The Wellcome Trust/Cancer Research UK Institute and the Department of Genetics, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QR, UK
¶ Author for correspondence (e-mail: ds139{at}mole.bio.cam.ac.uk)
Accepted 28 May 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: oskar, bicoid, Staufen, Polarity, Microtubules, Drosophila
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The localisation of bicoid and oskar mRNAs depends on
reciprocal signalling between the oocyte and the overlying somatic follicle
cells. In early stages of oogenesis, an MTOC is present at the posterior of
the oocyte and nucleates microtubules that extend through the nurse cells
(Theurkauf et al., 1992).
During stages 5-6, Gurken protein signals from the oocyte to induce the
adjacent somatic follicle cells to adopt a posterior fate
(Gonzalez-Reyes et al., 1995
;
Roth et al., 1995
). These
posterior cells are then thought to signal back to the oocyte to induce the
disassembly of the posterior MTOC, which leads to the formation of an AP
gradient of microtubules, in which most minus ends lie at the anterior of the
oocyte, with the plus ends extending towards the posterior pole
(Theurkauf et al., 1992
;
Clark et al., 1994
;
Clark et al., 1997
). This
polarised microtubule cytoskeleton defines the destinations of bicoid
and oskar mRNAs, and also directs the migration of the oocyte nucleus
and gurken mRNA from the posterior of the oocyte to the anterior
margin, where Gurken signals a second time to define the dorsal-ventral axis
(Neuman-Silberberg and Schüpbach,
1993
).
As the localisation of bicoid and oskar mRNAs depends on
the polarised microtubule cytoskeleton, a simple model is that these mRNAs are
transported to the anterior and posterior by minus- and plus-end-directed
microtubule motors, respectively (Clark et
al., 1994; Pokrywka and
Stephenson, 1995
). In support of this model, oskar mRNA
localisation requires the plus-end-directed motor, Kinesin 1
(Brendza et al., 2000
).
However, it is unclear whether Kinesin directly transports oskar mRNA
to the posterior, and if so, how it is coupled to its mRNA cargo
(Glotzer et al., 1997
;
Cha et al., 2002
;
Palacios and St Johnston,
2002
). The actin cytoskeleton may also be important in this
process, as some alleles of the actin-binding protein Tropomyosin II (TmII;
Tp2 - FlyBase) disrupt oskar mRNA localization
(Erdelyi et al., 1995
). How
bicoid mRNA is directed to the anterior of the oocyte is even less
well understood, because the minus ends of microtubules are found along the
lateral cortex as well as the anterior (Cha
et al., 2001
). Indeed, bicoid mRNA localises in a
microtubule-dependent manner to both the anterior and lateral cortex when it
is injected into the oocyte, and only localises specifically to the anterior
if it has been exposed to nurse cell cytoplasm. This has led to the proposal
that bicoid RNA is transported to the anterior along a specific
subpopulation of microtubules, and that nurse cell factors render it competent
to distinguish these microtubules from those nucleated laterally.
Very little is known about how the oocyte microtubule cytoskeleton becomes polarised, but three classes of mutants disrupt this organisation.
Genetic screens have identified a number of genes that play a role in the
localisation of bicoid and/or oskar mRNAs once the
microtubule network has been organised. Amongst these, staufen is
unique, as it is required for both bicoid and oskar mRNA
localisation. Staufen plays a central role in the regulation of oskar
mRNA, as it is required not only for its transport from the anterior to the
posterior of the oocyte, but also for the anchoring and translation of the RNA
once it has reached the posterior pole
(Ephrussi et al., 1991;
Kim-Ha et al., 1991
;
St Johnston et al., 1991
;
Rongo et al., 1995
;
Micklem et al., 2000
).
Furthermore, Staufen co-localises with oskar mRNA throughout
oogenesis, in both wild-type and all mutant conditions examined so far, and
this localisation requires oskar mRNA
(St Johnston et al., 1991
;
Ferrandon et al., 1994
).
Because Staufen is a dsRNA-binding protein, these results indicate that it
binds directly and stably to oskar mRNA
(St Johnston et al., 1992
;
Ramos et al., 2000
). In
freshly laid eggs, Staufen is also localised at the anterior, where it is
required to anchor bicoid mRNA
(St Johnston et al., 1989
;
Ferrandon et al., 1994
). The
anterior localisations of Staufen and bicoid mRNA are
mutually-dependent, again suggesting that Staufen interacts directly with the
RNA. However, it is unclear when Staufen first associates with bicoid
mRNA, as it is not enriched at the anterior of the oocyte at stage 10a, and
the later stages of oogenesis cannot be examined by antibody staining because
of the impermeable vitelline membrane that is deposited around the oocyte.
Several other genes are necessary for the anterior localisation of
bicoid mRNA at earlier stages. exuperantia is required for
the anterior accumulation of bicoid RNA from stage 7 onwards, and
appears to function in the nurse cells to render the RNA competent to localise
in the oocyte (Berleth et al.,
1988; Cha et al.,
2001
). In swallow mutants, the initial localisation of
bicoid mRNA is normal, but it is not anchored at the anterior of the
oocyte from stage 10b (Berleth et al.,
1988
; St Johnston et al.,
1989
). Swallow protein physically interacts with the Dynein light
chain, and localises to the anterior of the oocyte, but it is unclear whether
it plays a direct role in the anchoring of bicoid mRNA or functions
indirectly in the organisation of the microtubule cytoskeleton
(Schnorrer et al., 2000
). In
support of the latter view, Swallow interacts with
Tub37C and Grip75,
which are components of the
Tubulin ring complex that nucleates
microtubules; mutants in both of these genes also disrupt bicoid mRNA
anchoring (Schnorrer et al.,
2002
).
