|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 15 March 2006
doi: 10.1242/dev.02319
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Life Sciences and the National Institute for Biotechnology in
the Negev, Ben-Gurion University, Beer-Sheva 84105, Israel.
2 Howard Hughes Medical Institute, Department of Molecular Biology, Princeton
University, Princeton, NJ 08544, USA.
* Author for correspondence (e-mail: abdu{at}bgu.ac.il)
Accepted 9 February 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-like protein, and the Epidermal growth factor receptor (Egfr).
spn-F mutant females produce ventralized eggs similar to the
phenotype produced by mutations in the grk-Egfr pathway. We found
that the ventralization of the eggshell in spn-F mutants is due to
defects in the localization and translation of grk mRNA during
mid-oogenesis. Analysis of the microtubule network revealed defects in the
organization of the microtubules around the oocyte nucleus. In addition,
spn-F mutants have defective bristles. We cloned spn-F and
found that it encodes a novel coiled-coil protein that localizes to the minus
end of microtubules in the oocyte, and this localization requires the
microtubule network and a Dynein heavy chain gene. We also show that
Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show
that we have identified a novel protein that affects oocyte axis determination
and the organization of microtubules during Drosophila oogenesis.
Key words: Drosophila, Oogenesis, Microtubule, Bristle formation, spn-F
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Ruohola-Baker et al., 1994).
This communication is mediated by a single signaling system involving the
gurken (grk) and Epidermal growth factor receptor
(Egfr) genes (Gonzalez-Reyes et
al., 1995; Roth et al.,
1995). grk encodes a Tgf-like protein that is
expressed in the germline and is secreted from the oocyte
(Neuman-Silberberg and Schüpbach,
1993; Neuman-Silberberg and
Schüpbach, 1996). Egfr is expressed throughout the follicular
epithelium (Kammermeyer and Wadsworth,
1987; Sapir et al.,
1998), where it acts as a receptor for Grk. In earlier stages of
oogenesis, localized activation of Egfr at one end of the egg chamber
establishes posterior follicle cell fates. The follicle cells signal back to
the oocyte and this initiates the formation of the embryonic AP axis
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995). After the
oocyte nucleus has shifted to one side of the egg chamber at stage 8,
grk RNA accumulates in a crescent close to the nucleus, and the
resulting Gurken protein activates the receptor on one side of the egg chamber
to establish dorsal follicle-cell fates. The production and localization of
grk RNA and protein within the oocyte is tightly regulated at several
levels (for a review, see Nilson and
Schüpbach, 1999).
The asymmetric localization of grk mRNA within the developing egg
chamber during mid-oogenesis requires a network of polarized microtubules
(MTs). Before stage 7, a microtubule-organizing center (MTOC) is located at
the posterior of the oocyte, as revealed by the localization of the
unconventional kinesin Nod fused to ß-galactosidase (Nod:ß-gal)
(Clark et al., 1997). The first
grk signaling event leads to a repolarization of the oocyte MTs and
the migration of the oocyte nucleus to the dorsoanterior corner of the oocyte
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995). During
stage 8, the posterior MTOC disassembles and a diffuse MTOC forms at the
anterior. The anterior MTOC has been visualized by Nod:ß-gal
(Clark et al., 1997),
Centrosomin (Li and Kaufman,
1996) and the MT-nucleating factors, 
-Tubulin at 37C
(Tub37C) and the -Tubulin ring complex protein 75 (Grip75;
previously known as Dgrip75) (Schnorrer et
al., 2002). Injection of fluorescent grk mRNA into living
oocytes revealed that the mRNA is assembled into particles that move in two
distinct steps along two different MT arrays, first toward the anterior and
then, after an apparent turn, dorsally toward the oocyte nucleus
(MacDougall et al., 2003).
High-resolution imaging of Tau-GFP
(MacDougall et al., 2003;
Januschke et al., 2002) and
Nod:ß-gal distribution in the oocyte suggests that there is a distinct
network of MTs that are specifically associated with the oocyte nucleus, and
these MTs are responsible for the second step in grk RNA
localization. Moreover, recent work suggested that repositioning of the
nucleus and its tightly associated centrosome could control MT reorganization
in the Drosophila oocyte
(Januschke et al., 2006).
However, the precise nature and regulatory mechanisms that establish these
oocyte nucleus-associated MTs is still unknown.
