Max-Planck-Institut für Entwicklungsbiologie, D-72076 Tübingen, Germany
Localization of maternally provided RNAs during oogenesis is required for formation of the antero-posterior axis of the Drosophila embryo. Here we describe a subcellular structure in nurse cells and oocytes which may function as an intracellular compartment for assembly and transport of maternal products involved in RNA localization. This structure, which we have termed "sponge body," consists of ER-like cisternae, embedded in an amorphous electron-dense mass. It lacks a surrounding membrane and is frequently associated with mitochondria. The sponge bodies are not identical to the Golgi complexes. We suggest that the sponge bodies are homologous to the mitochondrial cloud in Xenopus oocytes, a granulo-fibrillar structure that contains RNAs involved in patterning of the embryo.
Exuperantia protein, the earliest factor known to be required for the localization of bicoid mRNA to the anterior pole of the Drosophila oocyte, is highly enriched in the sponge bodies but not an essential structural component of these. RNA staining indicates that sponge bodies contain RNA. However, neither the intensity of this staining nor the accumulation of Exuperantia in the sponge bodies is dependent on the amount of bicoid mRNA present in the ovaries. Sponge bodies surround nuage, a possible polar granule precursor. Microtubules and microfilaments are not present in sponge bodies, although transport of the sponge bodies through the cells is implied by their presence in cytoplasmic bridges. We propose that the sponge bodies are structures that, by assembly and transport of included molecules or associated structures, are involved in localization of mRNAs in Drosophila oocytes.
THE antero-posterior axis of the Drosophila embryo is determined by the localization of maternal
mRNAs in the egg. Two groups of genes are involved in the localization of the RNAs and thereby initiate
the formation of the segmented regions along the antero- posterior axis: the anterior system is necessary for the formation of head and thoracic structures, whereas the posterior system is responsible for abdomen formation and for
assembly of the germ plasm. Embryos from females homozygous mutant for any member of these two groups of
genes show pattern defects in the respective body regions
(for review see St Johnston and Nüsslein-Volhard, 1992 The key event in the anterior system is the localization of
the bicoid (bcd)1 mRNA to the anterior pole, which results
in a graded distribution of the transcription factor Bicoid
throughout the embryo (Frigerio et al., 1986 In addition to its role in the formation of the anterior
pattern, staufen is required for the localization of the oskar
(osk) mRNA to the posterior pole of the embryo, which is
necessary for abdominal segmentation (Ephrussi et al.,
1991 The localization of osk and bcd mRNA is initiated during oogenesis (Berleth et al., 1988 Translocation and localization of RNAs can occur via a
three-step pathway as proposed by Wilhelm and Vale (1993) Exu was proposed to be necessary for an association of
bcd mRNA with the microtubule-dependent localization
machinery (Pokrywka and Stephenson, 1991 In this paper we describe a subcellular structure in the
female germ line of Drosophila that contains RNA and is
strongly enriched for Exu protein. Our data suggest that
the so-called sponge bodies might form an intracellular
compartment for the assembly and transport of cis- and
trans-acting elements involved in RNA localization.
Fly Strains
The wild-type strain was OregonR. Unless noted otherwise we used flies
homozygous for the exuVL allele to obtain ovaries that express neither exu
RNA nor protein at detectable amounts (Hazelrigg et al., 1990 Antibody Generation and Preadsorption
The 3rd exon of the exu gene was amplified out of genomic DNA. The
fragment was cloned into the pQE12 vector (Qiagen, Chatsworth, CA). A
fusion protein containing six COOH-terminal His-residues and amino acids 100-532 of the exu gene product was expressed in Escherichia coli and
purified via a Ni-affinity column. The purified protein was used for subcutaneous injection of two rabbits. The sera of both rabbits were tested
against the preimmune sera on Western blots with bacterially expressed
Exu-6xHis fusion protein and extracts of wild-type or exu Drosophila
ovaries. Both sera showed very high specificity for the expected band of
~60 kD in extracts of wild-type but not exu ovaries. We therefore used
the unpurified rabbit serum for all further experiments. Before use, an aliquot of the serum was preadsorbed overnight at an equal volume of
paraformaldehyde-fixed exu ovaries or embryos.
Ovary Dissection and Fixation
Dissections were carried out in PBS at room temperature (or on ice,
which did not lead to different results). The ovaries were transferred into
a fixative consisting of 85 volume parts 4% paraformaldehyde in 200 mM
Pipes, pH 7, and 15 parts saturated picric acid (improved protocol of Stefanini et al., 1967 Embedding and Sectioning
For structural analysis, the ovaries were embedded in Epon 812 (Glycid-ether 100; Roth, Karlsruhe, Germany). Fixed (or freshly dissected for membrane analysis) ovaries were incubated in 1% OsO4 and 1% tannic acid in
PBS for 1 h on ice and stained with 1% aqueous uranyl acetate for 1 h at
4°C. These heavy metal treatments were omitted for samples used for
RNA detection (see below). The samples were subsequently dehydrated
through an ethanol series at room temperature. They were infiltrated
twice with a 1:1 mixture of ethanol and Epon and finally with pure Epon
resin overnight. After a second step in the pure resin for several hours,
they were embedded in flat wells and heat polymerized at 60°C for 48 h.
For immunogold and immunofluorescent labeling, the ovaries were embedded in the methacrylate resins Lowicryl K4M or HM20 (Polyscience Ltd, Eppenheim, Germany). (All pictures shown here are taken from sections embedded in HM20 since they yielded better contrast but similar labeling.) The ethanol dehydration for the Lowicryl resins was performed
with progressive lowering of the temperature (1 h in each ethanol step).
Infiltration with 2:1 and 1:1 mixtures of ethanol and resin was performed
at The sample blocks were trimmed on an LKB pyramitome. Sections of
50-100 nm were cut by an LKB ultramicrotome, and Lowicryl sections
were mounted either on pioloform-coated, carbon-coated copper grids, or
on polylysine-treated coverslips. Epon sections used for studying the ultrastructure were mounted on uncoated copper grids.
Immunofluorescence and Immunoelectron Microscopy
For postembedding labeling, the Lowicryl sections (on grids or coverslips)
were blocked with PBG (0.2% gelatine, 0.5% BSA in PBS) twice for 10 min. The first antibody was incubated in the same solution [1 h for rabbit
anti-Exu antiserum 1:100, for mouse anti- The immunofluorescently labeled sections were treated with 0.4 µg/ml
DAPI for 5 min and washed again with PBS before mounting in Moviol
4-88 (Hoechst) containing 25 mg/ml DABCO as antifading agent.
For immunoelectron microscopy, the grids were washed for several
times with distilled water after the antibody labeling. They were incubated
on 1% aqueous uranyl acetate for 10 min and subsequently washed again
thoroughly with water. A contrasting step with 0.4% lead citrate trihydrate
followed for 3 min. The immunolabeling was studied with a transmission
electron microscope (model CM10; Philips Electronics Instruments, Inc.,
Mahwah, NJ).