The localisation of oskar mRNA is also affected by mutations in a
number of other known genes. In barentsz mutants and hypomorphic
alleles of mago nashi and Y14, oskar mRNA fails to localise
to the posterior of the oocyte and accumulates along the anterior margin
instead (Newmark and Boswell,
1994; Micklem et al.,
1997
; Hachet and Ephrussi,
2001
). All three proteins transiently localise with oskar
mRNA at the posterior of stage 9 oocytes, suggesting that they are directly
involved in its transport (Newmark et al.,
1997
; Hachet and Ephrussi,
2001
; Mohr et al.,
2001
). Once oskar mRNA reaches the posterior of the
oocyte, it needs to be securely anchored. The actin cytoskeleton may be
important in this process as two actin-binding proteins, TmII and Dmoesin
(Moesin-like - FlyBase), are necessary for the anchoring of oskar
mRNA to the posterior cortex (Tetzlaff et
al., 1996
; Jankovics et al.,
2002
; Polesello et al.,
2002
). Oskar protein also plays an important role in anchoring, as
oskar mRNA is not maintained at the posterior in oskar
protein null mutants (Ephrussi et al.,
1991
; Kim-Ha et al.,
1991
). Mutations in genes necessary for oskar
translation, such as aubergine, therefore result in similar defects
in oskar mRNA anchoring (Harris
and Macdonald, 2001
). In addition, the homologue of the
Xenopus cytoplasmic polyadenylation element-binding (CPEB) protein,
ORB, binds oskar mRNA, and is required for its localisation and
translation (Lantz et al.,
1992
; Christerson and
McKearin, 1994
; Hake and
Richter, 1994
; Chang et al.,
1999
).
The majority of the mutants described above were identified in genetic
screens for maternal-effect lethal and female-sterile mutations
(Nüsslein-Volhard et al.,
1987; Schüpbach and
Wieschaus, 1989
;
Schüpbach and Wieschaus,
1991
). Although these screens were very successful at identifying
germline- specific factors required for oocyte polarisation and mRNA
localisation, they could only recover homozygous viable mutations, and
therefore missed many of the essential genes that also play a role in polarity
and mRNA localisation in other cell types. This problem can be overcome by
using the FLP/FRT/DFS system to perform screens in germline clones
(Xu and Rubin, 1993
;
Chou and Perrimon, 1996
). This
system allows the recovery of lethal mutations in essential genes, because it
selects for germline clones, while most somatic cells remain heterozygous.
Perrimon et al. successfully used this technique to screen a sample of 500
lethal P-elements recombined onto FRT autosomes, and identified maternal-
effect lethal factors involved in proper egg shell formation and patterning of
the cuticle (Perrimon et al.,
1996
).
A limitation of all of these screens is their use of the embryonic cuticle as a readout for AP patterning, as this precludes the identification of any mutants that affect AP axis formation in the oocyte but block development before cuticle formation. To circumvent this problem, we have designed a novel genetic screen in germline clones for mutations that alter the distribution of GFP-Staufen in living oocytes. Here, we report the results of this screen on chromosome arm 3R, and the characterisation of 23 new complementation groups that play a role in AP axis formation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following alleles were used for complementation tests:
osk54 (Lehmann and
Nüsslein-Volhard, 1991), cnc03921
(Guichet et al., 2001
),
spnEhls
157
(Gillespie and Berg, 1995
),
orbF343 (Lantz et al.,
1994
), orbmel
(Christerson and McKearin,
1994
), Ets97Dtne-4
(Schulz et al., 1993
),
tmIIgs1 (Erdelyi et
al., 1995
), btz2
(van Eeden et al., 2001
). The
ypsJM2 allele and the ypsJM2 orb
recombinant chromosomes were obtained from Dr Hazelrigg
(Mansfield et al., 2002
).
Mutagenesis and complementation
w; FRT82B males were starved for 6 hours before they were treated with 25
mM EMS (Sigma) in 1% sucrose for 18-24 hours to induce an average of one
lethal hit per chromosome arm. The number of lethal hits was estimated by
monitoring the number of X-linked lethals. Mutagenised males were mated with
w; Pr, Dr/TM3 virgin females. Single w; FRT82B, */TM3 virgin females (where
the asterisk indicates the mutagenised chromosome) were mated with y, w, hs-
FLP, GFP-Staufen; FRT82B, ovoD1/TM6B males. The progeny were
heat-shocked three times for 2 hours in a 37°C incubator during the third
larval instar and pupal stages. Ovaries from 3-5 females of the genotype y, w,
hs-FLP, GFP-Staufen/w; FRT82B, */FRT82B, ovoD1 were dissected and
screened for defects in GFP-Staufen localisation, using an inverted
fluorescence microscope (see Fig.
2). If a phenotype was observed, w; FRT82B, */TM6B males were
mated with w; Pr, Dr/TM3 virgin females and balanced stocks were established.
Mutants identified in the primary screen were re-screened under the confocal
microscope.
|
Two lines were considered allelic if no trans-heterozygous progeny were recovered, or if the trans-heterozygous females were sterile or maternal-effect lethal. We did not routinely test for weaker maternal- effect defects, such as a grandchildless phenotype.