To further understand the role of MTs in grk RNA localization, we
molecularly analyzed the spn-F locus. spn-F was first
identified as a maternal effect mutation that affects the DV polarity of the
eggshell (Tearle and
Nüsslein-Volhard, 1987). We found that the ventralization of
the eggshell in spn-F mutants is due to defects in the localization
and translation of grk mRNA during mid-oogenesis. Moreover, we found
that, in addition to the maternal effect, spn-F affects the
development of the bristles of the adult fly. We show that transport toward
the minus end of the MTs is affected in spn-F mutants, predominantly
in the subset of MTs that are required for the transport of grk RNA
from the anterior to the dorsal side of the oocyte towards the nucleus. We
cloned spn-F and found that it encodes a novel coiled-coil protein
that localizes to the minus end of MTs in the oocyte and in a punctate pattern
in the nurse cells. Treatment of ovaries with the MT-depolymerization drug
colcemid led to abrogation of Spn-F localization at the minus end of the MT
and to higher accumulation of the protein in the nurse cells. We show that
Spn-F interacts directly with the minus end-directed, motor-protein subunit
Dynein light chain. Moreover, Spn-F localization to the minus end of the MT
requires a Dynein heavy chain (Dhc) gene. Taken together,
our results identify a novel protein that is involved in the organization of
MTs in the oocyte during mid-oogenesis stages.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
),
spn-F2 (Drosophila Stock Center, Szeged; originally
designated l(3)s113003). The chromosome designated
l(3)s113003 failed to complement the spn-F bristle and
ovarian phenotypes. It had been suggested that this line carried two P-element
insertions: the first was mapped to 62E by in situ hybridization and the
second was uncovered by Df(3R)A177der21 [the second insertion site
appears to be a second site mutation where a P-element is no longer associated
with the semi-lethality (Deak et al.,
1997)]. Indeed, recombination allowed us to isolate a viable stock
containing the spn-F2 mutation. We used the kinesin
ß-Gal insertion line KZ503 and the Nod-ß-Gal insertion line
NZ143.2 (Clark et al.,
1997), as well as Tau-GFP
(Micklem et al., 1997).
Germline clones were generated using the ovoD-FLP technique and the FRT82B
(3R) (Chou and Perrimon, 1992).
CG12114 was expressed in the ovary using the nanos-Gal4-VP16
expression system (Van Doren et al.,
1998). The balancer chromosomes used in this study, as well as the
marker mutations, have been described previously (see
Lindsley and Zimm, 1992) (see
also FlyBase,
http://flybase.bio.indiana.edu).
Rescue construct
The entire coding sequence of CG12214 was amplified by PCR from
EST (LD01470), using modified primers to create a KpnI restriction
site at the 5' end and a NotI site at the 3' end. The PCR
product was then cloned into pUASp. P-element-mediated germ-line
transformation of this construct was carried out according to standard
protocols (Spradling and Rubin,
1982).
Sequencing of mutant alleles
Genomic DNA was prepared from flies of the genotype
spn-F*/spn-F* according to standard procedures
(Sambrook et al., 1989). The
coding region was sequenced and the sequences were compared with the wild-type
genomic sequence of the parental chromosomes using the MacVector (Kodak/IBI)
program.
Generation of anti-Spn-F (CG12114) monoclonal and polyclonal antibodies
The entire open-reading frame of CG12114 was cloned into pGEX-2T
(Pharmacia). The fusion protein was overexpressed in Escherichia coli
and the inclusion bodies were purified using a standard protocol
(Sambrook et al., 1989). The
CG12114 monoclonal antibodies were prepared by the Princeton monoclonal
facility and the polyclonal antibodies were raised in rabbits by PRF&L
Company, using the purified inclusion bodies as antigen.
In situ hybridization and antibody staining
RNA in situ hybridization on ovaries was performed using
digoxigenin-labeled probes according to standard protocols
(Roth and Schüpbach,
1994). Antibody staining of ovaries was carried out as described
(Queenan et al., 1999), using
mouse anti-Grk (1:10), mouse anti-CG12114 (1:10; clones 8C10 and 3F1), rabbit
anti-Oskar (1:500; kindly provided by P. MacDonald, University of Texas),
mouse anti--tubulin (1:250; Sigma), rabbit anti-ß-Gal (1:1000;
Promega), rabbit anti Centrosomin (1:500)
(Heuer and Kaufman, 1995)
(kindly provided by T. Kaufman). The secondary antibodies Alexa Fluor-488 goat
anti-rabbit IgG and Alexa Fluor-568 goat anti-mouse IgG (Molecular Probes)
were used at a dilution of 1:1000. For experiments involving -tubulin
and Tau-GFP staining, ovaries were fixed in 4% paraformaldehyde in PBS+0.1%
Triton X-100 for 20 minutes at room temperature and the ovaries were then kept
at room temperature to ensure that the MTs did not depolymerize. DNA was
stained with Hoechst (Molecular Probes) at a dilution of 1:10,000. Wheat Germ
Agglutinin (WGA-633, Molecular Probes, conjugated to Alexa Fluor-633) was used
at a dilution of 1:500 for 1 hour at room temperature. MT detection was
carried out as described (Januschke et
al., 2006). Briefly, ovaries were incubated in BRB80 buffer [80
mmol/l PIPES (pH 6.8), 1 mmol/l MgCl2, 1 mmol/l EGTA], containing
1% Triton X-100 (BRB-80-T) for 1 hour at 25°C without agitation. Then
ovaries were fixed in MeOH at -20°C for 15 minutes, rehydrated for 15
hours at 4°C in PBS 0.1% Tween, then blocked for 1 hour in PBS 0.1% Tween
containing 2% (w/v) bovine serum albumin (BSA) before incubation with primary
antibody overnight.
Drug treatment
Drugs were fed to 2- to 3-day-old adult females in a yeast paste, following
a one-hour starvation. Colchicine (Sigma) was used at a concentration of 25
µg/ml for periods of 24 hours. Antibody staining of the ovaries was carried
out as described above.
