the 16-cell cyst reaches region 3 of the
germarium and prepares to bud. We observed that the translocating mitochondria
in cysts just prior to region 3 form aggregates or `clouds' at the ring canals
that connect the last four nurse cells to the oocyte
(Fig. 3C, arrowhead). Shortly
thereafter, these mitochondrial clusters enter the oocyte, where they coalesce
into a single mass anchored at the large segment of fusome in the anterior of
the cell (Fig. 3D, arrow). We
term this mitochondrial mass a Balbiani body because it appears very similar
in the light and electron microscope (Fig.
3E, arrow) to the Balbiani bodies described in the early oocytes
of other species (Raven, 1961;
Guraya, 1979
). Moreover, as in
many other species, we found that the Drosophila Balbiani body
persists in the oocyte near the germinal vesicle during early follicle
development (see below).
An asymmetric fusome is essential for oocyte determination and for all
known aspects of cyst and follicle polarity. To ask whether the fusome is
functionally required for Balbiani body formation, we studied the behavior of
mitochondria in huli-tai-shao (hts) mutant flies.
hts encodes homologs of the mammalian spectrin-binding protein
Adducin, and is required to maintain the structural integrity of the fusome
(Lin et al., 1994) and to
organize microtubules in region 2 cysts
(Grieder et al., 2000
). We
found that in contrast to wild type, hts mutant cysts contain
mitochondria that are broadly distributed
(Fig. 3F,G). Mitochondrial
clumps remain around presumptive centrosomes in stem cells
(Fig. 3F, arrowheads), but this
is expected because hts flies retain functional centrosomes and
microtubules. Thus, the organized movement of mitochondria into the oocyte and
their association into a Balbiani body requires an intact fusome.
To investigate the relationship between Balbiani body formation and oocyte
determination, we analyzed mitochondria in egalitarian (egl)
mutant females. egl mutants initially specify all 16 cyst cells as
oocytes, but fail to maintain the oocyte fate, so that all the cells
eventually differentiate as nurse cells
(Schupbach and Wieschaus,
1991). Mitochondria behave normally in dividing egl cysts
(data not shown). However, in 16-cell cysts, they do not localize into a
single Balbiani body, even though centrosomes migrate along the fusome and end
up in one cell (Bolivar et al.,
2001
). Instead, most of the mitochondria in each cystocyte
aggregate on the fusome remnants at the ring canals
(Fig. 3H). These mitochondrial
aggregates are retained and grow in size during subsequent follicle
development (not shown).
Balbiani bodies contain Golgi vesicles
Balbiani bodies in other organisms contain additional organelles and
vesicles besides mitochondria (see Raven,
1961). Electron micrographs of Drosophila Balbiani bodies
in early region 3 oocytes revealed the presence of Golgi
(Fig. 4A), ER-like vesicles in
the residual fusome (Fig. 4A,B)
and centriole clusters (Fig.
4B,C). Using anti-
-mannosidase 2 antibody as a marker, we
confirmed that Golgi are present by confocal microscopy
(Fig. 4D). Furthermore, we
found that Golgi elements arrive by much the same pathway as mitochondria.
Golgi vesicles begin to associate with the fusome in region 2 (arrowhead),
become enriched near its center, and move into the oocyte in stage 1 (arrow).
The
-mannosidase 2-positive vesicles remain near the fusome and
Balbiani body within the oocyte anterior early in region 3, but soon disperse
throughout the oocyte cytoplasm. Some of the Golgi vesicles located in region
2 cysts never associate with the fusome or move to the oocyte, but remain
scattered throughout the 15 nurse cells.
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Balbiani bodies are associated with localized RNAs and proteins
In Xenopus the `METRO' region of the Balbiani body associates with
RNAs that will later become localized in germ plasm at the vegetal pole
(Forristall et al., 1995;
Kloc and Etkin, 1995
), and
similar links between Balbiani bodies and germ plasm constituents have been
noted in diverse species (Kobayashi and
Iwamatsu, 2000
; Tsunekawa et
al., 2000
; Bradley et al.,
2001
). Consequently, we investigated whether Drosophila
mRNAs that are known to localize to the early oocyte and participate in germ
plasm formation (reviewed by Saffmann and Lasko, 1999) associate with the
Drosophila Balbiani body using antibody/in situ hybridization double
labeling experiments. We found that osk mRNA is associated with the
center of the fusome as soon as it can be detected in region 2a cysts
(Fig. 5A). This is before
centrosomes and microtubules begin to move to the center. osk mRNA
remains on the fusome of the pro-oocyte where it associates with the newly
formed Balbiani body (Fig. 5B). In oocytes at this stage (Fig. 5B and
5B'), the fusome (blue, arrowhead) is the most anterior
structure, followed by the mitochondria (red, arrow), and finally the RNA
(green). The osk RNA (Fig.
5B, green) lies partially within the Balbiani body
(Fig. 5B, yellow line) and
partially outside its posterior side. However, this state is transient, as the
RNA moves to the oocyte posterior by the end of stage 1
(Fig. 5A, rightmost cyst).
orb RNA also localizes on the central fusome and associates with the
Balbiani body in early stage 1 cysts in a similar manner
(Fig. 5C-D'), although it
may associate with a slightly larger region of the fusome. While it was known
previously that hts is required to localize orb and
osk RNAs to a single cystocyte in region 2
(de Cuevas et al., 1996
;
Deng and Lin, 1997
), these are
the first indications that localized RNAs directly associate with the
fusome.
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Balbiani body-derived mitochondria preferentially associate with the
germ plasm
The previous experiments indicate that Drosophila eggs acquire
mitochondria from two sources. Oocytes receive an initial consignment of these
organelles from the Balbiani body at the time of follicle formation. However,
a large number of mitochondria are also transported into the oocyte from the
nurse cells much later in oogenesis, during nurse cell dumping in stage 11.
These dual sources raise the question of which mitochondrial subpopulation
furnishes the organelles that associate with the germ plasm and whose genomes
found the mitochondrial DNA of subsequent generations. To investigate whether
Balbiani body mitochondria are used preferentially in constructing germ plasm,
we studied the behavior of both mitochondrial populations during follicle
development using mito-GFP.
These studies revealed that Balbiani body-derived mitochondria preferentially associate with forming germ plasm. We could be sure of this because we found that the nurse cell mitochondria are blocked from moving through ring canals into the oocyte before nurse cell dumping, well after germ plasm has formed. This conclusion is based on studies of the movement of nurse cell mitochondria into the oocyte using movies of mito-GFP expressing follicles of various ages (Table 2). Before stage 11 nurse cell mitochondria build up in large masses just outside the oocyte ring canals instead of moving into the oocyte (Fig. 6B, arrowheads). Only at the time of nurse cell dumping do nurse cell-derived mitochondria enter the oocyte.
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DISCUSSION |
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Drosophila oocytes contain a Balbiani body
Drosophila oocytes were found to contain a typical Balbiani body
at the time follicles form in region 3 of the germarium. In a wide range of
animal species, including Xenopus
(Heasman et al., 1984), chick
(Ukeshima and Fujimoto, 1991
),
mouse (Pepling and Spradling,
2001
) and human (Hertig and
Adams, 1967
), young oocytes at a similar developmental stage
display these distinctive aggregates of mitochondria and other organelles near
their germinal vesicles (see Raven,
1961
). In a typical Balbiani body, centrioles and associated
cytoplasm are surrounded by a ring of Golgi bodies and encased in a large mass
of mitochondria. As the oocyte grows, the mitochondria first spread around the
nuclear periphery and later disperse throughout the oocyte cytoplasm.
Drosophila Balbiani bodies, like those described in other species,
were shown to contain clustered mitochondria, Golgi vesicles and centrioles.
Moreover, as young follicles develop from stage 1-6, the mitochondria move
around the germinal vesicle and disperse after microtubules re-organize in
stage 7. The fact that Balbiani bodies occur in a genetically tractable system
will now make it easier to decipher the function of these enigmatic
structures, which have been postulated to play an early role in RNA transport
and in patterning the mammalian egg (de
Smedt et al., 2000
).
The Balbiani body is assembled by regulated movement of organelles
along the fusome
The studies reported here provide new insight into the origin of Balbiani
bodies. Drosophila Balbiani bodies do not arise de novo within
oocytes, but are built by the transport of organelles from neighboring cells
within interconnected germline cysts. Our experiments make clear that many
components of oocyte cytoplasm arise in this manner. Centrioles were the first
cytoplasmic organelle whose movement through ring canals into the oocyte in
16-cell germarial cysts was described
(Mahowald and Strassheim,
1970). Studies of serial sectioned germaria in the electron
microscope also suggested that mitochondria move between cystocytes and toward
the oocyte in region 2b cysts (Mahowald
and Strassheim, 1970
;
Carpenter, 1975
;
Carpenter, 1994
). However,
despite a description of the mitochondrial cluster in the region 3 oocyte
(Mahowald and Strassheim,
1970
) and the proposal that it arose from transport, these
movements were not commonly viewed as a special process. Rather, they were
seen as just the start of an extended process of generalized `cytoplasmic
flow' from nurse cell to oocyte that formed an ongoing `nutrient stream'
responsible for the growth of the oocyte relative to the nurse cells
throughout oogenesis. (Little such growth takes place in the germarium.)
Nonetheless, these studies described the onset of mitochondrial movement,
their transient association with downstream ring canals, and the fact that
mitochondria appear to be constricted in diameter as they pass through ring
canals.
Our experiments document that virtually all of the newly formed
mitochondria in oocytes are derived from the Balbiani body. The great majority
are transported from other cystocytes along the fusome but 1/16th or more
might simply originate in the oocyte. Like oocyte determination itself,
Balbiani body formation was shown to depend on the fundamental cyst polarity
manifested in the fusome. Arising in embryonic germ cells
(Lin and Spradling, 1997), the
fusome builds up a framework of cyst polarity during the cystocyte divisions
(Lin and Spradling, 1995
;
de Cuevas and Spradling,
1998
). Fusome polarity probably acts directly to control centriole
migration (Grieder et al.,
2000
; Bolivar et al.,
2001
) and the meiotic gradient
(Huynh and St. Johnston,
2000
), and acts indirectly to differentiate and maintain the
oocyte by regulating the microtubule cytoskeleton
(Grieder et al., 2000
;
Huynh et al., 2001
).
Deciphering the molecular mechanisms that define fusome polarity and allow the
fusome to control microtubule organization remains a central issue for
understanding Balbiani body formation and oocyte development.
Oocytes develop from germline cysts or syncytia in diverse species
(reviewed by Pepling et al.,
1999) so Balbiani bodies may arise through intercellular transport
in a wide range of organisms besides Drosophila. In both
Xenopus (Al-Mukhtar and Webb,
1971
; Kloc and Etkin,
1995
) and the mouse (Pepling
and Spradling, 2001
), mitochondrial clouds present within
interconnected germ cells are thought to be precursors to the Balbiani bodies
that arise shortly after the cysts break down and form primordial follicles.
In Drosophila, the large chunk of fusome at the anterior of the early
stage 1 oocyte contains clustered centrioles and is likely to act as a
microtubule-organizing center (Grieder et
al., 2000
). It may attract and retain mitochondria, Golgi and
localized macromolecules as they enter the oocyte, thereby creating the
Balbiani body. Xenopus Balbiani bodies may arise in a similar fashion
as they have a similar organization consisting of a spectrin-rich zone,
mitochondria, Golgi and the Metro region containing RNAs in transit. However,
there has been insufficient study of the Xenopus larval ovary to
identify a fusome or some other material with microtubule organizing
properties that might play an analogous role. In most other systems whose
Balbiani bodies share the same basic structure in young oocytes, very little
is known about their origin during earlier stages of germ cell
development.
The Balbiani body may facilitate RNA localization
The Balbiani bodies in many species contain structures resembling germinal
granules. In Xenopus, these granules are found in a region containing
specific RNAs that are also destined to be localized in the egg and
incorporated in germ cells. Consequently, the Balbiani body has been proposed
to function as a messenger transport organizer (METRO) that organizes and
mediates the delivery of RNAs and germinal granules to the vegetal pole of the
egg (Forristall et al., 1995;
Kloc and Etkin, 1995
;
Kloc et al., 1998
). Specific
elements have been mapped in the 3' UTR of the Xcat2 mRNA that
are sufficient for localization to the Balbiani body (Zhao and King, 1996) or
to the germinal granules themselves (Kloc
et al., 2000
).
Our studies revealed that the Drosophila Balbiani body may play a related role. oskar RNA, a key component that is capable of inducing germ plasm formation, was associated with the posterior segment of the Balbiani body in early stage 1 oocytes, much as Xcat2 is localized in the Xenopus Balbiani body. A few hours later, towards the end of stage 1, osk RNA moves to the oocyte posterior along with the other Balbiani-associated RNAs and proteins we studied, presumably in response to the shift in microtubule polarity that occurs at this time. Thus, at least some molecules that participate in germ plasm assembly associate with the Balbiani body in early Xenopus and Drosophila oocytes.
We found that Drosophila RNAs that become associated with the Balbiani body, like organelles, first interact with the fusome during early stages of cyst development. However, there were significant differences in these fusome interactions that probably reflect different molecular mechanisms of delivery to the Balbiani body. Organelles associate next to the fusome along much of its length and subsequently move toward the center, in concert with microtubule minus ends. By contrast, the RNAs associate with one or a few cells at the center of the fusome from the earliest stages they could be detected, and are located within it, as well as nearby. These observations suggest that localized RNAs may read the fusome polarity directly, and need not rely on changes in microtubule organizing activity to get to the oocyte or be stabilized within it.