Stains and microscopy
The primary screen was performed on a Leica inverted fluorescence
microscope, using a broadband filter (Leica 13 513828) in which the GFP
fluorescence appears green, and yolk autofluorescence yellow. For live
observation, the ovaries were dissected in Voltalef 3S (screen) or 10S
(movies) oil (Elf Atochem) on a coverslip. The time-lapse movies were obtained
by collecting z series of five sections at 1 µm intervals every 30 seconds
for 20 minutes on a BioRad confocal MRC1024 microscope. The moving particles
were visualised by excitation with 568 nm light, and collection of the
emission through an OG515 filter (Palacios
and St Johnston, 2002). The Kalman images in
Fig. 7E-H were obtained using
the Kalman averaging function of the BioRad software to merge eight
consecutive scans taken at 3 second intervals. Static particles form dots in
these images, whereas moving particles appear as lines that represent the
direction and speed of movement. Actin was visualised by fixing ovaries in 4%
paraformaldehyde and staining with rhodamine-phalloidin (1:500; Molecular
Probes). In situ hybridisations were carried out using RNA probes labelled
with Digoxigenin-UTP (Roche). Immunohistochemical detection was performed with
alkaline phosphatase-conjugated anti-DIG (1:5000; Roche). For microtubule
staining, samples were fixed for 10 minutes in 8% paraformaldehyde and stained
with a FITC-conjugated monoclonal anti-
-tubulin antibody (1:400; Sigma)
(Theurkauf, 1994a
).
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We first crossed the mutants to alleles of all of the known genes on 3R
that affect oskar or bicoid mRNA localisation, and
identified two new alleles of oskar, four of orb, six of
spn-E, one of D-elg (Ets97D) and three of
cnc, but no alleles of tmII or barentsz were
identified (Ephrussi et al.,
1991; Kim-Ha et al.,
1991
; Christerson and
McKearin, 1994
; Erdelyi et
al., 1995
; Gajewski and
Schulz, 1995
; Gillespie and
Berg, 1995
; Tetzlaff et al.,
1996
; Guichet et al.,
2001
; van Eeden et al.,
2001
). As expected, all of these mutants affect oskar
mRNA localisation, and the three new alleles of cnc also affect the
localisation of bicoid mRNA. This indicates that the screen was
efficient, and achieved a reasonable degree of saturation.
We classified the remaining mutants on the basis of their phenotypes and used this as a guideline in the complementation tests (Table 1). As expected, we recovered mutants that affect only oskar mRNA localisation (74), only bicoid mRNA localisation (18), or the localisation of both mRNAs (40). To identify complementation groups, mutants within each phenotypic class were crossed to each other, and the progeny assayed for lethality, female-sterility and maternal-effect lethal phenotypes. In addition, mutants that affect both bicoid and oskar mRNAs were crossed to mutants that affect the localisation of only one mRNA. With this approach, we identified 23 novel complementation groups (Table 2).
|
|
|
vagabond is the only complementation group in this class that
gives the characteristic phenotype of mutants that disrupt oskar mRNA
transport, in which the mRNA remains at the anterior of oocyte
(Fig. 3C,D)
(van Eeden et al., 2001).
vagabond mutant oocytes display a range of additional phenotypes,
including a mislocalised nucleus and aberrant organisation of the follicle
cells, suggesting that the gene has other functions in addition to its role in
oskar mRNA transport. In this respect, it may be similar to mago
nashi and Y14 (Micklem et
al., 1997
; Newmark et al.,
1997
; Hachet and Ephrussi,
2001
; Mohr et al.,
2001
).
The third class of mutants consists of those in which oskar mRNA
localises to the posterior, but fails to adopt a wild-type pattern (19
mutants, of which nine fall into four groups). Mutants in this group are
unlikely to affect the transport of oskar mRNA or the polarisation of
the oocyte, as oskar mRNA can reach the posterior. Instead, they
probably disrupt the anchoring of oskar mRNA to the posterior cortex.
In most cases, oskar mRNA and GFP-Staufen initially localise to the
posterior normally (stage 9; Fig.
3G,H) but fail to be maintained there in later stages
(Fig. 3I,J). In
glissade (Fig. 3K,L)
and sedov, GFP-Staufen and oskar mRNA localise to the
posterior part of the oocyte but fail to restrict to a cortical crescent, a
phenotype very similar to that of the oskar protein- null allele
osk54, which suggests that mutants in this group could
also affect oskar translation
(Ephrussi et al., 1991;
Kim-Ha et al., 1991
). In
agreement with this, we found two novel oskar alleles in this class.
Western blot analysis failed to detect Oskar protein in these mutants,
suggesting that they are protein nulls (data not shown). Thus, most mutants in
this group probably affect the translation or anchoring of oskar
mRNA.
Mutants affecting bicoid mRNA localisation
Several mutants disrupt the anterior localisation of GFP-Staufen but have
little or no effect on posterior localisation, suggesting that they
specifically affect the localisation of bicoid mRNA
(Fig. 4). These mutants can be
separated into two distinct classes based on when the defect in GFP-Staufen
localisation is first observed. In the first class (five single alleles),
GFP-Staufen never concentrates at the anterior of late stage oocytes and
bicoid mRNA fails to accumulate at the anterior of the oocyte at any
stage (Fig. 4C,D), indicating
that these mutants affect an early step in the localisation of bicoid
mRNA. In the second class (13 mutants, six of which form three complementation
groups), bicoid mRNA localises to the anterior of the oocyte in stage
8-10 egg chambers, but GFP-Staufen fails to accumulate at the anterior of late
stage oocytes (Fig. 4E,F).