Scanning electron microscopy
Scanning electron microscopy was performed in the Materials Institute of
Princeton University on a Philips XL 30 PEG-SEM. Adult Drosophila
were fixed in 70% ethanol overnight and dehydrated through a series of
10-minute washes in 95% ethanol, 100% ethanol and acetone. The flies were
dried with Tetramethylsilane Ted-Pella for 10 minutes in capped tubes. The TMS
was allowed to evaporate, samples were mounted upon stubs and sputter coated
with gold.
GST pull-down assays
Expression of GST-Ddlc was performed as described previously
(Schnorrer et al., 2000). The
CG12114 was cloned into pHIS and protein induction performed overnight at
15°C. The recombinant protein samples were purified in phosphate-buffered
saline (PBS). A 10-µl bed volume of glutathione-Sepharose resin was added
to 30 µg of GST-Ddlc or GST alone, and the mixture was incubated on ice for
30 minutes before adding purified Spn-F. The mixture was incubated on ice
overnight. The resin was then washed four times with 500 µ
PBS. The
bound protein was eluted from the resin with 30 µl of Laemmli buffer and
boiled for 5 minutes. After centrifugation, the supernatant was subjected to
15% SDS-PAGE followed by staining with Coomassie brilliant blue-G250.
For GST and ovary extract pull downs (with GSH-Sepharose, Pharmacia) equal
amounts of ovary extracts were lysed, incubated and washed with 50 mM HEPES
(pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and a cocktail of a complete
protease inhibitor (Roche). Recombinant GST or GST-Ddlc protein (
5 µg)
was incubated with ovarian extracts for up to 4 hours at 4°C; the beads
were washed four times with lysis buffer and eluted in Laemmli buffer. Western
blots were performed using polyclonal anti-Spn-F antibodies at a 1:1000
dilution.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
)
(Fig. 1A,B,
Table 1). A fraction of the
eggs are fertilized and the embryos that form inside have a variety of pattern
abnormalities (Fig. 1). Apart
from ventralized embryos that resemble embryos produced by weak
gurken mutations, some bicaudal embryos are found inside the eggs,
ranging from embryos with the typical reduced head skeleton to rare
symmetrical bicaudals with several abdominal segments in mirror image symmetry
(Fig. 1C,D), and Filzkoerper
and telson at both ends. We analyzed the localization of Osk protein, whose
posterior concentration depends on endogenous kinesin heavy chain
function (Brendza et al.,
2002). The localization of oskar RNA in spn-F
mutants is similar to that observed in wild type (data not shown); however, we
found that the posterior association of Oskar protein with the cortex in
spn-F stage 10 oocytes is not as tight as in wild type
(Fig. 1E,F).
|
|
) (Fig. 1I). In
spn-F mutant ovaries, Grk is also restricted to the oocyte; however,
the levels of Grk are variably reduced throughout oogenesis, and in 67% of the
stage 9 egg chambers Grk protein was undetectable
(Fig. 1J). The DNA within the
oocyte nucleus is often fragmented or thread-like in appearance
(Fig. 1L), similar to the
phenotype produced by spindle class genes (e.g. okra, spn-A, spn-B,
spn-D) (Gonzalez-Reyes et al.,
1997; Ghabrial et al.,
1998; Abdu et al.,
2003; Staeva-Vieira et al.,
2003).
spn-F does not act through the meiotic checkpoint
Several of the spindle class genes, which are characterized by a variable
eggshell ventralization phenotype, were previously found to encode proteins
with homology to known DNA repair enzymes, and were shown to be required for
the repair of recombination-induced, double-strand DNA breaks during
Drosophila oogenesis (Ghabrial et
al., 1998; Abdu et al.,
2003; Staeva-Vieira et al.,
2003). Spindle A, B, C and D patterning defects
can be suppressed; for instance, by blocking the formation of double-strand
DNA breaks during meiosis using mutations in mei-W68
(Ghabrial and Schüpbach,
1999), or by eliminating the checkpoint by using mutation in the
checkpoint genes mei-41 and DmChk2 (lok - FlyBase)
(Ghabrial and Schüpbach,
1999; Abdu et al.,
2002). To determine whether the ovarian phenotype in
spn-F is also due to unrepaired double-strand DNA breaks, flies
double mutant for mei-W68 or DmChk2 and spn-F were
generated. Our results showed that neither ventralization nor the defects in
the appearance of the DNA within the oocyte nuclei of spn-F are
suppressed by mei-W68 or DmChk2 (data not shown). These
results strongly suggest that spn-F is not involved in the repair of
double-strand DNA breaks during meiosis. The DV patterning defects of
spn-F mutants therefore appear to arise by a mechanism that operates
either downstream or independently of the meiotic checkpoint.
Bristle defects in spn-F mutants
In contrast to the other spindle class genes, spn-F also affects
the development of the bristles (Fig.
2). When compared with wild type, spn-F mutants have
considerably shorter and thicker bristles. Scanning electron micrographs of
spn-F bristles showed that, in addition to the aberrant length and
width, the morphology of spn-F bristles was altered; the
spn-F bristles do not have finely tapered ends
(Fig. 2B), and, in some extreme
cases, the direction of bristle growth at some points along the bristle shaft
is altered (Fig. 2C,D).
|
-tubulin staining and localization of Tau-GFP,
which labels MTs (Micklem et al.,
1997) (Fig. 3). In
wild-type egg chambers at early stages, we found that MTs are equally
distributed in the oocyte cytoplasm (Fig.
