Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montréal, QC, H3A 1B1, Canada
Author for correspondence (e-mail:
beat.suter{at}izb.unibe.ch)
Accepted 22 November 2004
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SUMMARY |
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Key words: Drosophila, Posterior development, WD protein, Valois, Vasa, Oskar
![]() |
Introduction |
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The osk ribonucleoprotein (RNP) complex has been characterized,
and many conserved factors are known to function in mRNA localization and/or
translational control in different systems across phyla
(Farina and Singer, 2002;
Hachet and Ephrussi, 2004
;
Roegiers and Jan, 2000
;
Wilhelm et al., 2000
). Because
restriction of osk activity to the posterior is crucial for normal
development (Ephrussi and Lehmann,
1992
), both pre- and post-translational control mechanisms
regulate Osk protein accumulation. Osk protein is actively degraded by the
ubiquitin-proteasome pathway, but protected from it by phosphorylation by
Par-1 specifically at the posterior
(Riechmann et al., 2002
).
Translational control of osk involves the coordinate action of
repressors and derepressors interacting with discrete elements of osk
transcripts during transport and at the posterior pole
(Gunkel et al., 1998
;
Kim-Ha et al., 1995
;
Webster et al., 1997
;
Yano et al., 2004
). Additional
factors that do not function as derepressors are also required for stimulating
osk translation (Wilson et al.,
1996
). In addition, Oo18 RNA-binding protein (Orb) polyadenylates
osk transcripts at the posterior pole once derepression has been
achieved (Castagnetti and Ephrussi,
2003
).
Two isoforms of Osk (Long and Short Osk) are produced by initiation at two
different in-frame start codons. Short Osk has long been known as the active
isoform for pole plasm assembly which recruits downstream components of the
pathway such as Vasa (Vas) (Markussen et
al., 1995), and recently, Long Osk has been shown to be
responsible for anchoring osk mRNA and Short Osk at the posterior
(Vanzo and Ephrussi, 2002
).
Short Osk is likely to anchor Vas directly at the posterior
(Breitwieser et al., 1996
;
Vanzo and Ephrussi, 2002
). Vas
is an ATP-dependent RNA-helicase from the DEAD-box family and has been
implicated in translational activation of several maternal transcripts,
including osk (Styhler et al.,
1998
; Tinker et al.,
1998
; Tomancak et al.,
1998
). tudor (tud) acts downstream of
vas and is followed in the cascade by additional genes whose products
localize to the pole plasm and mark the separation of germline establishment
and abdominal patterning activities
(Golumbeski et al., 1991
).
Pole cell formation depends on the localization of germ cell less
(gcl) mRNA (Leatherman et al.,
2002
) and mitochondrial large ribosomal RNA
(Iida and Kobayashi, 1998
).
Abdominal patterning relies on the vas- dependent translation of
nanos (nos) mRNA at the posterior pole. This results in a
concentration gradient of Nos protein along the AP axis, which acts as the
primary posterior morphogen (Riechmann and
Ephrussi, 2001
).
One more posterior group gene, valois (vls), had been
identified in the initial screen for maternal-effect steriles
(Schupbach and Wieschaus,
1986), but has neither been cloned nor studied genetically in
detail yet. Based on three EMS-induced alleles of vls, it was
classified as a member of the `grandchildless-knirps-like' group that also
includes vas, stau and tud. Their phenotype is characterized
by a lack of pole cells at the posterior and various degrees of abdominal
segment deletions. Pole cell transplantation experiments demonstrated that
vls functions in the germline
(Schupbach and Wieschaus,
1986
) and vls mutants were shown to have a non-functional
pole plasm (Lehmann and Nusslein-Volhard,
1991
). Until now, the position of vls in the posterior
pathway has remained controversial. vls was tentatively placed
downstream of osk and vas, but upstream of tud.
This was based on the observation that osk mRNA and Vas protein are
initially correctly localized to the posterior of the oocyte in
vlsEMS mutants. Vas then detaches from the posterior of
the embryo soon after fertilization
(Ephrussi et al., 1991
;
Hay et al., 1990
;
Lasko and Ashburner, 1990
) and
Tud localization is disrupted in embryos from vls mothers
(Bardsley et al., 1993
).