These mutants therefore affect a later event in the anterior restriction of
bicoid mRNA. In agreement with this, bicoid mRNA is not
concentrated at the anterior of these mutants in late stage oocytes or freshly
laid eggs (data not shown; U. Irion, personal communication).
|
|
Two further complementation groups, mertz
(Fig. 5G,H) and
ellsworth, affect the localisation of both oskar and
bicoid mRNAs. In these mutants, the mRNAs localise to their
respective poles in much reduced amounts, or are diffusely distributed
throughout the oocyte. However, the oocyte nucleus is always positioned
normally, suggesting that the oocyte has been polarised correctly. These
mutants may therefore affect factors, such as staufen, that are
necessary for the transport of both oskar and bicoid mRNAs
(Ferrandon et al., 1994). In
agreement with this, oskar mRNA persists at the anterior of the
oocyte in ellsworth mutants, as it does in other mutants that
specifically disrupt oskar mRNA transport.
Other mutants
A number of mutants recovered for their defect in GFP-Staufen localisation
also display unexpected additional phenotypes. sorcière
(Fig. 6A) and gerlache
are dumpless mutants in which the bulk of GFP-Staufen remains in the nurse
cells, in or just anterior to the ring canals. Although this phenotype
suggests a defect in ring canal growth, the morphology and size of the ring
canals is indistinguishable from wild type, as assayed by rhodamine-phalloidin
staining. In trou mutants, the posterior of the oocyte detaches from
the follicle cells (Fig. 6B).
This detachment may interfere with signalling from the posterior follicle
cells, which could account for the variable defects in oskar and
bicoid mRNA localisation in these mutants. In sedov,
vagabond and wellman mutant egg chambers, excess follicle cells
are sometimes found between the oocyte and the nurse cells, indicating a
possible defect in the control of follicle cell migration or proliferation
(Fig. 6C). We also found
mutants that affect the growth of the oocyte (seven mutants, of which two form
the complementation group nain). As GFP-Staufen still accumulates in
one cell in these mutants, the oocyte appears to be correctly determined, but
fails to grow larger than the nurse cells. Finally, we found two mutants that
affect the number of germ cell divisions, as the egg chambers contain more
than 16 cells, and an oocyte with five or six ring canals
(Fig. 6E).
|
We examined the organisation of the microtubule network in three of the new
spn-E alleles (spn-E2A9-14,
spn-E4E2-14 and spn-E8D4-11) and found
that all display thick microtubule bundles at stage 9
(Fig. 7A,B). This microtubule
organisation resembles that observed in capu, spir or chic
mutants, which cause similar defects in oskar and gurken
mRNA localisation (Neuman-Silberberg and
Schüpbach, 1993;
Theurkauf, 1994b
;
Manseau et al., 1996
). As
these mutants also cause premature cytoplasmic streaming, we studied the
movement of autofluorescent vesicles in spn-E mutant oocytes. Whereas
wild- type oocytes show slow and chaotic cytoplasmic movements at stage 9,
40-60% of spn-E mutant oocytes show a very fast circular movement
(Fig. 7E,F; see Movie 1 at
http://dev.biologists.org/supplemental/).
Thus, spn-E is required for the formation of the polarised
microtubule network at stage 9 and for the regulation of cytoplasmic
streaming.
The role of ORB during mid-oogenesis has previously been studied using a
single hypomorphic allele, orbmel, in which oskar
mRNA fails to localise to the posterior and grk mRNA fails to
localise to the dorsoanterior corner of the oocyte
(Christerson and McKearin,
1994). As ORB binds oskar mRNA, these phenotypes have
been interpreted as a function of ORB in RNA transport and translation
(Christerson and McKearin,
1994
; Chang et al.,
1999
). ORB is also necessary for the determination of the oocyte,
as strong (orbF303) and null (orbF343)
alleles arrest oogenesis at early stages
(Lantz et al., 1994
). We
ordered the new orb alleles into an allelic series by analysing the
strength of their phenotype in trans-heterozygous combinations over
orbF343 (Fig.
7I). Whereas our stronger allele arrests at early stages of
oogenesis (orb9D6-3), the weaker alleles show similar
defects to orbmel: trans-heterozygous females lay
ventralised eggs with fused or no dorsal appendages, and the rare eggs that
are fertilised have a reduced number of abdominal denticle belts, indicating a
defect in pole plasm assembly.
Because orb mutants cause a similar defect in both AP and DV
patterning to spn-E mutants, we studied the organisation of the
microtubule cytoskeleton. Both anti--tubulin antibodies and Tau-GFP
reveal large cortical bundles of microtubules in over 90% of stage 9
orb mutant oocytes (Fig.
7A,C; data not shown). This phenotype was observed in germline
clones of all four new alleles, as well as in trans-heterozygous combinations
of these alleles and orbmel, over
orbF343. In addition, orb mutant oocytes display
a very fast circular cytoplasmic movement at stage 9 that is characteristic of
premature cytoplasmic streaming (Fig.
7E,G; see Movie 2 at
http://dev.biologists.org/supplemental/).
Thus, orb hypomorphs have the same effect on the microtubules and
cytoplasmic streaming as capu, spir, chic or spn-E mutants,
suggesting that this is the primary cause of the defect in oskar mRNA
localisation. Interestingly, ORB protein levels are dramatically reduced in
spn-E mutants, suggesting that SPN-E may be involved in the
post-transcriptional regulation of orb mRNA
(Fig. 7J).
Ypsilon Schachtel (YPS) is another RNA-binding protein that co-localises
with oskar mRNA at the posterior, and has been shown to act
antagonistically to ORB in oskar mRNA localisation and translation
(Mansfield et al., 2002).