3A). However, in spn-F early stages, the -tubulin
accumulated abnormally around the oocyte nuclear membrane
(Fig. 3B). In wild-type stage 9
egg chambers, -tubulin is enriched at the anterior region of the oocyte
with a slightly higher accumulation around the oocyte nucleus
(Fig. 3C). In spn-F
stage 9 egg chambers, -tubulin is abnormally associating with the
oocyte nuclear periphery (Fig.
3D). The oocyte nucleus does, however, move normally to the
dorsoanterior corner in the mutant egg chambers. Using Tau-GFP in stages 6-9
egg chambers (Fig. 3E-H), we
observed that, in early stages in wild type and mutants, MTs were uniformly
seen in the oocyte cytoplasm (Fig.
3E,F), whereas, at stage 9 of oogenesis, the MTs in both wild type
and mutants were most abundant at the anterior margin of the oocyte and
gradually diminished in concentration towards the posterior pole, as has been
previously reported (Micklem et al.,
1997) (Fig. 3E-H).
In living specimens, using Tau-GFP at a higher magnification and resolution,
it had been shown that, in wild-type egg chambers, a highly conserved array of
MTs exists at the anterior of the oocyte and that this anterior array of MTs
forms a basket around the oocyte nucleus
(MacDougall et al., 2003). In
GFP antibody-stained spn-F mutant egg chambers at stage 9, the
Tau-GFP fusion protein accumulated abnormally in discrete `dots' around the
oocyte nucleus, similar to as was observed with the -tubulin staining
(Fig. 3B,D).
|
As a next step, we investigated the function and polarity of the MTs in
spn-F mutant ovaries. First, the transport to the posterior pole of
the oocyte was analyzed using the MT plus-end marker kin:ß-gal
(Clark et al., 1997). We found
that kin:ß-gal localization in spn-F mutants is similar to that
observed in wild-type stage 9 egg chambers
(Fig. 4A), in that the fusion
protein is concentrated at the posterior end of the oocyte
(Fig. 4B).
To analyze transport to the anterior pole, we used the MT minus-end marker
Nod:ß-gal (Clark et al.,
1997). In wild-type stage 6 egg chambers, Nod:ß-gal is
concentrated at the posterior cortex of the oocyte
(Fig. 4C). By stage 8, the
protein is no longer detectable at the posterior of the oocyte but instead
concentrates along the anterior cortex of the oocyte with highest accumulation
flanking the oocyte nucleus (Fig.
4E). In spn-F mutant ovaries, neither the early posterior
localization (Fig. 4D) nor the
anterior ring localization in mid-oogenesis
(Fig. 4F) are detected. These
results indicate that transport to the minus end of the MTs or some aspect of
anchoring cargoes to the MT network is not functioning properly in
spn-F mutant egg chambers. To assay transport to the minus end of the
MTs in the anterior ring during mid-oogenesis versus transport to the
dorsoanterior corner of oocyte, we analyzed the localization of bicoid,
encore (Fig. 5) and
fs(1)K10 RNA (data not shown), which all accumulate in an anterior
ring, but are not further localized to the dorsal corner. We found that the
localization of these RNAs in spn-F mutants is similar to that
observed in wild type. We also found that Centrosomin, a MTOC component that
marks MT minus ends (Li and Kaufman,
1996), is localized to the anterior ring of the oocyte in
spn-F mutants, as in wild type (data not shown).
|
) that were initially mapped to this region to
spn-F234. One of the lines, l(3)s113003 failed to
complement the spn-F bristle and ovarian phenotypes. After we
recombined this insertion away from a second insertion on the same chromosome
(see Materials and methods), we were able to obtain a homozygous viable line
that contained a second spn-F allele (spn-F2).
Both hemizygous and homozygous spn-F2 individuals are
female sterile and show bristle defects similar to those observed in
spn-F234. We also found that there was no difference in
the severity of DV defects of the eggshell between the hemizygous
spn-F234 and spn-F2, suggesting that
both alleles are genetically null (Table
1).
|
To demonstrate that these mutations in the CG12114 gene are responsible for the defects observed in spn-F mutants, we expressed the entire CG12114 open reading frame in the germline under UAS control, using a nanos-Gal4 driver line in the spn-F234 and spn-F2 mutant backgrounds, and found that this transgene fully rescues the female sterility (data not shown). Also, expressing CG12114 using an actin-Gal4 driver rescued both the female sterility and the bristle defects. These results verify that spn-F indeed corresponds to the CG12114 gene.
Blast searches of protein databases identify proteins in Drosophila pseudoobscura (GA11408; 70% identity and 78% similarity) and in Anopheles gambiae (GenBank number: XP_311294; 34% identity and 49% similarity) as the proteins most closely related to CG12114. From sequence analysis, the spn-F gene is predicted to encode a 40 kDa protein with coiled-coil domains between amino acids 32-114 and 210-243. No homologous protein has been found in the databases outside the Dipteran species mentioned above.