Cytoskeletal Drug Treatment
OrR flies were starved overnight and subsequently fed on medium containing either 100 µM taxol or 20 µg/ml colchicine (or no drugs for control) for 2 d. The paired ovaries of one female were split; one half was further processed for Epon embedding (see above), whereas the other was
embedded in Lowicryl HM20 (see above). In parallel, ovaries from the
same batch of flies were processed for in situ hybridization with a DIG-
labeled bcd antisense RNA probe according to the protocol of Tautz and
Pfeifle (1989) RNA Staining
The RNA staining was performed by a modification of a protocol of Bernhard (1969) Exu Is Localized to Sponge-like Structures
To study the function of the Exuperantia protein (Exu) in
the female germ line, we investigated the intracellular protein distribution. On ultrathin methacrylate sections of ovaries, we observed a patchy distribution of Exu, as described
earlier for whole mounts and paraffin sections, and for an
Exu-GFP fusion construct (Fig. 1; Macdonald et al., 1991
To understand the nature of this patchy intracellular distribution pattern, we performed immunogold labeling of
Exu on the ultrathin Lowicryl sections for the transmission
electron microscope. We could observe a distinct, electron-dense structure in the cytoplasm in which Exu is highly enriched (Fig. 2). Strong immunogold labeling and high immunofluorescence were concentrated at the same position in the cells of the adjacent ultrathin sections. The distribution of these structures on the electron microscopic sections therefore directly corresponds to the patchy staining
pattern in nurse cells and oocyte seen by light microscope.
The densely Exu-labeled structure consists of elongated
elements embedded in an electron-dense matrix (as shown
in Fig. 2 [Lowicryl embedding] and in Fig. 3 [Epon embedding]). We will therefore refer to this structure as "sponge
body." The elongated elements of the sponge bodies are
formed by ER-like cisternae or small vesicles interspersed
between an electron-dense, amorphous material. These
ER-like tubules become most obvious after osmiumtetroxide treatment without previous fixation of the follicles,
thereby removing most of the soluble components of the
cytoplasm, including the electron-dense, amorphous matter of the sponge bodies (Fig. 4). However, the spacing between the tubules in these preparations appears larger
than between the elongated elements in unextracted follicles (compare Fig. 3 a and Fig. 4), so that we can not exclude the possibility that in addition to the membrane-bounded cisternae, other electron-translucent elements exist in the central regions of the sponge bodies that lack
bounding membranes and are removed by strong osmiumtetroxide fixation together with the electron-dense component of the sponge bodies. In addition to the elongated elements, occasionally small membrane-bounded
vesicles are present in the sponge body structures (Fig. 3
a). The outlines of the sponge body structures often appear indistinct, and they are not limited by a surrounding
membrane. Mitochondria are frequently associated with
the sponge bodies in the nurse cells, which is most obvious between stages 4 and 9 (Figs. 2 a and 3 a). In summary, we
define the sponge bodies as cytoplasmic aggregations of cisternae and vesicles embedded in amorphous material that
exclude most ribosomes. They are frequently situated in
close proximity to mitochondria.
Sponge Bodies Are Not a Specialized Golgi Apparatus
Since the sponge bodies include membrane-bounded cisternae and small vesicles, we were interested to know
whether these structures correspond to the Golgi apparatus of the cells. Only few typical Golgi consisting of ordered membranous stacks and small vesicles are present
within the ovarian germ line cells. However, the majority of Golgi complexes in nurse cells and oocytes consist of
aggregates of different sized vesicles and few membranous
stacks (Fig. 3, b and d). Golgi vesicles are frequently found
in proximity to the sponge bodies or even contained
therein. However, these Golgi vesicles are tightly packed
and never separated by electron-dense amorphous material like the cisternae of the sponge bodies. We can therefore conclude that the sponge bodies are not identical with
the Golgi apparatus of the Drosophila germ line cells.
Sponge Body Morphology Changes
with Progressing Development
Sponge bodies are first observed in the cytoplasm of stage
3-4 germ line cells and persist until stage 10-11. They are
found in the nurse cells as well as in the oocyte. The transport of the sponge bodies between the germ line cells is suggested by their presence in the cytoplasmic bridges (Fig. 5).
Throughout development, with the exception of the very
first sponge bodies forming that hardly label for Exu (in
stage 3-5 nurse cells, not shown), all sponge bodies contain
higher amounts of Exu than the surrounding cytoplasm
(Fig. 2).
The morphology of the sponge bodies differs slightly at
the different developmental stages and cell types. In nurse
cells (stage 4-9), the tight association of mitochondria with
the sponge bodies exists until mid-vitellogenesis (Figs. 2 a
and 3 a). At early stages (3-7), large aggregates of mitochondria exist, between which small sponge bodies are interspersed (not shown). Although there is a superficial
resemblance to fusomes, which are present in younger
stages, the fusomes never contain the electron-dense amorphous matter, and they show a wider spacing of the cisternae than the sponge bodies. Taken together with the presence of In the oocytes, mitochondrial aggregates like these in
the nurse cells are never observed. Instead, at stage 3-7
the cytoplasm is densely packed with small organelles (ER,
vesicles, lysosomes, Golgi, and microtubules), between
which small particles of electron-dense, amorphous material, resembling that of the later sponge bodies, are interspersed (Fig. 6). A clear distinction between sponge body
and non-sponge body material is still difficult, and Exu staining is rather uniform in these early oocytes. This early situation in the oocyte is terminated by the formation of
yolk granules that first aggregate in a central cluster (sometimes termed yolk nucleus). The forming sponge bodies
are restricted to lateral regions up to the time when the
yolk granules disperse throughout the oocyte (Fig. 2 d).
From then on, the sponge bodies are situated throughout
the cytoplasm of the oocyte, which accumulates organelles
and yolk granules of increasing number and size during the
vitellogenic stages (Fig. 3, c and d). By stage 10B, sponge bodies are no longer visible within this densely packed
ooplasm. However, Exu protein remains detectable up to
stage 12. It is then no longer restricted to a specific structure within the cytoplasm (Fig. 2 e).
Sponge Body Morphology Is Unchanged in Ovaries
Mutant for exu
Since Exu is highly enriched in the sponge bodies, we were
interested to see whether this protein is a structural component of the sponge bodies. We investigated ovaries from
females homozygous for the exuVL allele, which has a 700-bp
deletion due to imprecise P-element excision. Neither
RNA nor protein is detectable in these ovaries (exuVL ovaries) (Hazelrigg et al., 1990
RNA Is Present in the Sponge Bodies
To investigate whether the sponge bodies contain RNA,
we performed RNA staining on the ultrathin sections. This
staining procedure enhances the contrast of RNA-containing structures, as for example the euchromatin in the nuclei. Indeed, sponge bodies in sections treated with this
procedure are darkly contrasted in comparison to the surrounding cytoplasm (Fig. 8). This result suggests that the
sponge bodies do contain some RNA. Next, we studied
the contrast of the sponge bodies in follicles that lack bcd
mRNA. No bcd mutation exists that eliminates the expression of the RNA. However, ovaries lacking bcd mRNA can
be obtained taking advantage of mutations in the serendipity gene family. One of its members, serendipity
Exu Localization Is Not Dependent
on bcd Copy Number
We were interested to know whether the localization of
Exu in the sponge bodies depends on the presence of bcd
mRNA. We investigated ovaries that lack bcd mRNA (see
above). We find that the level and distribution of Exu in
the sponge bodies is similar to normal in these ovaries. Likewise, ovaries that have threefold-increased bcd mRNA
levels because of four additional copies of bcd show the
same distribution of Exu as wild-type ovaries (data not
shown). These experiments show that the localization of
Exu in the sponge bodies occurs independently of the
presence and amount of bcd mRNA.