Whereas oskar mRNA fails to localise to the posterior in over 90% of
orbmel/orbF303 oocytes, about 50% of
ypsJM2 orbmel/ypsJM2
orbF303 have wild-type amounts of oskar mRNA at the
posterior (Mansfield et al.,
2002
) (data not shown). This has been interpreted as a direct
antagonism between YPS and ORB in the transport and translation of
oskar mRNA (Mansfield et al.,
2002
). However, we do not detect significant changes in the level
of OSK protein in orb yps versus orb mutants, indicating
that YPS does not counteract the translational activation of oskar by
ORB (Fig. 7J). By contrast,
half of yps orb double- mutant oocytes have a wild-type microtubule
organisation and a normal pattern of cytoplasmic streaming, which indicates
that YPS acts antagonistically to ORB in the organisation of the microtubule
cytoskeleton (Fig. 7D,H; see
movies at
http://dev.biologists.org/supplemental/).
This observation suggests that the presence of oskar mRNA at the
posterior of yps orb double mutants reflects the restoration of a
wild-type microtubule network, rather than a direct effect of YPS on
oskar mRNA. YPS does not appear to exert its effect by altering the
levels of ORB protein, as ORB protein levels are not increased in yps
mutants (Fig. 7J).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Second, we performed the screen in germline clones, which allowed us to
recover lethal mutations. Indeed, 17 of the 23 new complementation groups are
essential for viability and would have been missed in female-sterile or
maternal-effect lethal screens. This suggests that about 70% of the genes
necessary for AP axis formation have essential functions in other cell types.
In the one case that we have examined in detail, we found that lkb1
is also required for epithelial polarity
(Martin and St Johnston,
2003). Thus, a proportion of the mutants identified in this screen
may be general factors regulating polarity in multiple cell types.
Surprisingly, four mutants that display clear defects in the localisation
of GFP-Staufen produce eggs that hatch and develop into normal larvae. Two of
these are only partially penetrant, but the others produce similar phenotypes
to barentsz mutants, in which the posterior localisation of
GFP-Staufen and oskar mRNA is strongly reduced. It has been suggested
that the defect in oskar mRNA localisation in barentsz
mutants is partially rescued by localised translation, resulting in a weak
grandchildless phenotype, and this may also be the case for these alleles
(van Eeden et al., 2001). This
illustrates another advantage of a direct visual screen that allows the
identification of mutations with subtle phenotypes that do not cause
lethality. The large size of the oocyte makes it a particularly good system in
which to detect defects in cellular organisation, and we recovered mutations
with a variety of polarity phenotypes that would have been very difficult to
identify in smaller cells.
Degree of saturation
One disadvantage of germline clone screens is that they are laborious,
which limits the number of lines that can be screened. We screened over 5000
mutagenised chromosomes and recovered new alleles for five of the seven known
genes on chromosome 3R required for oskar mRNA localisation
(oskar, spn-E, Ets97D, orb and cnc). However, we failed to
recover new alleles of barentsz and tmII. One reason for
this may be that these genes are not easily mutable by EMS, because all of the
existing alleles that disrupt oskar mRNA localisation are either
P-element insertions or deletions (Erdelyi
et al., 1995; Tetzlaff et al.,
1996
; van Eeden et al.,
2001
). Alternatively, hypomorphic alleles of these genes may exist
in our collection of mutants but have been overlooked in the complementation
tests, because they only cause a nonpenetrant grandchildless phenotype, as is
the case for btz1 (van
Eeden et al., 2001
). Thus, the screen was efficient in finding
mutants affecting the posterior localisation of GFP-Staufen but did not find
mutants in all genes that can be mutated to give this phenotype.
Another indication that the screen did not reach saturation is that 66
mutants (47%) could not be attributed to any complementation group. However,
the degree of saturation varies considerably between the different phenotypic
classes. It is likely that we missed many mutants that only affect the
anterior localisation of GFP-Staufen, as this localisation is more difficult
to score in living oocytes than the posterior localisation because of the
strong yolk autofluorescence at late stages of oogenesis. In addition,
GFP-Staufen only localises to the anterior in late oocytes and is therefore
visible in fewer egg chambers per ovary. Another class of genes for which we
probably did not reach saturation are those required at multiple stages of
oogenesis. orb, par-1 and 14-3-3, for example, not
only function to polarise the oocyte during mid-oogenesis, but are also
necessary for the determination of the oocyte in the germarium
(Lantz and Schedl, 1994
;
Cox et al., 2001
;
Huynh et al., 2001
;
Benton et al., 2002
). As
germline clones of null mutations in these genes produce no late egg chambers,
one can only recover hypomorphic mutations in a screen of this type. It is
therefore likely that many of the single alleles correspond to hypomorphic
mutants in such loci. However, it is now relatively straightforward to clone
genes for which only one mutant allele is available by meiotic mapping with
single nucleotide polymorphisms (Berger et
al., 2001
; Martin et al.,
2001
).
Although we may have missed a number of genes for these reasons, the screen was very successful in identifying new mutants that affect the posterior localisation of GFP-Staufen and oskar mRNA. For example, over 70% of the mutants with phenotypes similar to cnc belong to complementation groups, which suggests that we are approaching saturation for this phenotype. oskar mRNA localisation depends on the polarisation of the oocyte microtubule cytoskeleton, the transport of the mRNA to the posterior and its anchoring at the posterior cortex, and we identified new complementation groups that are required for each of these steps. The cloning of these genes should therefore provide valuable insights into the molecular mechanisms that underlie these processes.
Mutants affecting oocyte polarity
One of the surprising results of the screen was the large number of mutants
that appear to affect the polarity of the oocyte. These produce a range of
different phenotypes that can be classified into at least four classes.