Spn-F protein is localized to the minus end of the microtubule and interacts with the Dynein light chain Ddlc-1 (Cut up)
To analyze the intracellular localization of Spn-F, we generated monoclonal
antibodies against the CG12114-encoded protein
(Fig. 6). In wild-type ovaries,
during early stages of oogenesis, Spn-F protein is localized to the posterior
pole of the oocyte (Fig. 6A);
in later stages Spn-F becomes localized to the anterior margin, with higher
accumulation around the oocyte nucleus
(Fig. 6B). These results
strongly suggest that Spn-F is localized to the minus end of the MTs in the
oocyte. We also found that, in the nurse cells, Spn-F is found in a punctuate
pattern (Fig. 6B). The antibody
is specific for Spn-F, as such staining was not observed in spn-F
mutant ovaries.
We next investigated the role of the MT network in Spn-F localization
during oogenesis. Treatment of Drosophila females with colcemid, a MT
depolymerizing drug, simultaneously affects all MTs in the oocyte
(Pokrywka and Stephenson,
1995). We found that treatment of ovaries with colcemid abrogates
Spn-F localization at the minus end of the MTs both during early (data not
shown) and mid-oogenesis (Fig.
6C). Moreover, we found that in colcemid-treated ovaries, a more
punctate distribution of Spn-F protein accumulated in the nurse cells when
compared with wild-type ovaries (Fig.
6C). These results suggest that Spn-F is transported from the
nurse cells to the oocyte via the MT network, and requires MTs for its
localization to the posterior in early oocytes, and to the anterior in older
oocytes.
In a two-hybrid-based, protein-interaction map of the fly proteome, several
proteins were found to interact with Spn-F, among these, although with
somewhat low confidence, is cut up, which encodes a Dynein light
chain (Ddlc) (Giot et al.,
2003). Ddlc is a highly conserved light-chain subunit of
cytoplasmic Dynein, a MT minus-end motor protein that is thought to play a
fundamental role in both the assembly of the motor complex and the recruitment
of cargo. To investigate the direct association of Spn-F with Ddlc-1, a GST
pull-down assay was conducted. First, recombinant GST alone
(Fig. 6E, lane 1), GST-Ddlc
(Fig. 6E, lane 2) and His
tagged-Spn-F (Fig. 6E, lane 3)
proteins were purified and analyzed by gel electrophoresis. Next, GST-Ddlc-1
bound to GSH-agarose (Fig. 6E,
lane 4) or GST bound to GSH-agarose (Fig.
6E, lane 5) was incubated with purified Spn-F protein and a
pull-down assay was performed. We found that Spn-F binds efficiently to
GST-Ddlc-1 (Fig. 6E, lane 4),
but not to GST alone (Fig. 6E,
lane 5) or to any of the other GST-fusion proteins tested (data not
shown).
|
To test whether Spn-F localization to the minus end of the anterior of the
oocyte is dependent on Dynein, we studied the localization of Spn-F in
Dynein heavy chain (Dhc64) mutants. We tested the
localization of Spn-F in hypomorphic semi-viable dhc allelic
combinations (Gepner et al.,
1996) and found that, in 70% of the egg chambers, Spn-F protein
was not localized to the anterior minus end of the oocyte
(Fig. 6D), and a more punctate
distribution of Spn-F was found in the nurse cells. These results, together
with the direct interaction of Spn-F with Dynein light chains, suggest that
Spn-F transport from the nurse cells to the oocyte anterior is dependent on
the minus end-directed motor Dynein.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Saunders and Cohen, 1999;
Norvell et al., 1999). In
mutations of squid, which encodes an hnRNP, the mislocalized
grk mRNA along the anterior ring is translated, suggesting that Sqd
protein is involved in grk mRNA localization and translation
regulation (Norvell et al.,
1999). Although our results indicate that Spn-F is also involved
in grk mRNA localization, in spn-F, the mislocalized
grk mRNA is not relieved from its translational repression. We also observed a small fraction of bicaudal embryos, indicating that there was a problem with anterior posterior pattern formation; however, we did not observe ectopic Oskar protein at the anterior end of spn-F mutant oocytes, as might be expected, given the bicaudal phenotype. We did observe a minor mislocalization of Oskar protein at the posterior, which might have arisen as a result of the abnormalities in the MT or actin cytoskeleton, but this minor mislocalization cannot be the cause of the bicaudal phenotype. Given that only a small percentage of the embryos show the AP patterning abnormalities, it is likely that there is a small amount of ectopic Oskar protein present at the anterior in a few egg chambers that was not detected in our experiments.
What is the mechanism by which spn-F affects grk mRNA
localization? Various lines of evidence have suggested that there are two
separable steps in the localization of grk RNA. Mutations in several
genes, such as squid and fs(1)K10, allow the accumulation of
grk RNA at the anterior, but interfere with the localization to the
dorsal corner (Serano and Cohen,
1995; Neuman-Silberberg and
Schüpbach, 1993; Norvell
et al., 1999). Furthermore, after injection of labeled
grk RNA into live oocytes, it was observed that grk mRNA
particles move in two distinct steps, which are likely to involve two distinct
arrays of MTs. Initially the particles move towards the anterior, subsequently
they turn and move dorsally towards the oocyte nucleus
(MacDougall et al., 2003).
