INTRODUCTION
Oocytes provide virtually all the mitochondria of the zygote at
the time of fertilization. Embryonic viability and vitality
depend on the egg acquiring functional cellular components in
appropriate numbers and locations. Studies of mitochondria
during oogenesis hint that processes other than simple growth
and duplication may exist to address these needs. In particular,
mitochondria, Golgi bodies, endoplasmic reticulum and other
organelles form aggregates or ‘clouds’ in pre-follicular germ
cells and then congregate within a large structure known as the
Balbiani body in the early oocyte cytoplasm of many species
(reviewed by Raven, 1961; Guraya, 1979; de Smedt et al.,
2000). Mitochondrial clouds sometimes arise in interconnected
germ clusters shortly before cyst breakdown (Al-Mukhtar and
Webb, 1971) and the apoptotic death of many germ cells
(Pepling and Spradling, 2001). Balbiani bodies may also
contain RNAs and organelles destined for future germ cells,
leading to the idea that they play a role in the development of
germ plasm (see Matova and Cooley, 2001). However, it has
been difficult to test the function of mitochondrial clouds and
Balbiani bodies genetically, in part because they have not been
described in model organisms such as Drosophila or C.
elegans.
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
1579Development 130, 1579-1590
© 2003 The Company of Biologists Ltd
doi:10.1242/dev.00365
Maternally inherited mitochondria and other cytoplasmic
organelles play essential roles supporting the development
of early embryos and their germ cells. Using methods that
resolve individual organelles, we studied the origin of
oocyte and germ plasm-associated mitochondria during
Drosophila oogenesis. Mitochondria partition equally on
the spindle during germline stem cell and cystocyte
divisions. Subsequently, a fraction of cyst mitochondria
and Golgi vesicles associates with the fusome, moves
through the ring canals, and enters the oocyte in a large
mass that resembles the Balbiani bodies of Xenopus,
humans and diverse other species. Some mRNAs, including
oskar RNA, specifically associate with the oocyte fusome
and a region of the Balbiani body prior to becoming
localized. Balbiani body development requires an intact
fusome and microtubule cytoskeleton as it is blocked by
mutations in hu-li tai shao, while egalitarian mutant
follicles accumulate a large mitochondrial aggregate in all
16 cyst cells. Initially, the Balbiani body supplies virtually
all the mitochondria of the oocyte, including those used to
form germ plasm, because the oocyte ring canals
specifically block inward mitochondrial transport until the
time of nurse cell dumping. Our findings reveal new
similarities between oogenesis in Drosophila and
vertebrates, and support our hypothesis that developing
oocytes contain specific mechanisms to ensure that germ
plasm is endowed with highly functional organelles.
Key words: Mitochondria, Oogenesis, Fusome, Balbiani body,
Patterning, Germ plasm
SUMMARY
A Balbiani body and the fusome mediate mitochondrial inheritance during
Drosophila oogenesis
Rachel T. Cox and Allan C. Spradling*
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@ciwemb.edu)
Accepted 6 January 2003
1580
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.
Maternal organelles may play developmental roles, rather
than just accumulating passively in the oocyte. Mitochondria
are enriched in the germ plasm, a special region found in many
eggs that specifies germ cells. Mitochondria in some vertebrate
eggs associate with germ plasm precursors known as
granulofibrillar material or nuage beginning very early in
oogenesis (Al-Mukhtar and Webb, 1971; Heasman et al., 1984)
(reviewed by Saffman and Lasko, 1999). Specific RNAs
destined for germ plasm also associate with the Balbiani body
and/or forming germ granules (Forristall et al., 1995; Kloc and
Etkin, 1995; Bradley et al., 2001). In Drosophila eggs,
mitochondria associate closely with germ plasm constituents
known as polar granules (Mahowald, 1968). Moreover,
exported mitochondrial ribosomes and/or their component
rRNAs have been proposed to actively contribute to Drosophila
germ plasm function shortly after fertilization (Iida and
Kobayashi, 1998; Amikura et al., 2001).
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 bodyassociated
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.
MATERIALS AND METHODS
Drosophila strains and culture
Fly stocks were cultured at 22-25oC on standard food. The y; ry506
strain (Karpen and Spradling, 1992), the hts1 mutation (Yue and
Spradling, 1992) and eglwu50 (Schupbach and Wieschaus, 1991) have
been described. Other genes and balancer chromosomes are described
in FlyBase.