Potentially significant differences exist in the role of RNA transport
played by the Drosophila and Xenopus Balbiani bodies. The
Drosophila Balbiani body associates with germ plasm RNAs for only
5-10 hours during early stage 1. By contrast, Xenopus Balbiani bodies
associate throughout stage 1 of oogenesis, a process requiring many days, with
at least 11 RNAs. When the RNAs leave the Drosophila Balbiani body,
mitochondria mostly remain behind, only to follow much later in oogenesis. By
contrast, in Xenopus, both mRNAs and mitochondria are reported to
proceed together to the vegetal pole (Kloc
and Etkin, 1995). These differences may simply reflect differences
in the timing of cytoskeletal remodeling that control these events. Moreover,
our observation that a small subset of mitochondria recognized by COXI
antisera do translocate with the RNAs in stage 1 indicates that certain
Drosophila mitochondria may follow a Xenopus-like pattern.
However, it remains possible that RNAs in transit to the oocyte posterior may
simply pass through the Balbiani body without begin affected in any way.
Previously, Wilsch-Bräuninger et al.
(Wilsch-Bräuninger et al.,
1997) described sponge-like structures in the cytoplasm of stage
4-10 nurse cells that were associated with Exu protein, RNA, and (frequently)
mitochondria and nuage. They proposed that these structures were analogous to
classical Balbiani bodies and that they mediate transport of localized
transcripts such as bicoid RNA. Our results suggest that the ooctye
contains a true Balbiani body much earlier in stage 1 follicles. The
sponge bodies described by Wilsch-Bräuninger et al.
(Wilsch-Bräuninger et al.,
1997
) more likely represent transport complexes organized at the
surface of nurse cell nuclei that subsequently move through the follicle and
into the ooctye. However, there may be structural and molecular similarities
between nurse cell transport complexes and those mediating transport out of
the Balbiani body.
Ring canal behavior and fusome breakdown controls mitochondrial
movement
Our studies provide further evidence that the ring canals that join the
cystocytes play an important role in regulating Balbiani body formation.
Mitochondria appear to first enter the oocyte when fusome segments within the
adjoining ring canals break apart, unplugging the channels. Subsequently, a
novel mechanism blocks further mitochondrial passage through these canals,
because we observed large backups of mitochondria outside each oocyte ring
canal in young oocytes and documented a lack of mitochondrial movement into
the oocyte in movies. Mitochondria did not accumulate in the same manner
around the ring canals that join nurse cells, but were spread throughout the
cell and in the nuclear periphery. This behavior has the effect of limiting
the mitochondrial genotypes within the oocyte to those found in Balbiani body
mitochondria until well after mitochondria have begun to associate with the
germ plasm at the oocyte posterior pole. Despite the importance of these
regulatory steps, we still know very little about how movement through ring
canals is controlled.
Fusome-mediated transport may be selective
Our studies suggest that centrioles, mitochondria, Golgi, RNAs and other
key components of oocyte cytoplasm are added to the Drosophila oocyte
by a special mechanism that may have been widely conserved in evolution. It is
remarkable that in the oocyte, the lone cell that will contribute cytoplasm
for the next generation of organisms, many fundamental components of cytoplasm
do not arise by random partitioning among daughter cells. Rather, an elaborate
mechanism is used to transport materials from multiple cells and maintain them
in a large aggregate for an extended period of time. It is possible that
Balbiani bodies do not play a specific role in ooctye development, but
represent a byproduct of the unusual centrosome behavior in these cells.
However, we favor an alternative hypothesis.
One of the potentially most interesting reasons that oocyte organelles
might be delivered en mass via the fusome would be to increase organelle
fitness (Pepling et al., 1999;
Pepling and Spradling, 2001
).
Mitochondrial DNAs are known to accumulate mutations that have frequently been
postulated to affect the aging of cells and tissues
(Chinnery and Turnbull, 2001
)
(reviewed by Partridge and Gems,
2002
). If only mitochondria with functional genomes were able to
associate with the fusome and move into the oocyte, damaged genomes might be
weeded out when they still represent a small fraction of the total. Such a
system would be far more efficient than eliminating defective genomes by
inducing the apoptosis of entire germ cells. A purifying mechanism based on
organelle selection might be particularly important in organisms that need to
produce eggs with a high average viability, or that must support long
intergenerational life spans.
Several other observations may also be explained by the need to eliminate defective mitochondrial genomes. The exclusion of nurse cell mitochondria from passing through the oocyte ring canals prior to dumping would ensure that only the `selected' mitochondria in the Balbiani body populated the germ plasm. Mitochondria may break up into small, nearly round, organelles during this period so that each will contain a single genome whose fitness can be tested. The cytoplasmic streaming of the ooctye may serve to mix the two populations of organelles so each somatic cell type inherits at least some of the selected mitochondrial population. Finally, a requirement for translation on mitochondrial ribosomes in the early embryonic germ plasm might serve as a concluding selective step to ensure that viable germ cells are well supplied with intact mitochondrial genomes. If female germ cells do possess mechanisms to remove defective mitochondria, they would probably have contributed to the evolutionary conservation of germ line cysts and Balbiani bodies.
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ACKNOWLEDGMENTS |
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