Both steps of grk mRNA localization require intact MTs and
cytoplasmic Dynein (MacDougall et al.,
2003). We have analyzed the organization and polarity of MTs in
spn-F mutant oocytes through the imaging of Tau-GFP -tubulin
and Nod:ß-gal. We detected an abnormal accumulation of GFP-Tau protein
and tubulin in the periphery of the oocyte nucleus. We also tested the
accumulation of Nod:ß-gal, which is localized to the MT minus end during
early and mid-oogenesis in wild-type oocytes. We observed that this
accumulation does not occur in spn-F egg-chambers. However, a number
of probes that normally associate with the anterior cortex of the oocyte in a
MT-dependent manner, such as bicoid RNA and Centrosomin, appeared
unaffected in spn-F mutants. It is possible that molecules whose
anchoring at the anterior cortex is stabilized by interaction with other
molecules (Pokrywka and Stephenson,
1995; Schnorrer et al.,
2000) may accumulate seemingly normally at the anterior, whereas
molecules that depend solely on the MT network (Nod:ß-gal) are more
strongly affected in the spn-F mutant. It may be significant that
grk RNA itself, while localizing to the anterior, only forms a broad
and fuzzy ring at the anterior of spn-F mutants (which is different
from its accumulation in mutations such as squid), as grk
RNA has been shown to require continuous transport for its anchoring
(MacDougall et al., 2003). In
any case, the effect of spn-F on the organization of the minus ends
that are positioned along the anterior cortex appears to be more subtle,
whereas the effect on MTs around the oocyte nucleus is much more severe,
resulting in a visible disorganization and clumping of the network. The effect
of spn-F on the organization of the MT minus end in the oocyte seems
to be relatively specific, as no defects in the localization of kin:ß-gal
to the MT plus end were observed. We also found that Spn-F protein is
localized to MT minus end in the oocyte, and in a punctate form in the nurse
cells. We showed that the localization to the MT minus ends requires an intact
MT network and the subunit of the motor complex Dhc. We also observed
higher Spn-F accumulation in the nurse cells after colcemid treatment.
Given these results, one possible interpretation would be that spn-F mutants affect transport toward the minus end of MTs, predominantly in the subset of MTs that are required for the transport of grk RNA from the anterior to the dorsal side of the oocyte towards the nucleus. The observed interaction with the Dynein light chain would support such a mechanism. Another possibility is that Spn-F has a role in the organization of the minus end of MTs, with particularly strong effects on the MT network that surrounds the oocyte nucleus. The association of Spn-F with the Dynein light chain might be only transient under this model. Finally, given the collapsed aspect of the MT network in the vicinity of the oocyte nucleus, seen in the mutant egg chambers, it is also possible that Spn-F functions to provide a stabilizing connection between the minus ends of MTs and the actin cytoskeleton. However, to gain more insight into the direct role of Spn-F in MT organization, further experiments are needed. Analyzing the role of MT polymerization, MT polarity, and identifying further interaction partners of Spn-F will provide further insight into the function of the Spn-F protein.
In addition to the effects on the oocyte, we have found that spn-F
affects the development of the bristles. The highly elongated bristles of
Drosophila have proven to be a valuable model system for studying
cellular morphogenesis. Developing bristles in Drosophila pupae
contain 7-11 bundles of crosslinked actin filaments and a large population of
MTs. During bristle growth, the rate of cell elongation increases with bristle
length. It has been suggested that actin filaments and MTs play different
roles during bristle elongation in Drosophila
(Tilney et al., 2000). Whereas
actin assembly is crucial for bristle cell elongation, MTs must provide other
functions, such as providing bulk to the bristle cytoplasm, as well as playing
a role in vesicle transport. It has also been shown that MT antagonists such
as vinblastine and colchicine resulted in a decreased axial length and a
compensatory increase in width, so that the volume of the bristle was not
significantly changed (Fei et al.,
2002). These data suggest that the MT cytoskeleton is of central
importance for growth to be polarized in the axial direction
(Fei et al., 2002). Mutations
in the Dynein heavy chain resulted in shorter and thicker bristles
(Gepner et al., 1996);
mutations in kinesin also resulted in shorter and thicker bristles,
and the tips of bristles were often contorted, exhibiting flattened, flared or
twisted tips (Brendza et al.,
2000). Although the spn-F bristle phenotype is comparable
to the kinesin mutant bristles, the spn-F bristle phenotype is
nevertheless unique, suggesting a specific role in the process that is
slightly different from that of either Dynein or Kinesin.
In a global two-hybrid screen, Spn-F (CG12114) was found to interact with
the Ik2 (CG2615) protein (Giot et al.,
2003). Mutations in this protein have been isolated and
characterized by Shapiro and Anderson
(Shapiro and Anderson, 2006).