Microtubules Are Not a Component
of the Sponge Bodies
Pokrywka and Stephenson (1991)
In cappuccino (capu) or spire (spir) ovaries, unusually
thick, cortical bundles of MTs are found within stage 8-10
oocytes as described by Theurkauf (1994b)
Double immunolabeling of these mutant oocytes for
Further evidence against a direct association of sponge
bodies with MTs came by performing a drug treatment in
a similar manner as described by Pokrywka and Stephenson (1991)
Since no microtubules were detectable in the sponge
bodies, we wondered whether the second major component of the cytoskeleton, actin filaments, might be associated with the sponge bodies. Neither double fluorescence
of ovaries expressing the Exu-GFP fusion protein with
rhodamine-coupled phalloidin, nor immunogold labeling
of ultrathin sections with antiactin antibodies could reveal
actin cables present in the sponge bodies (data not shown).
The amount of detectable actin in the nurse cells or oocyte
is very low during midoogenesis. However, in stage 10 the
apical borders of the nurse cells are densely lined with actin. Sponge bodies are accumulated there at the same time
(Figs. 2, c and b), and at this location an association with
the microfilament network might occur.
Sponge Bodies Surround Nuage
During stage 7-10A, Exu is accumulated around the nurse
cell nuclei (Fig. 1 a), and sponge bodies are enriched on
the cytoplasmic face of the nuclear membrane as determined by electron microscopy (Figs. 2 b, 3 a, and 15 a). An
electron-dense structure called the nuage or fibrous body
has been described previously at this location (Eddy, 1975
In stage 9-10 follicles, a transient accumulation of Exu
at the posterior pole is observed in comparison to the uniform distribution of Exu in the remaining ooplasm (Wang
and Hazelrigg, 1994
The Nature of the Sponge Bodies
In this paper we describe a new subcellular structure in
Drosophila germ line cells. According to its sponge-like
appearance, we have called this structure "sponge body."
In wild-type ovaries, Exu is enriched in the sponge bodies,
as we observed by electron and light microscopy. We therefore assume that the sponge bodies correspond to the migrating fluorescent particles found in fly ovaries that express an Exu-GFP fusion protein (Wang and Hazelrigg, 1994 Comparison of the Sponge Bodies with the
Mitochondrial Cloud of Xenopus Oocytes
The mitochondrial cloud in Xenopus oocytes is also thought
to be homologous to Balbiani's vitelline bodies (Heasman
et al., 1984 Transport of the Sponge Bodies
In addition to their similarity in structure, the different
Balbiani's vitelline body homologues may involve similar
transport mechanisms. In Xenopus, both microtubule- and
microfilament-based transport mechanisms have been
shown to be required for localization of different RNAs to
the mitochondrial cloud (Kloc and Etkin, 1995 Bohrmann and Biber (1994) Association of Sponge Bodies with bcd mRNA
Many early transcripts and proteins are accumulated in
early Drosophila oocytes. In this respect it is an interesting
observation that these oocytes show small electron-dense
granules resembling the amorphous matter of the sponge
bodies, which form slightly later. RNA-rich regions of cells
usually show up as electron-dense areas in electron micrographs, therefore it can be assumed that these granules in
the early oocytes are accumulations of transcripts. These
might then assemble with the elongated elements of the
sponge bodies for subsequent transport or interaction with
other factors.
To fulfill its function in bcd mRNA localization, we expect Exu to interact directly or indirectly with bcd mRNA
at some point during oogenesis. Sponge bodies are good
candidates for an intracellular compartment where this interaction could occur. Nevertheless, we still do not know
whether the sponge bodies contain bcd mRNA and whether
they are essential for the function of Exu in bcd mRNA localization. The manner in which Exu functions in bcd mRNA localization appears to be very different from that of
Staufen. Staufen directly binds to double-stranded RNA,
and its localization to the anterior pole of the embryo depends on the amount of bcd mRNA present in the embryos (Ferrandon et al., 1994 Cotransport of Sponge Bodies and the Nuage
The movements of the Exu-GFP particles (Wang and Hazelrigg, 1994).
; Frohnhöfer
and Nüsslein-Volhard, 1986
; Berleth et al., 1988
; Driever
and Nüsslein-Volhard, 1988a
,b; Driever and Nüsslein-Volhard, 1989
; St Johnston et al., 1989
). Three genes, exuperantia (exu), swallow, and staufen, are involved in different
steps in the localization of the bcd mRNA (Berleth et al.,
1988
; Stephenson et al., 1988
; St Johnston et al., 1989
).
; Kim-Ha et al., 1991
). A second requirement for osk
mRNA localization exists in the formation of the germ
plasm at the posterior pole (Lehmann and Nüsslein-Volhard, 1986
). The germ plasm is characterized by the presence of electron-dense polar granules that contain Osk
protein (Mahowald, 1962
, 1992
; Breitwieser et al., 1996
). An
additional factor shown to be present in the polar granules is Vasa, one of the members of the posterior system, which
directly interacts with Oskar protein (Breitwieser et al.,
1996
; Hay et al., 1988a
,b). In addition to its location in the
polar granules, Vasa is concentrated in the nuage (Hay et al.,
1988a
,b; Liang et al., 1994
). This granular structure of the
nurse cells has been proposed to be a stage in the life cycle
of the polar plasm, and it, as well as the polar granules,
contains RNA (Mahowald, 1971b
; Eddy, 1975
). Since
vasa encodes for an ATP-dependent RNA helicase of the
D-E-A-D family of proteins, it might act on the RNAs included in the nuage and polar granules, and therefore it might mediate their localization to these cytoplasmic organelles (Mahowald, 1971a
; Hay et al., 1988a
,b; Lasko and
Ashburner, 1988
, 1990
).