However, conflicting data were reported subsequently. Assembly of an ectopic
pole plasm at the anterior of the oocyte, caused by overexpressing
osk (6xosk) (Smith et
al., 1992
) or by targeting osk transcripts specifically
to the anterior margin (osk-bcd3'UTR)
(Ephrussi and Lehmann, 1992
),
results in progeny embryos with ectopic pole cells and duplication of the
abdomen at the anterior. vls function was found to be required for
the expression of the 6xosk phenotype, confirming its position
downstream of osk, but not for the expression of the
osk-bcd3'UTR phenotype
(Ephrussi and Lehmann, 1992
;
Smith et al., 1992
).
Here, we report the cloning and characterization of vls. We have created a null mutant for vls that shows stronger phenotypes than the presently available vlsEMS alleles. In contrast to previous models, this tool allows us to demonstrate that vls acts upstream of vas. Furthermore, vls dramatically affects the levels of Osk protein, even though localization of osk mRNA and initial accumulation of Osk do not require vls function. vls encodes a novel protein with significant similarity to WD domain proteins. The presented data suggest that Vls may act as a co-factor in assembling protein-protein and/or protein-RNA complexes.
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Materials and methods |
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Generation of anti-Vls antibody
A partial vls cDNA was recovered by PCR amplification from an
ovarian cDNA library (Larochelle and
Suter, 1995). Rabbit polyclonal antibodies were raised against an
E. coli expressed Vls polypeptide (amino acids 166-367;
Fig. 1) that contained an
N-terminal 6xHis tag. The fusion protein was purified by affinity
chromatography and gel electrophoresis prior to injection. Antiserum was
affinity purified with a MBP::Vls fusion protein (expressed from the same
vls cDNA) that was coupled to CnBr-activated sepharose beads
(Pharmacia Biotech).
|
RNA in situ and immunostaining
In situ hybridizations to osk mRNA on ovaries were performed as
described previously (Suter and Steward,
1991). The osk probe was generated by random priming with
the DIG High Prime digoxigenin labeling system (Roche Applied Science).
Immunostaining on ovaries were performed as described previously
(Findley et al., 2003
) with
-Osk at 1:3000 and secondary Alexa Fluor anti-rabbit 488nm.
Vls-eGFP and Vas-eGFP observations
A Zeiss confocal microscope was used for Vls-eGFP and a Leica confocal
microscope for Vas-eGFP observations. Two- to three-day-old females were used
for ovaries and embryo collections. For live observation of Vls-eGFP, ovaries
were dissected in halocarbon oil 27, separated and dragged onto a cover slip.
They were then covered with petriPERM 50 hydrophobic membrane dishes
(Vivascience). Samples were used for no more than 15 minutes after dissection.
vas-eGFP ovaries were dissected in Ringer's buffer, fixed for 20
minutes in phosphate-buffered saline (PBS) with 4% paraformaldehyde, rinsed
three times, washed twice for 5 minutes in PBST (PBS + 0.2% Tween 20) and
mounted in 60% glycerol. Embryos were collected for 1 hour periods, aged
accordingly, dechorionated by rolling them over double-sided sticky tape and
mounted on slides in halocarbon oil 27. Background signal was evaluated for
all types of samples by using yw118 ovaries or embryos as
a control. The wavelength window of detection was adjusted to reduce
background signal produced by autofluorescent particles.
![]() |
Results |
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Overlapping genes and repression of chk2 by vls
Interestingly, chk2 and vls are encoded by opposite
strands and cDNA sequence data shows that their 3'UTRs are complementary
over 127 nucleotides (Fig. 1).
chk2 is translationally repressed by orb during oogenesis
(Masrouha et al., 2003), and
because translational control often relies on the binding of
trans-acting factors to sequences in the 3'UTR of mRNAs, we
were curious to know whether vls could also play a role in
chk2 translational control. Indeed, Chk2 levels increase about 6-fold
in vlsPG65/HC33 and vlsPG65/RB71
ovaries compared with wild type (Fig.