Interestingly, ik2 mutants share many phenotypes with spn-F,
including a very similar bristle phenotype and specific effects on MT
organization in oogenesis. However, ik2 mutants are lethal, whereas
spn-F homozygotes survive. In addition, whereas spnF
mutations have only mild effects on Oskar protein localization and a low
frequency of bicaudal phenotypes, such effects are more pronounced in the
ik2 mutants. Nevertheless, the striking similarities strongly suggest
that the two genes function in a common pathway that affects certain types of
MT more strongly than others. In normal mitotic cells, the minus ends of MTs
are usually focused by the centrosomes in the interior of the cell, and plus
ends contact the cortex. However, in specialized cells, such as the Drosophila
oocyte, there are minus ends that make contact with the cortex
(Cha et al., 2002). It is
therefore possible that Spn-F and Ik2 are required for providing a stable
connection between such cortical MT minus ends and cortical actin for subsets
of MTs involved in specialized transport processes. Future experiments will
address the interactions of Spn-F and Ik2 directly, and will determine
whether, for instance, Spn-F might be a target of Ik2.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abdu, U., Brodsky, M. and Schüpbach, T. (2002). Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk. Curr. Biol. 12,1645 -1651.[CrossRef][Medline]
Abdu, U., Ghabrial, A., Gonzalez-Reyes, A. and Schüpbach,
T. (2003). The Drosophila spn-D gene encodes a
RAD51C-like protein that is required exclusively during meiosis.
Genetics 165,197
-204.
Brendza, R. P., Sheehan, K. B., Turner, F. R. and Saxton, W.
M. (2000). Clonal tests of conventional kinesin function
during cell proliferation and differentiation. Mol. Biol.
Cell 11,1329
-1343.
Brendza, R. P., Serbus, L. R., Saxton, W. M. and Duffy, J. B. (2002). Posterior localization of dynein and dorsal-ventral axis formation depends on kinesin in Drosophila oocytes. Curr. Biol. 12,1541 -1545.[CrossRef][Medline]
Cha, B.-J., Serbus, L. R., Koppetsch, B. S. and Theurkauf, W. E. (2002). Kinesin I-dependent cortical exclusion restricts pole plasm to the oocyte posterior. Nat. Cell Biol. 4, 592-598.[Medline]
Chou, T. B. and Perrimon, N. (1992). Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131,643 -653.[Abstract]
Clark, I. E., Jan, L. Y. and Jan, Y. N. (1997). Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle. Development 124,461 -470.[Abstract]
Deak, P., Omar, M. M., Saunders, R. D., Pal, M., Komonyi, O., Szidonya, J., Maroy, P., Zhang, Y., Ashburner, M., Benos, P. et al. (1997). P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F. Genetics 147,1697 -1722.[Abstract]
Fei, X., He, B. and Adler, P. N. (2002). The
growth of Drosophila bristles and laterals is not restricted to the
tip or base. J. Cell Sci.
115,3797
-3806.
Gepner, J., Li, M., Ludmann, S., Kortas, C., Boylan, K., Iyadurai, S. J., McGrail, M. and Hays, T. S. (1996). Cytoplasmic dynein function is essential in Drosophila melanogaster.Genetics 142,865 -878.[Abstract]
Ghabrial, A. and Schüpbach, T. (1999). Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1, 354-357.[CrossRef][Medline]
Ghabrial, A., Ray, R. P. and Schüpbach, T.
(1998). okra and spindle-B encode components of
the RAD52 DNA repair pathway and affect meiosis and patterning in
Drosophila oogenesis. Genes Dev.
12,2711
-2723.
Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B.,
Li, Y., Hao, Y. L., Ooi, C. E., Godwin, B., Vitols, E. et al.
(2003). A protein interaction map of Drosophila melanogaster.Science 302,1727
-1736.
Gonzalez-Reyes, A., Elliott, H. and St Johnston, D. (1995). Polarization of both major body axes in Drosophila by gurken-torpedo signalling. Nature 375,654 -658.[CrossRef][Medline]
Gonzalez-Reyes, A., Elliott, H. and St Johnston, D. (1997). Oocyte determination and the origin of polarity in Drosophila: the role of the spindle genes. Development 124,4927 -4937.[Abstract]
Hawkins, N. C., Van Buskirk, C., Grossniklaus, U. and Schüpbach, T. (1997). Post-transcriptional regulation of gurken by encore is required for axis determination in Drosophila. Development 124,4801 -4810.[Abstract]
Heuer, J. G., Li, K. and Kaufman, T. C. (1995). The Drosophila homeotic target gene centrosomin (cnn) encodes a novel centrosomal protein with leucine zippers and maps to a genomic region required for midgut morphogenesis. Development 121,3861 -3876.[Abstract]
Januschke, J., Gervais, L., Dass, S., Kaltschmidt, J. A., Lopez-Schier, H., St Johnston, D., Brand, A. H., Roth, S. and Guichet, A. (2002). Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation. Curr. Biol. 12,1971 -1981.[CrossRef][Medline]
Januschke, J., Gervais, L., Gillet, L., Keryer, G., Bornens, M.
and Guichet, A. (2006). The centrosome-nucleus complex and
microtubule organization in the Drosophila oocyte.
Development 133,129
-139.
Kammermeyer, K. L. and Wadsworth, S. C. (1987).
Expression of Drosophila epidermal growth factor receptor homologue
in mitotic cell populations. Development
100,201
-210.
Li, K. and Kaufman, T. C. (1996). The homeotic target gene centrosomin encodes an essential centrosomal component. Cell 85,585 -596.[CrossRef][Medline]
Lindsley, D. L. and Zimm, G. G. (1992).The Genome of Drosophila melanogaster . New York: Academic Press.
MacDougall, N., Clark, A., MacDougall, E. and Davis, I. (2003). Drosophila gurken (TGFalpha) mRNA localizes as particles that move within the oocyte in two dynein-dependent steps. Dev. Cell 4,307 -319.[CrossRef][Medline]
Micklem, D. R., Dasgupta, R., Elliott, H., Gergely, F., Davidson, C., Brand, A., Gonzalez-Reyes, A. and St Johnston, D. (1997). The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr. Biol. 7, 468-478.[CrossRef][Medline]
Neuman-Silberberg, F. S. and Schüpbach, T. (1993). The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. Cell 75,165 -174.[CrossRef][Medline]
Neuman-Silberberg, F. S. and Schüpbach, T. (1996). The Drosophila TGF-alpha-like protein Gurken: expression and cellular localization during Drosophila oogenesis. Mech. Dev. 59,105 -113.[CrossRef][Medline]
Nilson, L. A. and Schüpbach, T. (1999). EGF receptor signaling in Drosophila oogenesis. Curr. Top. Dev. Biol. 44,203 -243.[Medline]
Norvell, A., Kelley, R. L., Wehr, K. and Schüpbach, T.
(1999). Specific isoforms of squid, a Drosophila hnRNP, perform
distinct roles in Gurken localization during oogenesis. Genes
Dev. 13,864
-876.
Pokrywka, N. J. and Stephenson, E. C. (1995). Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev. Biol. 167,363 -370.[CrossRef][Medline]
Queenan, A. M., Barcelo, G., Van Buskirk, C. and Schüpbach, T. (1999). The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89, 35-42.[CrossRef][Medline]
Roth, S. and Schüpbach, T. (1994). The relationship between ovarian and embryonic dorsoventral patterning in Drosophila. Development 120,2245 -2257.[Abstract]
Roth, S., Neuman-Silberberg, F. S., Barcelo, G. and Schüpbach, T. (1995). cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81,967 -978.[CrossRef][Medline]
Ruohola-Baker, H., Jan, L. Y. and Jan, Y. N. (1994). The role of gene cassettes in axis formation during Drosophila oogenesis. Trends Genet. 10, 89-94.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sapir, A., Schweitzer, R. and Shilo, B. Z. (1998). Sequential activation of the EGF receptor pathway during Drosophila oogenesis establishes the dorsoventral axis. Development 125,191 -200.[Abstract]
Saunders, C. and Cohen, R. S. (1999). The role of oocyte transcription, the 5'UTR, and translation repression and derepression in Drosophila gurken. Mol. Cell 3, 43-54.[CrossRef][Medline]
Schnorrer, F., Bohmann, K. and Nusslein-Volhard, C. (2000). The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes. Nat. Cell Biol. 2, 185-190.[CrossRef][Medline]
Schnorrer, F., Luschnig, S., Koch, I. and Nusslein-Volhard, C. (2002). Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during Drosophila oogenesis. Dev. Cell 3, 685-696.[CrossRef][Medline]
Schüpbach, T. (1987). Germ line and soma cooperate during oogenesis to establish the dorsoventral pattern of egg shell and embryo in Drosophila melanogaster. Cell 49,699 -707.[CrossRef][Medline]
Serano, T. L. and Cohen, R. S. (1995). A small predicted stem-loop structure mediates oocyte localization of Drosophila K10 mRNA. Development 121,3809 -3818.[Abstract]
Shapiro, R. S. and Anderson, K. V. (2006).
Drosophila Ik2, a member of the I
B kinase family, is required
for mRNA localization during oogenesis. Development
133,1467
-1475.
Spradling, A. C. and Rubin, G. M. (1982).
Transposition of cloned P elements into Drosophila germ line
chromosomes. Science
218,341
-347.
Staeva-Vieira, E., Yoo, S. and Lehmann, R. (2003). An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control. EMBO J. 22,5863 -5874.[CrossRef][Medline]
Tearle, R. and Nüsslein-Volhard, C. (1987). Tübingen mutants stock list. Dros. Inf. Serv. 66,209 -226.
Tilney, L. G., Connelly, P. S., Vranich, K. A., Shaw, M. K. and Guild, G. M. (2000). Actin filaments and microtubules play different roles during bristle elongation in Drosophila. J. Cell Sci. 113,1255 -1265.[Abstract]
Van Doren, M., Williamson, A. L. and Lehmann, R. (1998). Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 8, 243-246.[CrossRef][Medline]
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  A. M. Jaramillo, T. T. Weil, J. Goodhouse, E. R. Gavis, and T. Schupbach The dynamics of fluorescently labeled endogenous gurken mRNA in Drosophila J. Cell Sci., March 15, 2008; 121(6): 887 - 894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Barbosa, N. Kimm, and R. Lehmann A Maternal Screen for Genes Regulating Drosophila Oocyte Polarity Uncovers New Steps in Meiotic Progression Genetics, August 1, 2007; 176(4): 1967 - 1977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Song, L. Fee, T. H. Lee, and R. P. Wharton The Molecular Chaperone Hsp90 Is Required for mRNA Localization in Drosophila melanogaster Embryos Genetics, August 1, 2007; 176(4): 2213 - 2222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Shapiro and K. V. Anderson Drosophila Ik2, a member of the I{kappa}B kinase family, is required for mRNA localization during oogenesis Development, April 15, 2006; 133(8): 1467 - 1475. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||