; St Johnston et al., 1989
;
Ephrussi et al., 1991
; Kim-Ha et al., 1991
). A Drosophila
egg chamber consists of 16 germ line cells surrounded by
follicle cells of somatic origin. The germ line cells arise
from a common stem cell by four rounds of incomplete divisions and remain interconnected by cytoplasmic bridges
(for a review of oogenesis see Spradling, 1993
). One cell of
this arising cluster is specified during development to form the oocyte. The other 15 cells develop into nurse cells with
highly polyploidized nuclei. Most of the maternal mRNAs,
as well as the factors required for RNA localization, are
produced in the nurse cells and then transported into the
oocyte. An extensive cytoskeletal network (for review see
Cooley and Theurkauf, 1994
) extending throughout the
germ line cells and through the ring canals may allow this
transport. Transport along microtubules is necessary for
both bcd and osk mRNA localization (Pokrywka and Stephenson, 1995
). The polarity of the microtubule network
within the oocyte, in combination with plus and minus end-
directed motor molecules, might explain the different target sites of the RNA transport (Theurkauf et al., 1992
,
1994a). However, the motor molecules involved have not
been identified so far, nor has a direct association of the
RNAs with microtubules been shown.
.
In this model, the formation of RNP particles precedes the
transport along cytoskeletal elements and subsequent anchoring of the RNA. In the case of the bcd mRNA, the RNP
particles could be formed by the binding of trans-acting
factors to the 3
untranslated region, which is essential for
the localization process (Macdonald and Struhl, 1988
; Macdonald et al., 1993
; Ferrandon et al., 1994
). A possible candidate for such a trans-acting factor is the Exuperantia protein (Exu). After an initial phase in oogenesis of wild-type flies when bcd mRNA is present throughout the oocyte, Exu is required to mediate bcd mRNA localization in
an anterior ring in stage 6-9 oocytes (King, 1970
; St
Johnston et al., 1989
). Subsequently, Swallow, as a possible component of the cytoskeleton, seems to anchor the
bcd mRNA at the cortex of the oocyte during stage 10B-11 (Stephenson et al., 1988
; St Johnston et al., 1989
; Chao et
al., 1991
; Hedge and Stephenson, 1993
). The third protein
known to be involved, Staufen, does not act on bcd mRNA
localization until stage 12 of oogenesis; it then releases the
transcript from the cortex into the anterior cytoplasm of
the embryo, where it is localized until degradation at cellularization (St Johnston et al., 1989
, 1991
). bcd mRNA fails
to be localized during oogenesis of females homozygous
mutant for exuperantia (exu females). As a result, the bcd
mRNA is initially equally distributed in the egg (Berleth
et al., 1988
; St Johnston et al., 1989
), although a weak gradient forms later by destabilization of the bcd mRNA at
the posterior pole by nanos activity (Berleth et al., 1988
; Driever and Nüsslein-Volhard, 1988a
; Wharton and Struhl,
1989
). The intermediate Bcd protein levels extending
throughout the anterior two thirds of the egg result in a
lack of head structures and extended thoracic body regions in exu embryos (Frohnhöfer and Nüsslein-Volhard,
1987
; Driever and Nüsslein-Volhard, 1988a
).
). It is not
known so far whether Exu binds directly to bcd mRNA.
No significant RNA-binding domains or other functional
domains within the Exu sequence have been identified that would help to understand the mode of its function.
The protein does not accumulate at the anterior end of the
oocyte (Macdonald et al., 1991
; Marcey et al., 1991
). In contrast to Swallow and Staufen, which are still present in
early embryos, Exu is present only during oogenesis (Macdonald et al., 1991
; Marcey et al., 1991
). It is first detectable in stage 4 oocytes. Starting at stage 7, it is accumulating in the nurse cells, where a high concentration can be
observed during midoogenesis. The content of Exu in the oocyte increases again by stage 10, when the protein is
transported from the nurse cells into the oocyte. Subcellularly, the protein shows a patchy distribution. Wang and
Hazelrigg (1994)
have shown a fusion protein of Exu and
green fluorescent protein (GFP) to be present in particles
that are transported through the germ line cells, but the
nature of these particles has not been resolved.
Materials and Methods
). Transgenic flies carrying six additional copies of the bcd locus were constructed by T. Berleth (Berleth et al., 1988
). The serendipity flies, which in transheterozygous combination give rise to ovaries not expressing bcd mRNA
(Payre et al., 1994
), were a kind gift of A. Vincent (University of Toulouse, France). The transgenic flies that express an Exu-GFP fusion gene
under the pCaSper promoter were constructed by Wang and Hazelrigg
(1994)
.
) for immunohistochemistry. For structural analysis, the
fixative also contained 2% glutaraldehyde. The ovaries were fixed for 15-30 min and afterwards washed several times with PBS.
35°C, as well as the final infiltration steps (overnight/several hours)
with the pure resin. Polymerization was performed by UV irradiation at
366 nm and
35°C in closed 750-µl reaction tubes (Sarstedt, Nümbrecht-Rommelsdorf, Germany) for oxygen exclusion.
-tubulin IgG 1:125 (Sigma Chemical Co., St. Louis, MO], and for mouse anti-actin IgG 1:300 [Cedarlane
Laboratories, Hornby, Ontario, Canada]; and 10 min for mouse anti-Vasa
mAb46F11 1:100, as described in Hay et al. [1988a]). Sections were washed with PBG six times for 5 min and incubated with the secondary antibody
in PBG for 1 hr (Cy3-conjugated anti-rabbit IgG 1:500 [Jackson ImmunoResearch, West Grove, PA]; protein A-15-nm gold [noncommercial];
Cy3-conjugated anti-mouse IgG 1:500 [Jackson ImmunoResearch]; anti-
mouse IgG 18-nm gold-conjugated 1:20 [Jackson Immunoresearch]; Cy3-conjugated anti-mouse IgM 1:500 [Jackson ImmunoResearch]; 10-nm
gold-conjugated anti-mouse IgM 1:10 [Janssen Biotech N.V., Olen, Belgium]). Six washes with PBG followed, and two with PBS.
.
. Epon sections were mounted on pioloform-coated, carbon-coated copper grids. They were treated with 5% aqueous uranyl acetate
for 10 min. One half of the sections was directly processed with lead citrate (2.5 min) as controls, whereas the other half was partially destained
again by incubation in 0.2 M EGTA for 15 min. These grids were then
contrasted with lead citrate for 2.5 min as well.
Results
;
Marcey et al., 1991
; Wang and Hazelrigg, 1994
).
Fig. 1.
Exu distribution
in wild-type ovaries. Ovaries
embedded in Lowicryl HM20
were sectioned (100 nm) and
afterwards immunofluorescently labeled with anti-Exu
antiserum. The staining was
studied by light microscopy.
(a) Stage 6 and 8 follicles
showing a punctate distribution pattern of Exu (frequently perinuclear) in the nurse cells (arrowhead) and an accumulation of Exu in
the oocyte (arrows). The follicle cells show no staining. (b) Stage 10A follicle with apical accumulation of Exu in the nurse cells (arrowheads) and transient enrichment of Exu at the posterior end of the oocyte (arrow). (c) Stage 10B follicle after the onset of the bulk flow
of cytoplasm. The follicle is sectioned at a slightly oblique angle; therefore, the oocyte appears smaller than the area covered by nurse
cells. The nurse cell-derived cytoplasm containing high amounts of Exu enters the oocyte, and the yolk-rich (darker) cytoplasm is restricted to more central regions of the oocyte. A cortical enrichment of Exu in the oocyte can be observed (arrow). The columnar follicle cells covering the oocyte do not stain for Exu and therefore are not visible. Bar, 100 µm.