2) and this is close to the 10-fold upregulation reported for
orb mutants (Masrouha et al.,
2003
). This indicates that vls is also involved in the
regulation of Chk2 levels. However, orb does not simply function to
control Vls levels because these are normal in orb mutants (data not
shown).
|
Valois belongs to a family of divergent WD domain proteins
vls encodes a novel protein and PROSITE predicted the existence of
two WD domains. Database searches reveal the best sequence similarity with the
human methylosome protein 50 (MEP50; 20.4% identity;
Fig. 3 and
Table 1) and alignment of Vls
and MEP50 shows that the two predicted WD domains of Vls correspond closely to
the predicted WD domains 2 and 3 of MEP50. With the exception of the WD domain
5, the predicted WD domains of MEP50 show elevated similarity with
corresponding Vls regions compared to the alignment of the entire proteins
(Table 1). This suggests that
Vls may have five to six domains that have a similar structure or function as
WD domains, and it may mean that Vls has evolved from a WD domain protein. The
six WD domains of MEP50 are thought to fold into a ß-propeller structure,
which serves as a platform for recruiting the Arg-methyltransferase JBP1/PRMT5
and its substrates, the Sm proteins
(Friesen et al., 2002). This
event is required for assembling the splicing machinery prior to import into
the nucleus (Friesen et al.,
2001
).
|
|
Valois is a maternal product
Northern analysis detected a transcript of 1.5 kb for vls
expressed in ovaries, early embryos and adult females, but absent from pooled
larval instars and adult males (Butler et
al., 2001). In situ hybridization to OreR ovaries with a
vls probe detected signal throughout the germ cell cytoplasm from
early oogenesis onwards. The signal showed no specific localization pattern.
Surprisingly, we detected an equally strong signal in
vlsPG65/RB71 ovaries, and only in the
vlsnull ovaries the signal is at background levels (data
not shown).
On Western blots, polyclonal anti-Vls antibodies do not detect any Vls in vlsnull, vlsPG65, vlsRB71 and vlsHC33 ovary extracts (Fig. 4A). This shows that the antibody specifically recognizes the Vls protein and that the EMS mutants do not make significant levels of stable full length Vls. However, because we do not know which epitopes are recognized by the polyclonal antibody, it is still possible that the EMS alleles produce truncated forms of Vls. In wild-type flies, Vls is abundant in ovaries, early embryos and adult females, but reduced in adult males (Fig. 4B). The fact that it is present in ovaries and in 0- to 1-hour-old embryos indicates that Vls is a maternally provided protein and this is consistent with the maternal-effect phenotype of vls mutants.
|
|
Posterior localization of Osk protein in late oogenesis depends on vls
To investigate further the position of vls in the pathway, we
examined the distribution of posterior products in vlsnull
ovaries. osk mRNA is efficiently localized at the posterior of
vlsnull mutant oocytes
(Fig. 6A), consistent with
previous reports for embryos from vlsPE36 mothers
(Ephrussi et al., 1991). Osk
protein accumulates at the posterior pole of the oocyte during stages 8-10.
During this phase, we observe similar patterns in wild type and vls
mutants (Fig.
6B-E,B'-E'). However, at later stages (stage 11,
Fig. 6B''-E''), Osk
levels at the posterior seem somewhat reduced in vlsnull
oocytes compared with OreR and vlsnull vls+,
and we often do not detect any signal for Osk in vlsnull
oocytes. This reduction of Osk levels at the posterior is also observed in
hemizygous vlsPG65, albeit to a lesser extent
(Fig. 6D'').
|
|
vls is essential for posterior localization of Vasa
Vas protein is the next factor in the posterior pathway to localize to the
posterior end of the oocyte after osk mRNA and protein. This
osk-dependent Vas localization remains stable at the posterior pole
during the early stages of embryogenesis and Vas is later incorporated into
pole cells (Lasko and Ashburner,
1990). In vlsnull ovaries and in embryos from
vlsnull mothers, anti-Vas antibody staining showed very
little or no accumulation of Vas at the posterior end (data not shown). This
observation was further confirmed by analyzing the distribution of Vas-eGFP
(Styhler et al., 1998
) in
vlsnull and vlsPG65 ovaries and
embryos. Although the early localization pattern of Vas in nuage of the mutant
nurse cells is normal (Fig.