The diversity of phenotypes described above indicates that the polarisation of the microtubule cytoskeleton is a complex process that can be disrupted in several different ways. It is therefore likely that the signal from the posterior follicle cells is not transduced in a simple linear manner, but impinges on several parallel pathways that act together to generate the polarised microtubule array.
The role of orb,yps and spn-E in the
localisation of oskar mRNA
ORB protein has been shown to bind oskar mRNA, and is required for
its localisation and translation at the posterior of the oocyte
(Christerson and McKearin,
1994; Lantz et al.,
1994
; Chang et al.,
1999
). Although this suggests that ORB plays a direct role in both
processes, our results indicate that its effects on mRNA localisation are
indirect. All hypomorphic orb alleles disrupt the organisation of the
microtubule cytoskeleton and cause premature cytoplasmic streaming, and this
can account for the dramatic reduction in the amount of localised
oskar mRNA. Indeed, other mutants that cause premature cytoplasmic
streaming, such as capu and spir, cause an identical
oskar mRNA localisation phenotype
(Theurkauf, 1994b
;
Manseau et al., 1996
). Thus,
ORB presumably controls the organisation of the microtubules by regulating the
translation of some other mRNA(s), and the principal function of its
interaction with oskar mRNA is to regulate its translation by
promoting the formation of a long polyA tail
(Chang et al., 1999
;
Castagnetti and Ephrussi,
2003
). This function is very similar to that of its
Xenopus homologue, CPEB, which binds cytoplasmic polyadenylation
elements (CPE) in the 3'UTRs of several maternal mRNAs to stimulate
their polyadenylation and translation
(Lantz et al., 1992
;
Hake and Richter, 1994
).
The RNA-binding protein YPS has also been proposed to play a direct role in
the localisation and translation of oskar mRNA, because it
co-localises with the mRNA to the posterior of the oocyte and antagonises the
effects of ORB (Mansfield et al.,
2002). Our results again suggest that this effect is indirect.
yps mutants partially rescue the microtubule and premature streaming
phenotypes of orb hypomorphs but have little or no effect on the
levels of Oskar protein. Thus, YPS presumably counteracts the effects of ORB
on the translation of RNAs that regulate the microtubule organisation. This is
consistent with the biochemical characterisation of YPS as a component of a
multi-protein complex, containing EXU and ME31B, that has been proposed to
repress the translation of many oocyte mRNAs
(Wilhelm et al., 2000
;
Nakamura et al., 2001
).
Mutants in spn-E produce very similar microtubule and premature
streaming phenotypes to orb mutants, and have strongly reduced ORB
protein levels. SPN-E is an RNA-helicase, raising the possibility that it
exerts its effect by regulating the processing of orb mRNA
(Gillespie, 1995). Alternatively, as ORB is known to regulate its own
translation, ORB and SPN-E may function together to regulate the translation
of common target RNAs, including that of orb itself
(Tan et al., 2001). These
results reveal a novel regulation of the microtubule network at the level of
RNA translation, and it will be important in future to identify the RNA
substrates of ORB, YPS and SPN-E.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* These authors contributed equally to this work
Present address: Columbia University, Department of Microbiology, 701 W
168th Street, New York, NY 10032, USA
Present address: IBMC UPR CNRS 9022, 15 rue René Descartes, 67084
Strasbourg, France
Present address: P. A. Consulting, Cambridge Technology Centre, Melbourn
SG8 6DP, UK
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benton, R., Palacios, I. M. and St Johnston, D. (2002). Drosophila 14-3- 3/PAR-5 is an essential mediator of PAR-1 function in axis formation. Dev. Cell 3,659 -671.[Medline]
Berger, J., Suzuki, T., Senti, K. A., Stubbs, J., Schaffner, G. and Dickson, B. J. (2001). Genetic mapping with SNP markers in Drosophila. Nat. Genet. 29,475 -481.[CrossRef][Medline]
Berleth, T., Burri, M., Thoma, G., Bopp, D., Richstein, S., Frigerio, G., Noll, M. and Nüsslein-Volhard, C. (1988). The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 7,1749 -1756.[Abstract]
Brendza, R. P., Serbus, L. R., Duffy, J. B. and Saxton, W.
M. (2000). A function for kinesin I in the posterior
transport of oskar mRNA and Staufen protein.
Science 289,2120
-2122.
Castagnetti, S. and Ephrussi, A. (2003). Orb
and a long poly(A) tail are required for efficient oskar translation
at the posterior pole of the Drosophila oocyte.
Development 130,835
-843.
Cha, B. J., Koppetsch, B. S. and Theurkauf, W. E. (2001). In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule- dependent axis specification pathway. Cell 106,35 -46.[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]
Chang, J. S., Tan, L. and Schedl, P. (1999). The Drosophila CPEB homolog, Orb, is required for Oskar protein expression in oocytes. Dev. Biol. 215,91 - 106.[CrossRef][Medline]
Chou, T. B. and Perrimon, N. (1996). The
autosomal FLP-DFS technique for generating germline mosaics in Drosophila
melanogaster. Genetics
144,1673
-1679.
Christerson, L. B. and McKearin, D. M. (1994). Orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis. Genes Dev. 8, 614-628.[Abstract]
Clark, I., Giniger, E., Ruohola-Baker, H., Jan, L. Y. and Jan, Y. N. (1994). Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr. Biol. 4,289 -300.[Medline]
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.