[View Larger Version of this Image (38K GIF file)]
Fig. 2.
Ultrastructural distribution of Exu. Electron
micrographs (a-e) and schematic drawings (a-e
) of ultrathin Lowicryl sections on
which Exu was indirectly immunolabeled by 15-nm colloidal gold particles. (a and
a
) Nurse cell, stage 9. Exu is
highly enriched in the sponge
body in comparison to the
surrounding mitochondria and cytoplasm. The nuage
particles surrounded by the
sponge body contain no Exu.
(b and b
) Nurse cell, stage
10. Exu is accumulated in the
elongated sponge body, which
extends from the nucleus into the cytoplasm. Nuage particles
next to the nucleus are partly surrounded by higher amounts
of Exu present in small sponge
bodies. (c and c
) Nurse cell,
stage 10. A large sponge body
is present at the apical border of the nurse cell. A small
fragment of the overlying follicle cell can be seen at the
upper right edge of the micrograph. The gap between
these cells is due to the embedding. (d and d
) Oocyte,
stage 8. Sponge bodies with
high concentrations of Exu
are present in the ooplasm.
However, the central region of the oocyte where the yolk
granules first accumulate (yolk
nucleus) contains only very
little Exu or sponge bodies.
(e and e
) Oocyte, stage 10B.
Thick parallel bundles of microtubules run at this stage in
the cortical layer of the oocyte.
No sponge bodies are present,
but Exu is equally distributed
between the large yolk granules. No accumulation of Exu
on the microtubules can be
observed either. g, Golgi vesicles; l, lipid droplet; m, mitochondria (hatched); mt, microtubule; na, nuage (middle gray); nc, nurse cell; nu, nucleus; ooc, oocyte; sb,
sponge body (light gray); y, yolk granule (dark gray); yn, yolk nucleus. Each black dot in the drawing corresponds to a 15-nm gold particle in the micrograph. Bar, 1 µm.
[View Larger Version of this Image (104K GIF file)]
Fig. 3.
Ultrastructural distribution of the sponge bodies. Electron micrographs (a-d) and schematic drawings (a-d
) of ultrathin Epon sections of glutaraldehyde- and osmiumtetroxide-fixed wild-type follicles. (a and a
) Nurse cell, stage 9. The sponge
body consists of electron-translucent, elongated elements that are
interspersed between an electron-dense amorphous mass. It
hardly contains ribosomes in contrast to the surrounding cytoplasm. Small vesicles are present within the sponge body. The
sponge body is situated close to the nucleus and is surrounded by
mitochondria. (b and b
) Nurse cell, stage 9-10. Adjacent to the
flattened follicle cells, sponge bodies are accumulated in the apical regions of the nurse cell. ER tubules and Golgi vesicles are
present between the sponge body clusters. (c and c
) Oocyte,
stage 9-10. The large sponge body can be distinguished from the
surrounding ooplasm by the accumulation of elongated elements
in an electron-dense matter, excluding ribosomes and other organelles. (d and d
) Oocyte, stage 10A. Most sponge bodies have
disappeared by this stage; however, some small clusters are left in
the cytoplasm, which is densely packed with yolk granules, ER
tubules, mitochondria, and Golgi vesicles. er, endoplasmic reticulum; fc, follicle cells; g, Golgi complex; l, lipid droplet; m, mitochondria (hatched); na, nuage (middle gray); nc, nurse cell; nu,
nucleus; ooc, oocyte; sb, sponge body (light gray); y, yolk granule
(dark gray). Bar, 1 µm.
[View Larger Version of this Image (111K GIF file)]
Fig. 4.
Ultrastructure of sponge bodies after extraction. Micrographs (a and b) and schematic drawings (a and b
) of ultrathin
Epon sections of wild-type follicles that were only fixed with osmiumtetroxide, not with aldehyde, for better visualizing the membranes in the sponge bodies. (a and a
) Nurse cell, stage 9. The
sponge body contains ER-like cisternae. No amorphous matter is
visible between these due to the strong extraction of the cytoplasm. However the electron-dense nuage particle contained in
the sponge body is not affected by this procedure. (b and b
)
oocyte, stage 9. The lumen of the cisternae of the sponge body in
the oocyte appears larger than that in the nurse cells. As in the
nurse cells no amorphous matter is left after the extraction. g,
Golgi complex; l, lipid droplet; m, mitochondria; na, nuage; nc,
nurse cell; nu, nucleus; sb, sponge body; y, yolk granule. Bar, 1 µm.
[View Larger Version of this Image (91K GIF file)]
Fig. 5.
Sponge bodies are
present in ring canals. Micrograph and schematic drawing
of a stage 10 follicle that was
treated as described for Fig.
3. The cytoplasmic bridge,
connecting a nurse cell (to the left) with a yolk-containing oocyte, includes a sponge
body as well as other cytoplasmic organelles (ER and
lipid droplets). A multivesicular body containing electron-dense, yolk-like material is present in close
proximity to the ring canal.
ir, inner ring; m, mitochondria; mvb, multivesicular
body; sb, sponge body; y, yolk. Bar, 1 µm.
[View Larger Version of this Image (107K GIF file)]
-spectrin in fusomes but not in sponge bodies
(Lin et al., 1994
; Lee et al., 1997
), this observation argues
against the sponge bodies being fusome-derived structures. In the nurse cells of stage 7-9, the mitochondrial aggregates, which earlier have been restricted to one or two
large areas close to the nucleus, fractionate into smaller
clusters still including sponge bodies. In stage 10 nurse cells, the association of sponge bodies with mitochondria
has mostly disappeared (Figs. 2, b and c, and 3 b). The
sponge bodies in these cells are mostly elongated (Fig. 2 b)
(in comparison to their rounded shape earlier on) and
form apical clusters next to the thin layer of follicle cells
(Figs. 2 c and 3 b). At this stage, the amorphous material
in the sponge bodies is hardly visible between the ER-like
cisternae (Fig. 3 b); however, Exu protein is still enriched
in these structures (Fig. 2 c). As development proceeds,
the sponge bodies dissociate further and the distribution of Exu becomes uniform in the nurse cells (Fig. 1 c, not
shown).
Fig. 6.
Cytoplasm of previtellogenic oocyte. Micrograph and schematic drawing
of a stage 5 oocyte that was
treated as described for Fig.
3. The microtubule network
of the oocyte (ooc) is organized by the centrioles (ce) at
the posterior pole adjacent to
the follicle cells (fc). The cytoplasm is packed with short
microtubules (mt), ER-like
tubules (er), ribosomes, and
electron-dense granule (am, amorphous matter; some of
the granules were marked by
arrows in the micrograph).
The appearance of these
granules resembles the amorphous matter in the sponge
bodies. nu, nucleus. Bar, 1 µm.
[View Larger Version of this Image (96K GIF file)]
). However, the morphology of the sponge bodies shows no obvious difference to that of
the wild type (Fig. 7). The same result was obtained with
the EMS-induced alleles exuSC and exuXL1, which do not
produce detectable Exu (Hazelrigg et al., 1990
; Marcey et al.,
1991
; data not shown). This implies that Exu is not an essential structural component of the sponge bodies, although it is closely associated with these structures in wild-type ovaries.
Fig. 7.
Sponge bodies in
exuVL/exuVL ovaries. Micrograph and schematic drawing
of a stage 9 exuVL/exuVL
nurse cell that was treated as
described for Fig. 3. No obvious difference in the morphology of sponge bodies
that do not contain Exu can
be observed compared with
sponge bodies in wild-type
follicles by electron microscopy. As in wild-type follicles, these sponge bodies exclude ribosomes and consist
of electron-translucent elements in an amorphous matter with some small vesicles
in between. er, endoplasmic reticulum; g, Golgi; m, mitochondria;
nc, nurse cell; sb, sponge body. Bar, 1 µm.
[View Larger Version of this Image (108K GIF file)]
, is required for viability, as well as for the activation of transcription of bicoid. Replacement of two domains within
the Serendipity
protein by the corresponding region of
the closely related Serendipity
protein rescues the zygotic phenotype of serendipity
but not the activation of
bcd transcription (Payre et al., 1994
). In ovaries of such
flies, bcd mRNA levels are strongly reduced or absent (at
18°C). However, no difference in the contrast of the sponge
bodies can be observed in these follicles containing no bcd
mRNA. The same result was obtained for follicles containing additional copies of bcd mRNA, transcribed from multiple copies of the gene (not shown). This implies that bcd
mRNA, if present in the sponge bodies, is not the only or
predominant RNA component of the sponge bodies. However, this method is not suited to directly detect bcd mRNA
in the sponge bodies. So far, we have failed to visualize
bcd mRNA in the nurse cells under conditions sufficiently preserving the structure of the sponge bodies. Therefore,
we cannot directly show whether bcd mRNA is present in
the sponge bodies. However, the distribution of the bcd
mRNA in stage 10 nurse cells is patchy (Fig. 9, and as described in St Johnston, 1989), which might indicate an accumulation of bcd mRNA on some subcellular structure.
Fig. 8.
RNA staining of wild-type stage 9 nurse cells. (a) Electron micrograph of an ultrathin Epon section with normal contrast. DNA-rich regions of heterochromatin in the nucleus (nu)
are darkly stained (arrowheads), whereas the extended euchromatin and sponge bodies show little contrast (arrows) in comparison to the ribosomes in the cytoplasm. (b) Modified Bernhard staining of an ultrathin Epon section. Note the dark contrast of the
RNA rich regions (arrows) of the nucleus (nu) and the sponge
bodies in contrast to the bright regions of the heterochromatin
(arrowheads). m, mitochondria. Bars, 1 µm.
[View Larger Version of this Image (153K GIF file)]
Fig. 9.
Patchy bcd mRNA localization in the nurse cells. bcd
mRNA in a whole wild-type stage 10 follicle was fluorescently labeled by in situ hybridization. In the nurse cells, a patchy distribution of bcd mRNA is observed at the apical side of the cells
and around the nuclei (arrows). The nurse cells adjacent to the
oocyte are depleted of most of the bcd mRNA (as it was described by Wang and Hazelrigg [1994] for the Exu-GFP fusion
protein).
[View Larger Version of this Image (77K GIF file)]
reported that in stage 10 oocytes, taxol-induced, additional microtubules result in
ectopic bcd mRNA localization in wild-type but not in exu
ovaries. Wang and Hazelrigg (1994)
observed that Exu is
also ectopically localized close to the taxol-induced microtubules. These experiments suggest a function of Exu in
connecting bcd mRNA to the microtubular molecular motor. We stained microtubules (MTs) within the ovaries
with an anti-
-tubulin antibody in order to investigate the
possibility of a colocalization of Exu and MTs. However,
no accumulation of
-tubulin in Exu-labeled sponge bodies could be observed either by double immunofluorescence of ultrathin sections (Fig. 10) or by confocal microscopy of double-labeled ovaries (not shown). In rare cases,
MTs can be observed running close to the sponge bodies,
but even here MTs are never present near the center of the
sponge bodies (Fig. 11). Clearly, the majority of sponge
bodies in the cells is not associated with MTs. At stage
10B, MT bundles form at the oocyte cortex (Fig. 2 e).
Again, no colocalization can be observed, but rather Exu
accumulates cortically (Figs. 1 c and 2 e). Consistent with
these data, double immunogold labelings with differently
sized gold particles did not reveal a colocalization of
-tubulin and Exu (data not shown).
Fig. 10.
Comparison between Exu (a-c) and -tubulin
(d-f) distribution in wild-type
follicles. Ultrathin Lowicryl
sections were immunofluorescently labeled with either
anti-Exu serum (a-c) or anti-
-tubulin antibodies (d-f) and
observed by fluorescent light
microscopy. Follicle cells harbor much higher amounts of
microtubules than the germ
line cells, but no Exu is present
in the follicle cells. (a and d) Stage 4 and 6. High amounts
of
-tubulin are found at the
borders of the nurse cells and
in the oocyte (arrows), but the punctate Exu staining is not enriched at these sites. (b and e) Stage 9. Many sponge bodies (b, bright
dots) are present but not accumulated at the borders of the nurse cells or in the oocyte (arrows) where tubulin is enriched. (c and f)
Stage 10. Hardly any microtubules are labeled, but sponge bodies labeled by Exu (c, bright dots) are accumulated in apical patches in
the nurse cells. In the oocyte, Exu is not accumulated along the microtubules running parallel to the anterior border of the oocyte (arrows). Bar, 50 µm.
[View Larger Version of this Image (87K GIF file)]
Fig. 11.
Subcellular localization of sponge bodies versus microtubules. Micrograph
and schematic drawing of a
wild-type stage 8 oocyte that
was treated as described for
Fig. 3. Microtubule bundles (mt) run across the cytoplasm. Although sponge bodies (sb) are present close by,
no microtubules are present
in these structures. m, mitochondria; y, yolk. Bar, 1 µm.
[View Larger Version of this Image (93K GIF file)]
and Emmons
et al. (1995)
. These two genes are required for the transport of all posterior factors to the posterior pole of the oocyte and for transport of Nanos protein from the posterior
pole to its site of action in the abdomen (Manseau and
Schüpbach, 1989
). Theurkauf (1994b)
interpreted the thick
MT bundles in capu or spir ovaries as premature formation of the thick MT bundles leading to the cytoplasmic
streaming of wild-type stage 10B oocytes. We observed
not only thick bundles of MT forming at stage 8 (instead of
stage 10B) parallel to the oocyte cortex but also much
higher amounts of MT bundles in later stages, compared
with wild-type follicles (Fig. 12 b), although Emmons et al.
(1995)
could not detect higher concentrations of
- or
-tubulin in capu or spir ovaries.
Fig. 12.
Exu is not accumulated on the thick microtubules forming in cappuccino
oocytes. Adjacent ultrathin
Lowicryl sections of capuRK/
capuRK follicles were immunofluorescently stained for Exu
(a) or -tubulin (b). Unusual
thick bundles of microtubules
run in the cortical cytoplasm
of the stage 10A oocyte. However, in the same oocyte Exu
is equally distributed. The
same pattern can be observed
in spire follicles. Bar, 50 µm.
[View Larger Version of this Image (163K GIF file)]
-tubulin and Exu protein did not reveal any additional
accumulation of Exu at these ectopic microtubules, on neither light- nor electron microscopic levels (Fig. 12, not
shown). An association of sponge bodies with these microtubule bundles is not observed either (not shown). Consistent with these data, a change in the bcd mRNA localization in mutant ovaries compared with wild type has not
been found (Manseau et al., 1996
).
. After feeding flies for several days with either
colchicine- or taxol-containing medium, large sponge bodies were observed (Fig. 13). In both cases, these structures
still contained high amounts of Exu (Fig. 14, e and f), although in comparison to untreated follicles a clear difference in the tubulin distribution was observed (Fig. 14, g, b,
and c), and the colchicine feeding had completely abolished bcd mRNA localization in sibling ovaries (Fig. 14, c
versus a). However, we were not able to reproduce the ectopic localization of bcd RNA nor of Exu in follicles of
taxol-treated flies (Fig. 14, b and e), which has been described by Pokrywka and Stephenson (1991)
and by Wang
and Hazelrigg (1994)
.
Fig. 13.
Ultrastructure of
sponge bodies in ovaries after taxol or colchicine treatment. Micrographs of stage
9-10 nurse cells of flies that
have been fed with cytoskeletal drugs for several days.
The follicles were treated as
described for Fig. 3. (a)
Taxol; (b) colchicine. er, endoplasmic reticulum; g,
Golgi; m, mitochondria; na,
nuage; nu, nucleus; sb,
sponge body. Bars, 1 µm.
[View Larger Version of this Image (94K GIF file)]
Fig. 14.
Effects of taxol or
colchicine on localization of
maternal factors. Ovaries of
flies fed without any drug (a,
d, and g), with taxol (b, e, and
h) or colchicine (c, f, and i)
were stained for bcd mRNA
(a-c), Exu (d-f) or -tubulin
(g-i). (a-c) bcd mRNA in situ hybridization on whole
ovaries. After taxol treatment (b) no striking difference is to be seen compared
with wild type (a), however
after colchicine treatment
(c) the localization of bcd mRNA is completely abolished. Note the central position of the oocyte nucleus in
c, which can serve as a control for the MT destabilization. (d-f) Ultrathin Lowicryl sections of ovaries treated with taxol (e) or colchicine (f) were immunofluorescently labeled for Exu and compared with the wild-type staining (d). Neither treatment eliminates the
punctate distribution pattern of Exu; however, in colchicine-treated follicles the staining seems to be preferentially close to the nucleus.
The size difference of the sponge bodies observed here does not exceed the variance in the size of sponge bodies observed in different
wild-type follicles. (g-i) Adjacent ultrathin Lowicryl sections of d-f stained for
-tubulin. After taxol treatment (h), clear microtubules
are visible while the staining is diffuse and strongly reduced after colchicine treatment (i) compared with the wild-type staining (g). (The
vitelline membrane in i is stained by cross-reaction of the secondary antibody.)
[View Larger Version of this Image (88K GIF file)]
;
Mahowald, 1962
; Mahowald, 1971a
; Mahowald and Kambysellis, 1980
). The nuage of Drosophila follicles shows an
electron-dense granular or fibrous structure similar to the polar granules at the posterior pole of the oocyte, and in
contrast to the sponge bodies it does not contain elongated
elements. In addition to their conspicuous perinuclear location, smaller nuage particles are present in the cytoplasm further away from the nuclear membrane (as previously described by Mahowald, 1971a
) for germarial stages
and frequently observed by us in vitellogenic stages (Figs. 2 a and 4 a). Interestingly, the nuage is surrounded by
sponge bodies in the nuclear lobes and in particular in the
cytoplasm of the nurse cells (Figs. 2 a, 4 a, and 15 a). The
nuage does not contain Exu (Fig. 2 a). Vasa protein is
present in the nuage at the nuclear membrane of the nurse
cells and in the polar granules at the posterior pole of the
oocyte (Hay et al., 1988a
,b; Liang et al., 1994
). Vasa is required for the assembly of the polar granules and is a member of the posterior system. We used Vasa as a marker for
nuage particles and found Vasa-staining nuage material
surrounded by sponge bodies in the cytoplasm away from the nuclei (Fig. 15), where localization of Vasa in nuage
particles had not been reported previously. Mahowald and
Kambysellis (1980)
reported some nuage particles to enter
the oocyte through the ring canals. We confirmed this observation and, in addition, found a few, small Vasa-labeling nuage particles surrounded by sponge bodies anterior
to the position where the polar granules form in stage 9 oocytes (Fig. 15 b).
Fig. 15.
Vasa distribution
in nuage particles. Micrographs (a and b) and schematic drawings (a and b
) of
follicles embedded in Lowicryl. Vasa protein was indirectly immunolabeled by
10-nm colloidal gold particles.
(a) Nurse cell, stage 9. Vasa
is accumulated in the nuage
(na), which is present next to
the nuclear membrane (nu, nucleus) and delaminating
into the cytoplasm. The electron-dense, compact nuage
particles are surrounded by
sponge bodies (sb). (b) Oocyte, stage 9. Small Vasa-
labeled nuage particles (na) are found at low frequency in
the sponge bodies (sb) in the
anterior cytoplasm of the oocyte. m, mitochondria; y,
yolk. Bars, 1 µm.
[View Larger Version of this Image (86K GIF file)]
; Fig. 1 b). However, Exu is excluded
from the polar granules that are forming in the posterior
cytoplasm of the oocyte at this same time (Fig. 16). Since
the nuage and polar granules are surrounded by regions
enriched for Exu in wild-type follicles, Exu might be required for the organization and distribution of these structures. However, we could not detect a change in the Vasa
distribution in exu ovaries or an altered number of pole
cells in exu embryos (data not shown). Therefore, Exu is
not necessary for the accumulation of Vasa in polar granules and related organelles.
Fig. 16.
Transient posterior localization of Exu. Micrograph
and schematic drawing of an ultrathin Lowicryl section of the posterior pole of a stage 10 oocyte that was immunogold-labeled for
Exu (15-nm gold). The gold particles are accumulated in the posterior cytoplasm next to the vitelline membrane (vm), whereas
the cytoplasm between the large yolk granules (y) is hardly labeled for Exu. The polar granules (p) at the posterior pole are
free of Exu. m, mitochondria. Bar, 1 µm.