8A-D), the posterior localization in stage 10 oocytes is not
observed in the null mutants, and appears very weak in
vlsPG65 hemizygotes
(Fig. 8A'-D').
|
![]() |
Discussion |
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Nature of vls mutants
Specification of the germline in Drosophila is more sensitive to
pole plasm activity than is abdominal patterning. This is illustrated by the
fact that weak alleles of posterior group mutants display a grandchildless
phenotype caused by the lack of pole cells, while stronger alleles cause
additional abdominal patterning defects that result in embryonic lethality
(Lehmann and Nusslein-Volhard,
1991). In our hands, the hemizygous EMS alleles
vlsPG65, vlsRB71 and
vlsHC33 are only partially maternal-effect lethal and 100%
grandchildless. vlsnull, however, is 100% maternal-effect
lethal. The stronger phenotype of the null mutant suggests that the EMS
alleles may be hypomorphs. However, the initial work on vls produced
strong genetic evidence that the EMS alleles are actually nulls (Schupbach,
1986). It is therefore also possible that the EMS allele stocks accumulated
maternal-effect modifiers that allow them to survive to adulthood.
vls mRNA escapes NMD
Although vlsEMS alleles contain premature stop codons
in the vls ORF, the corresponding mutant mRNAs seem to escape
nonsense-mediated mRNA decay mechanisms (NMD). Even though premature stop
codons are recognized differently in Drosophila and vertebrates, the
NMD components are conserved (Gatfield et
al., 2003). Given that vls+ is translated
during oogenesis, it seems unlikely that the mutants are protected because of
lack of translation (Dreyfuss et al.,
2002
). It would thus be interesting to find out why
vlsEMS transcripts accumulate to normal levels.
Collapse of the pole plasm in the absence of vls function
Because all aspects of the vls mutant phenotype observed in
embryos, including abdominal segment deletions, lack of pole cells,
gastrulation defects and weak ventralization are rescued completely by a
vls transgene and not even partially by a chk2 transgene, we
concluded that vls alone has a developmental requirement.
Furthermore, we have demonstrated elsewhere that chk2 function is
only clearly required upon activation of cell cycle checkpoints
(Masrouha et al., 2003). The
vls phenotypes are reminiscent of a collapse of pole plasm assembly
that seems to occur around stage 10 of oogenesis in our
vlsnull mutants. vas is crucial for the pole
plasm to assemble properly and recruit the mRNAs and proteins required for
pole cell specification and abdominal patterning. Genetic evidence implicates
vas in the translational activation of several targets during
oogenesis, including osk, grk and, in particular, nos at the
posterior pole of the embryo (Carrera et
al., 2000
; Gavis et al.,
1996
; Johnstone and Lasko,
2004
; Markussen et al.,
1995
; Rongo et al.,
1995
; Styhler et al.,
1998
; Tomancak et al.,
1998
). Vas levels directly correlate with pole plasm activity,
pole cell formation being more vulnerable to decreased Vas levels than
abdominal patterning is (Ephrussi and
Lehmann, 1992
). Previous immunostaining for Vas has been reported
to show indistinguishable Vas accumulation at the posterior pole of
vls mutant and wild-type oocytes, and young embryos. These studies,
performed with the homo- and hemizygous EMS mutants, showed a loss of
posterior localization in the embryos from vls mothers sometime
between fertilization and pole cell formation
(Hay et al., 1990
;
Lasko and Ashburner, 1990
). We
used vas-eGFP transgenes to assess the posterior localization of Vas
in vlsnull and hemizygous EMS alleles in detail. Maximal
localization was still very weak and was found in oocytes and embryos from
vlsEMS mothers. In vlsnull mutants we
observed a nearly complete failure to localize Vas-eGFP at the posterior pole.