Cox, D. N., Lu, B., Sun, T. Q., Williams, L. T. and Jan, Y. N. (2001). Drosophila par-1 is required for oocyte differentiation and microtubule organization. Curr. Biol. 11,75 -87.[CrossRef][Medline]
Driever, W. (1993). Maternal control of anterior development in the Drosophila embryo. In The Development of Drosophila melanogaster, Vol.1 (ed. M. Bate and A. Martinez-Arias), pp.301 -324. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by Oskar. Nature 358,387 -392.[CrossRef][Medline]
Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). Oskar organizes the germ plasm and directs localization of the posterior determinant Nanos. Cell 66, 37-50.[Medline]
Erdelyi, M., Michon, A. M., Guichet, A., Glotzer, J. B. and Ephrussi, A. (1995). Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature 377,524 -527.[CrossRef][Medline]
Ferrandon, D., Elphick, L., Nüsslein-Volhard, C. and St Johnston, D. (1994). Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 79,1221 -1232.[Medline]
Gajewski, K. M. and Schulz, R. A. (1995). Requirement of the ETS domain transcription factor D-ELG for egg chamber patterning and development during Drosophila oogenesis. Oncogene 11,1033 -1040.[Medline]
Gillespie, D. E. and Berg, C. A. (1995). Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DEH family of RNA-dependent ATPases. Genes Dev. 9,2495 -2508.[Abstract]
Glotzer, J. B., Saffrich, R., Glotzer, M. and Ephrussi, A. (1997). Cytoplasmic flows localize injected oskar RNA in Drosophila oocytes. Curr. Biol. 7, 326-337.[Medline]
Golic, K. G. and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59,499 -509.[Medline]
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.
Guichet, A., Peri, F. and Roth, S. (2001). Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg. Dev. Biol. 237,93 -106.[CrossRef][Medline]
Hachet, O. and Ephrussi, A. (2001). Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport. Curr. Biol. 11,1666 -1674.[CrossRef][Medline]
Hake, L. E. and Richter, J. D. (1994). CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation. Cell 79,617 -627.[Medline]
Harris, A. N. and Macdonald, P. M. (2001). aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C. Development 128,2823 -2832.[Medline]
Huynh, J. R., Shulman, J. M., Benton, R. and St Johnston, D.
(2001). PAR- 1 is required for the maintenance of oocyte fate in
Drosophila. Development
128,1201
-1209.
Jankovics, F., Sinka, R., Lukacsovich, T. and Erdelyi, M. (2002). Moesin crosslinks actin and cell membrane in Drosophila oocytes and is required for Oskar anchoring. Curr. Biol. 12,2060 -2065.[CrossRef][Medline]
Kim-Ha, J., Smith, J. L. and Macdonald, P. M. (1991). oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell 66, 23-35.[Medline]
Lane, M. E. and Kalderon, D. (1994). RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization. Genes Dev. 8, 2986- 2995.[Abstract]
Lantz, V. and Schedl, P. (1994). Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis. Mol. Cell. Biol. 14,2235 -2242.[Abstract]
Lantz, V., Ambrosio, L. and Schedl, P. (1992). The Drosophila orb gene is predicted to encode sex-specific germline RNA-binding proteins and has localized transcripts in ovaries and early embryos. Development 115, 75-88.[Abstract]
Lantz, V., Chang, J. S., Horabin, J. I., Bopp, D. and Schedl, P. (1994). The Drosophila Orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity. Genes Dev. 8,598 -613.[Abstract]
Le Hir, H., Gatfield, D., Braun, I. C., Forler, D. and
Izaurralde, E. (2001). The protein Mago provides a link
between splicing and mRNA localization. EMBO Rep.
2,1119
-1124.
Lehmann, R. and Nüsslein-Volhard, C. (1991). The maternal gene nanos has a central role in posterior pattern formation in the Drosophila embryo. Development 112,679 -691.[Abstract]
Manseau, L., Calley, J. and Phan, H. (1996).
Profilin is required for posterior patterning of the Drosophila
oocyte. Development 122,2109
-2116.
Mansfield, J. H., Wilhelm, J. E. and Hazelrigg, T.
(2002). Ypsilon Schachtel, a Drosophila Y-box protein, acts
antagonistically to Orb in the oskar mRNA localization and
translation pathway. Development
129, 197-
209.
Martin, S. G. and St Johnston, D. (2003). A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity. Nature 421, 379- 384.[CrossRef][Medline]
Martin, S. G., Dobi, K. C. and St Johnston, D. (2001). A rapid method to map mutations in Drosophila. Genome Biol. 2, RESEARCH0036.
Micklem, D. R., Adams, J., Grünert, S. and St Johnston,
D. (2000). Distinct roles of two conserved Staufen domains in
oskar mRNA localization and translation. EMBO
J. 19,1366
-1377.
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.[Medline]
Mohr, S. E., Dillon, S. T. and Boswell, R. E.
(2001). The RNA-binding protein Tsunagi interacts with Mago Nashi
to establish polarity and localize oskar mRNA during
Drosophila oogenesis. Genes Dev.
15, 2886-
2899.
Nakamura, A., Amikura, R., Hanyu, K. and Kobayashi, S.
(2001). Me31B silences translation of oocyte-localizing RNAs
through the formation of cytoplasmic RNP complex during Drosophila
oogenesis. Development
128,3233
-3242.
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.[Medline]
Newmark, P. A. and Boswell, R. E. (1994). The
mago nashi locus encodes an essential product required for germ plasm
assembly in Drosophila. Development
120,1303
-1313.
Newmark, P. A., Mohr, S. E., Gong, L. and Boswell, R. E.