[View Larger Version of this Image (84K GIF file)]
Discussion
). By light microscopic staining methods, structures
have been detected previously in Drosophila ovaries that
show a distribution within the germ line cells that is similar
to that in our sponge bodies (Hsu, 1952
, 1953
). Hsu termed
these structures yolk flakes, judging them to be proteid
yolk precursors. Although the distribution of the sponge
bodies parallels that of the yolk flakes, the sponge bodies
are not yolk precursors. Yolk granules are formed in distinct, so-called multivesicular bodies in young oocytes and
some nurse cells (Fig. 5). Furthermore, the central region
of young oocytes, where the first accumulation of yolk
granules can be observed (yolk nucleus), hardly contains
sponge bodies (Fig. 2 d). However, in ovaries of many invertebrates and vertebrates, a structure with a morphology
that is strikingly similar to the sponge bodies has been described, which is termed Balbiani's vitelline body (Henneguy, 1887
; Guraya, 1979
). It shares the presence of a
structured, electron-dense mass and ER within an area enriched in mitochondria and Golgi with the sponge bodies
of Drosophila. Although Guraya was not able to identify a
corresponding structure in insect ovaries, we assume that
our sponge bodies are the Balbiani's vitelline bodies of
Drosophila. The high variability in the morphology might be one possible reason why the sponge body structure had
not been described earlier. So far we have not been able to
determine precisely which factors are responsible for the
variance in size and consistency of the sponge bodies, but
genetic background is definitely involved, as well as to a minor degree feeding conditions of females and temperature.
). The morphology of the mitochondrial cloud,
which consists of granulo-fibrillar material and surrounding mitochondria, closely resembles that of the sponge
bodies. The mitochondrial cloud migrates from an early
position next to the germinal vesicle to the vegetal pole of
Xenopus oocytes while partitioning into large islands. RNA molecules like Vg1, Xcat2, Xwnt11 mRNAs, and
Xlsirts, thought to be involved in axis determination in
Xenopus, comigrate with this structure (Forristall et al.,
1995
; Kloc and Etkin, 1995
). The granular material of the
mitochondrial cloud is thought to form the germinal granules in later stages of Xenopus embryogenesis, which contain a Vasa-related protein of the D-E-A-D family of proteins (Watanabe et al., 1992
). Interestingly the sponge
bodies surround the granular, Vasa-containing nuage particles, which are putative polar granule precursors. It is
tempting to speculate that homologous molecules are
transported and localized via conserved structures in insect and vertebrate ovaries.
). In Drosophila, involvement of both microtubule- and microfilament-based transport in RNA localization is suggested by
the finding that a cytoplasmic tropomyosin (cTmII) is required for osk mRNA localization (Erdelyi et al., 1995
), in
addition to a previously shown microtubule dependency
(Pokrywka and Stephenson, 1995
). So far, no effect of microfilament-destabilizing drugs (cytochalasin B and D) has been observed on osk or bcd mRNA localization (Pokrywka and Stephenson, 1995
), but the microfilament network in general seems to be less sensitive to inhibitor treatment than the microtubule network (Emmons et al., 1995
).
In contrast to osk mRNA localization, no hint at an involvement of an actin-based transport or anchoring has been
described for bcd mRNA localization. Consistent with this,
no actin is detectable in the Exu-containing sponge bodies. However, we were not able to confirm a microtubule-
dependent transport of the sponge bodies or Exu either,
since a clear colocalization of Exu or sponge bodies with
microtubules was not observed. Nevertheless, it is still possible that some proportion of Exu that is not localized to
the sponge bodies is connected with the microtubule network, although we consider this to be rather unlikely.
have shown that intracellular transport of cytoplasmic particles within the germ line
cells is dependent on microtubules, but microfilaments are
involved in the transport through the ring canals. In addition, Pelham et al. (1996)
have recently shown that some
organelles are able to move along both microtubules and
actin filaments depending on the concentration of a tropomyosin isoform in cultured rat epithelial cells. Furthermore, there is now growing evidence that microtubules
can be required to organize actin bundles and vice versa in
cultured cells (Challacombe et al., 1996
; Fishkind et al.,
1996
). A similar link between the two systems is indicated
by the organization of the microtubule and microfilament
network by cappuccino or chickadee gene products in Drosophila germ line cells (Manseau et al., 1996
). Therefore, the sponge bodies might migrate both along microtubules
and microfilaments depending on their cytoplasmic environment in nurse cells and oocyte and they might require
the two systems for different steps of their transport.
). In contrast, the formation
of the sponge bodies and the Exu distribution in the germ
line cells is not dependent on the presence of bcd mRNA, and no direct interaction of RNA and Exu has been shown.
In exu mutants, the bcd mRNA localization is disrupted
within the oocyte, but the transport into the oocyte is not,
and therefore Exu (unlike Staufen) might not be involved
in the actual transport of the bcd mRNA. Since Exu is
mostly localized on the sponge bodies in the nurse cells at
the time of its action, we assume that its function is performed at this location. The function of Exu could somehow be to modulate a bcd mRNA-binding protein or bcd
mRNA itself (e.g. by connecting it to a trans-acting factor),
thereby allowing an Exu-independent but microtubule-
dependent transport of the bcd mRNA to the anterior
pole after entry into the oocyte. This assumption would be
able to account for the different effect of additional microtubules on the bcd mRNA localization in wild-type and
exu ovaries that was observed by Pokrywka and Stephenson (1991)
without involving a colocalization of Exu and
microtubules. While this model does not specify how the
bcd mRNA is transported into the oocyte, it does not rule
out a function for the observed migration of the sponge
bodies in the transport of other factors.
) and the presence of the sponge bodies in the
ring canals (Fig. 6) imply that these structures migrate
through the germ line cells. The remarkable accumulation
of mitochondria next to the sponge bodies might provide
the energy for this migration. Golgi complexes are found
close to or in the sponge bodies. These can serve to transport molecules from the ER to their site of secretion. In a
similar manner, the ER-like tubules within the sponge bodies might function as storage containers for transported proteins. In addition, it is an interesting observation that
the sponge bodies surround the nuage, as this implies that
the nuage could migrate in concert with the sponge bodies,
at least on its way from the nuclei into the cytoplasm of the
nurse cells. Because of their size and abundance, there is
less evidence for a simultaneous migration of these two structures into and within the oocyte, although the transient,
posterior localization of some proportion of Exu argues in
favor of this possibility. This localization is not observed
after colchicine treatment (Fig. 14 f), and polar granule-like
particles do not localize to the posterior pole of colchicine-treated follicles (Clark et al., 1994
; Pokrywka and Stephenson, 1995
). Therefore the migration of the nuage and
the sponge bodies might be mediated by a shared motor
molecule. Exu has no effect on the formation of the polar
granules nor is Vasa involved in the anterior system. Therefore, we cannot deduce so far whether the association of
the nuage and sponge bodies results from a common transport mechanism, which might be required for the function
of these structures in localization of the maternal mRNAs.
Received for publication 28 May 1997 and in revised form 21 July 1997.
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