This failure coincides with the collapse of the pole plasm and is probably the
cause for the various embryonic phenotypes mentioned above. Consistent with
this, the observed Vas localization defects parallel the severity of the
phenotypes that we report for these vls alleles. The weak
accumulation of Vas at the posterior of vlsPG65 hemizygous
oocytes gives rise to a grandchildless phenotype, whereas the almost complete
absence of Vas from the posterior of vlsnull oocytes
results in a fully penetrant maternal-effect lethal phenotype.
vls is thus required during oogenesis for the localization (transport or anchoring) of Vas to the posterior cortex of the oocyte. The fact that Vls is not specifically enriched at the posterior may suggest that it acts to modify or transport pole plasm components before they reach the posterior pole. Preliminary experiments also failed to produce evidence that Vls and Vas are part of the same protein complex (not shown). This suggests that the mode of action of vls on Vas localization is transient or indirect. The fact that osk mRNA and protein are initially correctly localized implies that oocyte polarity is normal in vls mutants and that vls is not required for osk mRNA localization. Levels of Osk protein isoforms are then reduced in later stages and western analysis reveals a much more drastic decrease of overall Osk levels than immunostaining does for both types of vls alleles. This suggests that most of the drop in Osk levels occurs during the late stages of oogenesis, when the vitelline membrane prevents antibody staining for oocyte Osk. Therefore, it seems that shortly after initiating pole plasm assembly, Osk fails to be maintained at the posterior of vls mutants and progressively disappears, concurrent with a complete collapse of the pole plasm.
vls acts upstream of Vas and Osk
Several lines of evidence implicate the Short Osk isoform in directly
anchoring Vas. Short Osk interacts strongly with Vas in the two-hybrid system
and recruits Vas when ectopically localized in the oocyte
(Breitwieser et al., 1996;
Cha et al., 2002
;
Vanzo and Ephrussi, 2002
).
Because Vas-eGFP mis-localization patterns in stage 10 oocytes are
indistinguishable in vls and osk54 mutants (not
shown), vls could act directly at the level of Osk accumulation (e.g.
in stimulating translation of osk), which is necessary for anchoring
Vas at the posterior pole. On the other hand, it is also possible that
vls acts primarily on Vas protein localization. Because Vas also
seems to act in a positive feedback loop back on Osk protein accumulation
(Markussen et al., 1995
), the
lack of Vas localization in vls mutants would then also preclude
maintenance of posterior accumulation of Osk protein. In vls mutants,
Osk levels appear to decrease just slightly after Vas should have localized to
the posterior pole, thus it appears that the failure to localize Vas could be
the cause of the pole plasm collapse in vls mutants. To investigate
these issues further, we compared Osk levels in vas and tud
mutants with those in vls mutants by western analysis where we detect
a more significant drop than by immunostaining. This analysis revealed
generally stronger phenotypes for vls than for vas and
tud mutants. We observed a comparable decrease of Short Osk levels on
western blots of vls, vas and tud mutant extracts, but with
slight differences in the extent of reduction of the hyper- and
hypophosphorylated forms, both of which are more severely affected in
vls mutants. In addition, we observed a clear reduction of Long Osk
levels in vls, a minor reduction in tud, but none in
vas mutant extracts (Fig.
7). However, this analysis is complicated by the fact that the
vas and tud alleles that are useful and available,
respectively, for these experiments are not nulls
(Bardsley et al., 1993
;
Hay et al., 1988
;
Lasko and Ashburner, 1990
).
Their residual activity may therefore maintain Osk at the posterior for a
longer period of time. These data are thus consistent with the idea that
vls acts on either pathway target, Vas or Osk, in a process which
could involve additional intermediates that remain to be identified.
Why is vls not required for expression of the osk-bcd 3'UTR phenotype?
vls was tentatively placed downstream of vas in the
posterior pathway based on studies reporting that Vas localization is correct
initially in vlsEMS mutants
(Hay et al., 1990;
Lasko and Ashburner, 1990
),
and because vls was found to be required for the expression of the
6xosk phenotype (Smith et al.,
1992
). Surprisingly, however, vls is not required for the
expression of the osk-bcd 3'UTR phenotype
(Ephrussi and Lehmann, 1992
).
As the 3'UTR is present in the 6xosk transgenes but not in the
osk-bcd 3'UTR transgene, one explanation for this
discrepancy could be that vls is required to relieve translational
repression mediated by the osk 3'UTR.
It is also possible that differences in osk mRNA levels and
concentration at the anterior between the two systems might explain the
discrepancy. In fact, Vas protein accumulation at the posterior pole and the
number of pole cells that develop afterwards correlate directly with the
osk gene copy number (Ephrussi and
Lehmann, 1992). Besides, 6xosk produces lower levels of
osk mRNA at the anterior than osk-bcd3'UTR
(Smith et al., 1992
).
Therefore, the ectopic pole plasm induced by
osk-bcd3'UTR mRNA is probably more resistant to
defects in localization/anchoring of downstream components such as Vas or to
defects in the maintenance of Osk itself. By contrast, the 6xosk
system seems to represent a more sensitized background where the collapse of
an ectopic pole plasm is more likely to occur in the absence of vls.
Supporting this idea, the bicaudal phenotype of the progeny from transgenic
mothers is 100% penetrant with osk-bcd 3'UTR
(Ephrussi and Lehmann, 1992
),
but only 73% penetrant with 6xosk
(Smith et al., 1992
).
vls might thus function as an enhancer of pole plasm assembly, which
is dispensable when osk pole plasm-inducing activity is already
extensively deployed at the anterior. This is consistent with our observation
that vls dose also correlates with pole plasm activity in the same
way that osk does. One copy of a wild-type vls+
transgene rescues almost completely the phenotypes described for
vlsnull, but we sometimes noted minor defects compared
with wild-type flies, and reduced hatching rates of embryos (not shown).
Speculations on the molecular function of Vls
vls differs in many respects from the other long-known members of
the posterior pathway and seems to encode a co-factor acting on Osk protein
accumulation, Vas localization and possibly on another, yet unknown, component
of this pathway. Two lines of evidence suggest that vls facilitates
the process of pole plasm assembly but is not absolutely essential: some
residual Vas localization is possible even in the null mutant; and an ectopic
pole plasm can assemble in the absence of vls function provided that
the system is set up excessively or through different 3'UTR control
elements (osk-bcd3'UTR vs. 6xosk). How could
Vls perform this function at the molecular level?
Vls is a divergent WD domain protein. The ß-propeller structure of WD
proteins is thought to arise from the folding of at least four WD domains and
to promote several simultaneous protein-protein interactions
(Smith et al., 1999). Because
computer predictions only found two or three such domains in Vls, we tested in
preliminary experiments whether Vls forms homodimers. However, we did not
detect any untagged Vls in immunoprecipitations performed with functional
Vls-eGFP and Vls-6xHis fusion proteins (not shown). Whether Vls forms
heterodimers with other WD domain-containing proteins remains to be tested.
Sequence alignments point to a more likely interpretation. Vls and a whole
family of Drosophila WD domain proteins show similarities to MEP50,
which contains six WD domains and facilitates the interactions between a
methyltransferase and its substrates, the Sm proteins
(Friesen et al., 2002
).
Notably, the regions corresponding to the WD domains of MEP50 are better
conserved than the others, suggesting that these domains are under greater
selection pressure and may therefore fold in similar structures that can
fulfill similar functions. This sequence comparison also shows that Vls might
not be the ortholog of MEP50 and that different members of this family might
fulfill the function of MEP50 in different Drosophila tissues.
It is therefore possible that Vls also acts as a mediator of molecular interactions between proteins and possibly also mRNAs. Future experiments will have to focus on identifying the interactors of Vls to determine how precisely vls facilitates the pole plasm assembly process. The Vls interactions may turn out to represent an activating step in pole plasm assembly that involves a methyltransferase or another protein modification enzyme and their substrates. With this information it should then also be possible to clarify how directly this mechanism acts on the targets Vas and Osk.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Institute of Cell Biology, University of Berne,
Baltzerstrasse 4, 3012 Bern, Switzerland
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