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 (1×PBS, 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 α-Drosophila ATP synthase, β subunit (1:350, gift
from Dr Rafael Garesse), mouse α-human cytochrome c1 oxidase,
subunit I (COX1, 1:500, Molecular Probes), rabbit α-rat mannosidase
2 (1:300, Dr Kelley Moremen), rabbit α-Drosophila α-Spectrin
(1:300) (deCuevas et al., 1996), mouse α-Drosophila 1B1 (specific
for Hts protein, 1:100) (Zacci and Lipshitz, 1996), rabbit and rat αDrosophila
Vasa (1:2000 and 1:200, respectively, gift of Dr Paul
Lasko), mouse α-Drosophila Orb (1:100, Dr Paul Schedl,
Developmental Studies Hybridoma Bank), rat α-Drosophila Cup
(1:1000) (Keyes and Spradling, 1997), mouse and rat anti-α-tubulin
(1:350, Sigma; 1:20, Oxford Biotechnology, respectively), mouse αphosphotyrosine
PY20 (1:1000 ICN Biomedicals), and mouse αDrosophila
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 αrabbit
and α-mouse AlexaFluor488, AlexaFluor568, AlexaFluor546
and AlexaFluor633 (1:200, Molecular Probes); and goat α-rabbit, αmouse
and α-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)
R. T. Cox and A. C. Spradling
1581Balbiani body and oocyte organelles
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.
RESULTS
Visualizing individual Drosophila mitochondria
using confocal microscopy
Mitochondria in developing female germ cells have been
studied previously by electron microscopy (Mahowald and
Strassheim, 1970; Carpenter, 1975) (Fig. 1B). To facilitate
more detailed studies, we developed methods to visualize these
organelles in fixed or living tissues using confocal microscopy.
An antibody to Drosophila ATP synthase β-subunit was found
to label mitochondria in both germline and somatic cells (Fig.
1C), as the labeled organelles appear similar in size and
number to those revealed by electron microscopy. In addition,
a mitochondrial-GFP marker (‘mito-GFP’) constructed by
fusing the human COX VIII mitochrondrial targeting signal to
the N terminus of EGFP and expressing the construct from the
UASp promoter (see Materials and Methods) produced an
indistinguishable pattern of labeling. By visualizing
mitochondria within living ovarian tissue from strains
expressing mito-GFP (Fig. 1D) or after incubation with
fluorescent molecules such as ‘Mitotracker’ (Fig. 1E), we
could study their dynamic behavior. Time-lapse confocal
movies of mitochondrial behavior could be obtained using a
special mounting technique and a spinning disc confocal
microscope with a relatively low rate of sample bleaching (see
Materials and Methods).
Mitochondria change shape and distribute equally
during asymmetric stem cell and cystocyte
divisions.
Germline stem cells (GSCs) and their progeny divide
Fig. 1. High resolution detection of mitochondria. (A) The
germarium in cross-section; at the anterior tip (region 1, left) reside
two germline stem cells (blue) as well as cystoblasts and growing
cysts (gray). Region 2a contains 16-cell germline cysts that enter
meiosis and begin centrosome migration, while in region 2b, more
mature cysts associate with follicle cells (green), and stretch to span
the entire width of the germarium. Subsequently, in region 3, a new
follicle with a well-defined oocyte (white) prepares to bud off. The
fusome (red) grows, branches and eventually breaks down, leaving a
remnant in the oocyte; it provides a distinctive marker for each stage.
(B) A transmission electron micrograph showing the nucleus (nu)
and mitochondria (m) of a region 1 cystocyte. The inset shows a
cross-section of a ring canal containing a fusome plug surrounded by
mitochondria. (C) Another region 1 cystocyte sectioned facing a ring
canal (arrowhead): mitochondria (anti-ATP synthase) are green, the
fusome (1B1 antibody) is blue and ring canal actin (rhodamine
phalloidin) is red. (D,E) Mitochondria within living 16-cell
cystocytes (broken circles) are labeled with the mito-GFP fusion
gene (D) or using Mitotracker (E). Scale bars: in E, 1 µm for B-E;
100 nm in inset in B.
1582
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.
Cystocytes fail to double in size prior to mitosis during their
rapid, synchronous cell cycles. Consequently, the overall size
of a newly completed cyst is only about fourfold larger than a
stem cell, despite containing 16 times as many cells (King,
1970). To determine if mitochondria follow a similar pattern,
we measured their number and shape within growing cysts.
Mitochondrial number increases about eightfold and
mitochondrial volume rises 14- to 20-fold during cyst
formation, compared with only a 3.6-fold gain in total
cytoplasm (Table 1). Hence mitochondrial capacity
approximately doubles prior to each mitosis, despite the
cleavage-like cystocyte cell cycle. Mitochondrial growth must
be regulated differently than total cytoplasmic volume.
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
R. T. Cox and A. C. Spradling
Fig. 2. Equal distribution of mitochondria during cyst formation.
(A) Asymmetric fusome behavior during stem cell and cystocyte
divisions. The fusome (red) remains in the stem cell (GSC) following
mitosis (green, mitotic spindle; blue, chromosomes), while a smaller
fusome grows in the cystoblast (CB) before cell separation is
complete. The fusome branches during subsequent divisions that
produce two-, four-, eight- and 16-cell cysts. The letters under each
diagram correspond to figure panels illustrating the indicated stage.
(B) A germline stem cell (broken circle) in G2 phase. Mitochondria
(green) vary in length but most associate with the fusome (blue).
Green, ATPsynthase; red, phalloidin; blue, 1B1. (C) Another GSC
(broken circle), revealing the clustering of microtubules (green)
around the fusome (red). Green, tubulin; red, 1B1; blue, DNA. (D) A
mitotic GSC (broken circle). Round mitochondria associate with the
outer region of the mitotic spindle (red) and segregate equally (see
Table 1). Green, ATP synthase; red, tubulin; blue, DNA. (E) A stem
cell (broken circle, left) and daughter cystoblast (broken circle, right)
in S phase after mitosis remain connected via a ring canal (red)
containing the fusome (blue). Mitochondria aggregate around the
stem cell fusome (upper left), but show little or no association with
the cystoblast fusome (lower right). Green, ATPsynthase; red,
phalloidin; blue, 1B1. (F) Spherical mitochondria (green) within an
eight-cell cyst (broken circle) do not associate with the fusome (blue).
Green, ATPsynthase; red, phalloidin; blue, 1B1. (G) The anterior
region of a living germarium labeled with Mitotracker to reveal the
position and movement of mitochondria. Mitochondria in region 1
cysts do not associate with the fusome (arrowhead) while
mitochondria in region 2 do (arrow). B-F are projections of multiple
confocal z-sections. Scale bars: in F, 5 µm for B-F; 10 µm for G.
1583Balbiani body and oocyte organelles
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.
Directed mitochondrial migration toward the center of the
fusome was associated with significant reductions in random
intracellular movement. Drosophila germaria were labeled
using the fluorescent dye ‘Mitotracker’ (Fig. 2G) and movies
that spanned up to 3 hours of developmental time were
recorded with a confocal microscope (see Materials and
Methods). At all stages of germ cell development,
mitochondria unassociated with the fusome are mobile, and
sometimes undergo bursts of directed motion within a cell (Fig.
2G, arrowhead). By contrast, mitochondria associated with the
fusome in stem cells or developing cysts show little net
movement within the cytoplasm (Fig. 2G, arrow). They remain
on the fusome but their translocation toward its center occurs
too slowly to be clearly visualized in these experiments.
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-taishao
(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),
Table 1. Mitochondrial numbers and concentrations in germline cysts
Mitochondrial
Number of Number of Volume/ Mitochondrial
diameter (nm)*
mitochondria/ mitochondria/ Cyst cytoplasmic Cell cytoplasmic mitochondrion volume§/cell
Stage (number of cells) Major axis Minor axis cyst† cell volume (µm3) volume‡ (µm3) (µm3) (µm3)
Germline stem cell (10) n.d. n.d. 140±32 140 1600 1600±520 0.20 28±12
Cystoblast (10) n.d. n.d. 150±23 150 1600 1600±630 0.27 40±17
Two-cell cyst (16) n.d. n.d. 280±46 140 2600 1300±110 0.096 27±17
Four-cell cyst (24) 430±43 340±36 410±72 100 3800 950±130 0.26 26
Eight-cell cyst (25) 460±30 380±29 810±31 100 4500 560±110 0.30 29
16-cell cyst (25) 550±36 460±34 1100±43 69 5800 360±99 0.53 36
*Mitochondrial diameters were measured by applying NIH image software to confocal images of individual mitochondria from germaria stained with ATP
synthase.
†Mitochondrial number was determined using ATP synthase staining and confocal microscopy.
‡Cell cytoplasmic volume was determined by measuring major and minor axes, as outlined by phalloidin staining, and then subtracting the nuclear volume, as
determined by either the region without Vasa staining or enclosed by Lamin staining. Calculations assumed the structures were ellipsoids. The cell radii were
very similar to those we measured in our transmission electron micrographs. Cyst cytoplasmic volume was determined by extrapolation for any unmeasured cells.
§Mitochondrial volume was measured for germline stem cells, cystoblasts and two-cell cysts using confocal microscopy of germaria stained with ATP synthase
and using NIH Image software. Total pixel volume was determined at the highest setting where labeling remained confined to mitochondria. For four-, eight- and
16-cell cysts, which contain ellipsoidal mitochondria, volume was calculated from the mitochondrial diameters.
Values without standard errors are calculated from the other data.
1584
ER-like vesicles in the residual fusome (Fig. 4A,B) and
centriole clusters (Fig. 4B,C). Using anti-α-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 αmannosidase
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.
A subset of mitochondria may be enriched in the
Balbiani body
To investigate whether more than one population of mitochondria
might be present in pre-follicular cysts, we examined the staining
of a variety of other antibodies to mitochondrial proteins. We
observed that crossreacting antibodies against human cytochrome
c1 oxidase subunit I (COXI) label round cytoplasmic structures
that associate with the fusome in region 2 and move into the
oocyte in stage 1, much like mitochondria and Golgi vesicles
(Fig. 4E). Double-labeling experiments showed that the small
COXI reactive particles represent a subset of germ cell
mitochondria because they co-localize with ATP-synthase
staining (Fig. 4F) but are distinct from Golgi (Fig. 4G). However,
it was not clear what property might set these mitochondria apart
from others. Very few mitochondria in region 1 or in somatic cells
were labeled by the anti-COXI antisera, and it was difficult to
explain the pattern of labeling based on signal strength or
accessibility to staining (somatic cells should be more
accessible). Moreover, two other antisera, anti-human COXII and
anti-yeast porin, produced a similar pattern. These results suggest
that some of the mitochondria entering the oocyte in the Balbiani
body have distinctive properties.
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
R. T. Cox and A. C. Spradling
Fig. 3. Fusome-dependent formation of a Balbiani body. (A-D) Green, mitochondria (ATPsynthase); red, fusome (1B1). (A) Mitochondria in a
16-cell cyst (broken outline) midway in region 2a begin to associate with the fusome (red); in an older adjacent cyst (not outlined), they move
toward the center of the fusome (yellow). (B) Mitochondrial movement along the fusome has progressed further in these two region 2b cysts
(broken outline). (C) In cysts just entering region 3, clouds of mitochondria (arrowhead) accumulate near the ring canals that connect to the
oocyte (small broken circle). (D) In region 3 follicles and young budded egg chambers, a Balbiani body containing many aggregated
mitochondria (arrow) is visible in the anterior of the oocyte (small broken circle). (E) Electron micrograph of a region 3 follicle (large outline)
reveals mitochondria entering the oocyte (small outline) via a ring canal (arrowheads) to form the Balbiani body (arrow). (F,G) Balbiani bodies
fail to form in cysts from hts mutant females that lack fusomes. Arrowheads show mitochondrial clusters around presumptive centrosomes in
the stem cells. Green, mitochondria (ATPsynthase); red, germ cells (Vasa); blue, DNA. (G) A stage 1 hts cyst (broken outline) showing the
absence of a Balbiani body or mitochondrial clusters near presumptive ring canals. (H) Cysts mutant for egalitarian (egl) contain a normal
fusome (red) but mitochondrial aggregates (green) of reduced size arise in all 16 cells at stage 1. Green, mitochondria (ATPsynthase); red,
fusome (1B1). Scale bars: in H, 10 µm for A-D,F-H; in E, 2 µm for E.
1585Balbiani body and oocyte organelles
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.
Several proteins, including Orb and Cup,
also localize within the future oocyte in
developing region 2-3 cysts. We examined the
relationship between these proteins and the
Balbiani body using specific antibodies (Fig. 5E-I). Cup
protein (Fig. 5E, red) is located on and adjacent to the central
fusome (Fig. 5E, blue) within the pro-oocyte in late region 2b
cysts, near the clustered mitochondria (Fig. 5E, green). As the
mitochondria enter the ooctye early in region 3 to form a
Balbiani body, Cup associates briefly along the posterior edge
(Fig. 5H) but soon moves towards the oocyte posterior (Fig.
5F). The behavior of Orb protein is very similar at these stages
(Fig. 5G,I), but Orb is always slightly more diffuse. These
proteins may be associated with some of the localized mRNAs
that follow a similar path. Our experiments show that at least
for a brief period the Drosophila Balbiani body is regionally
organized, with fusome material located at the anterior and
specific RNAs and proteins located toward the posterior.
Balbiani body-derived mitochondria preferentially
associate with the germ plasm
The previous experiments indicate that Drosophila eggs
Fig. 4. Multiple organelles associate with the
Balbiani body. (A) An electron micrograph of the
Balbiani body in the anterior region of a young
oocyte. The residual fusome (white arrow), still
rich in ER-like vesicles, lies at the anterior near
the ring canals (white arrowheads) it formerly
occupied. Numerous mitochondria are located
adjacent to the fusome, while scattered Golgi
stacks lie further below (asterisks). (B,C) Electron
micrographs showing nearby sections of a single
ring canal (white arrowhead) connecting a nurse
cell (NC) and a young ooctye (O). Five centrioles
or centriole pairs (numbered) lie within the
material moving in to form the Balbiani body.
(D) The anterior portion of an ovariole stained
with anti-α-mannosidase 2 to reveal Golgi
vesicles (green) and with 1B1 to show the fusome
(red). The Golgi elements associate with the
fusome beginning in region 2b (yellow,
arrowhead), move into the oocyte with the
Balbiani body (arrow), but in a slightly older
oocyte (broken line) spread throughout the
ooplasm. (E) COXI-reactive vesicles (green)
associate with the fusome (red) in region 2b
(yellow, arrowhead) and move into the oocyte as
part of the Balbiani body (arrow). (F) COXIpositive
particles (green) appear to be
mitochondria because they are co-labeled with the
mitochondrial ATP synthase marker (red). Broken
circle, oocyte. (G) COXI-positive particles (green)
are not co-labeled with a Golgi marker (red, αmannosidase
2). Broken circle, oocyte. Scale bars:
in E, 10 µm for D,E; in A, 1 µm for A; in C, 1 µm
for B,C; in G, 1 µm for F,G.
1586
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.
To examine when Balbiani body-derived mitochondria begin
to associate with the germ plasm, we carried out double
labeling experiments using germ plasm markers. In early
follicles, the Balbiani body mitochondria remain clustered
around the germinal vesicle, often at its posterior (Fig. 6A).
After the microtubules re-organize at stage 7, Balbiani body
mitochondria disperse throughout the cytoplasm (Fig. 6B). By
stage 9, they begin to contact the forming germ plasm at the
posterior pole (Fig. 6C). Later, in stage 10A, many more
R. T. Cox and A. C. Spradling
Fig. 5. Localized RNAs and
proteins associate with the
fusome and Balbiani body. (A) In
situ hybridization reveals that osk
RNA (green) associates with the
central fusome (red) within the
future oocyte beginning in region
2 (arrowhead). The RNA moves
to the posterior of the oocyte
during stage 1 (rightmost
follicle). (B) An early region 3
oocyte (broken line) shown at
higher magnification, reveals that
osk RNA (green) partially
overlaps (yellow) the Balbiani
body on the posterior side of the
mitochondrial mass (red, antiATP
synthase). The arrowhead
indicates the fusome (blue, 1B1).
B′ is the same image as B without
the green channel, in order to
better show the Balbiani body
(arrow). (C) orb RNA (green),
like osk RNA, associates with the
center of the fusome (red)
beginning early in region 2
(arrowheads), and then moves to
the oocyte posterior in stage 1
(rightmost follicle). (D) Like osk
RNA, orb RNA (green) also overlaps with the Balbiani body (red). D′ is the same as D without the green channel, in order to better show the
Balbiani body (arrow). (E,F,H) Cup protein (red) associates with the center of the fusome (blue) and with mitochondria (green) in region 2
cysts. (G,I) Orb protein (red) localizes to a similar position as Cup (green, mitochondria). (H,I) Both Cup (red, H) and Orb (red, I) briefly
associate with the edge of the Balbiani body mitochondria (green) in early region 3 oocytes and then move to the oocyte posterior (not shown).
Scale bars: in G, 10 µm for A,C,E-G; in D′ 1 µm for B,B′,D,D′; in I, 1 µm for H,I.
Table 2. Nurse cell mitochondria do not enter the oocyte
until stage 11
Rate of ring Ooplasmic
canal transit* streaming†
Stage 8 0.1/minute 0/10
Stage 9 1.5/minute 0/7
Stage 10A/B 3.6/minute 1/8
Stage 11‡ ӷ60/minute 4/4
*Mitochondrial movement through the ring canals was measured by
counting the number of organelles that crossed the nurse cell/oocyte boundary
in movies of the indicated stages during a 5 minute period. No movement was
seen prior to stage 8, and the measured rates prior to stage 11 probably
overestimate true nurse cell/oocyte transfer because some organelles only
appeared to cross the boundary because of the twisting of the egg chamber in
the observation chamber, rather than because of true translocation.
†The number of egg chambers with ooplasmic streaming (see Materials and
Methods)/the total number of egg chambers analyzed at the indicated stage.
‡The measured rate during stage 11 (dumping) strongly underestimates the
true value of mitochondrial translocation because individual frames required
2-3 seconds of exposure, during which time most mitochondria became
blurred and could not be followed.
1587Balbiani body and oocyte organelles
mitochondria associate with the expanded region
containing germ plasm (Fig. 6D,D′, arrowheads).
Although these studies show a gradual association
of Balbiani body mitochondria with the forming
germ plasm beginning in stage 9 and 10A, we
cannot rule out that a small subpopulation of these
mitochondria, such as those labeled by COXI
antisera, associate with germ plasm precursors at an
earlier time. At stage 11, most of the oocyte
cytoplasm begins to undergo vigorous cytoplasmic
streaming, mixing the two mitochondrial
populations (Table 2). However, streaming does not
dislodge the germ plasm, so the Balbiani bodyderived
mitochondria in this location are likely to
remain associated at the oocyte posterior and
eventually be inherited by forming germ cells.
DISCUSSION
Tools for a genetic analysis of
mitochondrial inheritance
Our experiments revealed that mitochondria behave
in a distinctive but previously unknown manner
during Drosophila oogenesis. They associate with
the spindle to facilitate equal inheritance, fragment
into small spheres and associate with the fusome,
damping their normal intracellular movement. One
major subpopulation moves along the fusome to
form an oocyte Balbiani body, while another group
is blocked from entering the oocyte by ring canals.
Each of these biological events can now be
visualized and analyzed dynamically using the
spinning disk confocal microscope, which will
make it possible to screen for genes that
developmentally control these behaviors.
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
Fig. 6. Balbiani body-derived mitochondria associate with the germ plasm prior
to nurse cell dumping. (A) Mitochondria labeled with anti-ATP synthase (green)
congregate on the posterior side of the GV (arrowhead) in stage 6-7 egg
chambers. Green, ATP synthase; red, germ cells (Vasa); blue, ring canals (antiPhosphotyrosine).
(B) By contrast, mitochondria (green) in the nurse cells
adjacent to the oocyte accumulate (arrowheads) at the ring canals (blue), but fail
to enter the oocyte until nurse cell dumping (see Table 2). Green, ATPsynthase;
red, Vasa; blue, phosphotyrosine. (C) At stage 9, a few mitochondria (green)
associate with the germ plasm, which has begun to coalesce at the oocyte
posterior as revealed by Vasa staining (red). Green, ATPsynthase; blue,
Phosphotyrosine. (D,D′). By stage 10A, mito-GFP-labeled mitochondria (green)
are abundant in the germ plasm (arrowheads in D′). D′ shows a higher
magnification. Green, mito-GFP; red, Vasa; blue, actin-rich membranes
(phalloidin). (E) A summary of the structure and assembly of the Drosophila
Balbiani body. Green arrows indicate direction of movement along the fusome
(red) towards the oocyte. Scale bars: in C, 10 µm for A-D; in D′, 5 µm for D′.
1588
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
R. T. Cox and A. C. Spradling
1589Balbiani body and oocyte organelles
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.
We thank Dr Raphael Garesse for providing the ATP synthase
antibody and Mike Sepanski for assistance with the transmission
electron microscopy. R.T.C. was supported by the Helen Hay Whitney
Foundation and the Howard Hughes Medical Institute.
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