(1997). mago nashi mediates the posterior follicle
cell-to-oocyte signal to organize axis formation in Drosophila.
Development 124,3197
-3207.
Nüsslein-Volhard, C., Frohnhofer, H. G. and Lehmann, R. (1987). Determination of anteroposterior polarity in Drosophila. Science 238, 1675- 1681.[Medline]
Palacios, I. M. and St Johnston, D. (2002).
Kinesin light chain-independent function of the Kinesin heavy chain in
cytoplasmic streaming and posterior localisation in the Drosophila
oocyte. Development 129,5473
-5485.
Perrimon, N., Lanjuin, A., Arnold, C. and Noll, E.
(1996). Zygotic lethal mutations with maternal effect phenotypes
in Drosophila melanogaster. II. Loci on the second and third chromosomes
identified by P-element-induced mutations. Genetics
144,1681
-1692.
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]
Polesello, C., Delon, I., Valenti, P., Ferrer, P. and Payre, F. (2002). Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis. Nat. Cell Biol. 4,782 -789.[CrossRef][Medline]
Ramos, A., Grunert, S., Adams, J., Micklem, D. R., Proctor, M.
R., Freund, S., Bycroft, M., St Johnston, D. and Varani, G.
(2000). RNA recognition by a Staufen double-stranded RNA-binding
domain. EMBO J. 19,997
-1009.
Riechmann, V. and Ephrussi, A. (2001). Axis formation during Drosophila oogenesis. Curr. Opin. Genet. Dev. 11,374 -383.[CrossRef][Medline]
Rongo, C., Gavis, E. R. and Lehmann, R. (1995).
Localization of oskar RNA regulates oskar translation and requires
Oskar protein. Development
121,2737
-2746.
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.[Medline]
Schnorrer, F., Bohmann, K. and Nüsslein-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.[Medline]
Schuldt, A. J., Adams, J. H., Davidson, C. M., Micklem, D. R.,
Haseloff, J., St Johnston, D. and Brand, A. H. (1998).
Miranda mediates asymmetric protein and RNA localization in the developing
nervous system. Genes Dev.
12,1847
-1857.
Schulz, R. A., The, S. M., Hogue, D. A., Galewsky, S. and Guo, Q. (1993). Ets oncogene-related gene Elg functions in Drosophila oogenesis. Proc. Natl. Acad. Sci. USA 90,10076 -10080.[Abstract]
Schüpbach, T. and Wieschaus, E. (1989).
Female sterile mutations on the second chromosome of Drosophila
melanogaster. I. Maternal effect mutations.
Genetics 121,101
-117.
Schüpbach, T. and Wieschaus, E. (1991).
Female sterile mutations on the second chromosome of Drosophila
melanogaster. II. Mutations blocking oogenesis or altering egg
morphology. Genetics
129,1119
-1136.
Shulman, J. M., Benton, R. and St Johnston, D. (2000). The Drosophila homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localization to the posterior pole. Cell 101,377 -388.[Medline]
St Johnston, D., Driever, W., Berleth, T., Richstein, S. and Nüsslein- Volhard, C. (1989). Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. Development 107, 13-19.[Medline]
St Johnston, D., Beuchle, D. and Nüsslein-Volhard, C. (1991). staufen, a gene required to localize maternal RNAs in the Drosophila egg. Cell 66, 51- 63.[Medline]
St Johnston, D., Brown, N. H., Gall, J. G. and Jantsch, M. (1992). A conserved double-stranded RNA-binding domain. Proc. Natl. Acad. Sci. USA 89,10979 -10983.[Abstract]
Tan, L., Chang, J. S., Costa, A. and Schedl, P.
(2001). An autoregulatory feedback loop directs the localized
expression of the Drosophila CPEB protein Orb in the developing
oocyte. Development 128,1159
-1169.
Tetzlaff, M. T., Jackle, H. and Pankratz, M. J. (1996). Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation. EMBO J. 15,1247 -1254.[Abstract]
Theurkauf, W. (1994a). Immunofluorescence analysis of the cytoskeleton during oogenesis and early embryogenesis. In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology. Vol. 44 (ed. L. Goldstein and E. Fyrberg), pp. 489-506. London: Academic Press.
Theurkauf, W. E. (1994b). Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes. Science 265,2093 -2096.[Medline]
Theurkauf, W. E., Smiley, S., Wong, M. L. and Alberts, B. M.
(1992). Reorganization of the cytoskeleton during
Drosophila oogenesis: implications for axis specification and
intercellular transport. Development
115,923
-936.
Tomancak, P., Piano, F., Riechmann, V., Gunsalus, K. C., Kemphues, K. J. and Ephrussi, A. (2000). A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation. Nat. Cell Biol. 2,458 -460.[CrossRef][Medline]
van Eeden, F. and St Johnston, D. (1999). The polarisation of the anterior- posterior and dorsal-ventral axes during Drosophila oogenesis. Curr. Opin. Genet. Dev. 9, 396-404.[CrossRef][Medline]
van Eeden, F. J., Palacios, I. M., Petronczki, M., Weston, M. J.
and St Johnston, D. (2001). Barentsz is essential for the
posterior localization of oskar mRNA and colocalizes with it to the
posterior pole. J. Cell Biol.
154,511
-523.
Wilhelm, J. E., Mansfield, J., Hom-Booher, N., Wang, S., Turck,
C. W., Hazelrigg, T. and Vale, R. D. (2000). Isolation of a
ribonucleoprotein complex involved in mRNA localization in Drosophila
oocytes. J. Cell Biol.
148,427
-440.
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Related articles in Development: