European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Author for correspondence (e-mail:
ephrussi{at}embl-heidelberg.de)
Accepted 29 September 2004
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SUMMARY |
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Key words: Drosophila, bicoid, Par-1, Exn, Polarity, mRNA localisation
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Introduction |
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Recently, it has been reported that par-1 is also involved in
localisation of the anterior determinant bicoid mRNA
(Benton et al., 2002).
bicoid mRNA localisation occurs in two phases
(St Johnston et al., 1989
;
Schnorrer et al., 2002
): in
the early phase, the mRNA is transported to the anterior margin of the oocyte,
resulting in its distribution in a ring-shaped pattern. Anterior localisation
of bicoid mRNA is microtubule dependent
(Pokrywka and Stephenson,
1991
), and is most probably mediated by plus end-directed motors.
It is unclear, however, how the bicoid mRNA localisation complex
distinguishes between microtubules nucleating from the lateral cortex and
those nucleating from the anterior cortex of the oocyte. It has been
speculated that anterior-nucleating microtubules might be qualitatively
different from lateral microtubules and that this difference might be
perceived by the bicoid mRNA-transport complex
(Cha et al., 2001
). In the late
phase, bicoid mRNA redistributes along the entire anterior cortex,
resulting in a disc-shaped distribution of the mRNA. This localisation is
dependent on the function of
Tub37C and Dgrip75
(Grip75 FlyBase), suggesting that it requires the formation
of an anterior microtubule organising centre (MTOC)
(Schnorrer et al., 2002
). In
hypomorphic par-1 mutants, the early phase of bicoid mRNA
localisation is disrupted, such that the mRNA is not restricted to the
anterior of the oocyte, but rather is distributed down the lateral cortex
towards the posterior pole (Benton et al.,
2002
).
Another gene essential for bicoid mRNA localisation is
exu. In exu mutants, bicoid mRNA localisation is
affected beginning from the early phase, when the mRNA fails to localise
specifically at the anterior and is dispersed throughout the oocyte cytoplasm
(Berleth et al., 1988;
St Johnston et al., 1989
). Exu
protein contains no known domains, with the exception of a region with weak
homology to an RNA-binding motif
(Macdonald et al., 1991
;
Marcey et al., 1991
). However,
this domain is dispensable for the bicoid mRNA localisation function
of Exu (Wang and Hazelrigg,
1994
). By electron microscopy, Exu is detected in large
electron-dense structures in the nurse cells, the sponge bodies, where Exu is
thought to associate with bicoid mRNA
(Wilsch-Bräuninger et al.,
1997
). A series of RNA injection experiments has led to the
hypothesis that, in the nurse cells, Exu promotes the recruitment of
anterior-targeting factors to bicoid mRNA. This assembly of the
bicoid mRNA localisation complex would render the mRNA competent for
transport to the anterior margin of the oocyte during the early phase
localisation (Cha et al.,
2001
).
We have identified Exu in a proteomic screen for direct targets of Par-1 kinase. We show that Exu is a phosphoprotein whose phosphorylation is dependent on Par-1. In the nurse cells, Exu and Par-1 colocalise in patches that also contain bicoid mRNA. We have determined Par-1 phosphorylation sites in Exu, and show that mutation of these sites abolishes the ability of Exu to mediate bicoid mRNA localisation during mid-oogenesis. These mutants, which specifically affect phosphorylation of Exu protein, allow us differentiate between two phases of exu-dependent bicoid mRNA localisation: an early phase during mid-oogenesis, which is strictly dependent on Exu phosphorylation; and a late phase at the end of oogenesis, in which the requirement for Exu phosphorylation is less stringent.
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Materials and methods |
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Whole-mount in situ staining
Antibody staining of ovaries and embryos was performed as described
(Tomancak et al., 2000). In
situ hybridisation was performed as described by Wilkie et al.
(Wilkie et al., 1999
), and an
RNA probe corresponding to full-length bicoid mRNA was generated by
in vitro transcription using an Ambion Megascript kit. Double staining for
bicoid mRNA and Exu GFP was performed as described by Vanzo and
Ephrussi (Vanzo and Ephrussi,
2002
) using a mouse anti-GFP antibody (Roche) in a 1:200 dilution.
For double staining for Exu-GFP and Par-1, the same antibody was used in
combination with a rabbit anti-Par-1 antibody
(Tomancak et al., 2000
) at a
1:40 dilution. Anti-Bicoid (Kosman et al.,
1998
) antibody was used at a 1:600 dilution.
Western blotting
Preparation of ovarian extracts and western blotting was performed as
described (Riechmann et al.,
2002). An anti-Exu antibody from rabbit was used at a 1:10.000
dilution. For the blot shown in Fig.
1B, Exu was detected with Enhanced Chemiluminescence using an
anti-rabbit antibody coupled to horseradish peroxidase. For the blot in
Fig. 4E, a fluorochrome-coupled
anti-rabbit antibody was used, and signal was detected using an Odyssey
scanner (LI-CORE).
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Fly stocks
Following fly stocks were used: exuXL and
exuVL (protein null alleles), par-1W3
(a null allele), and par-19A and
par-1574 (hypomorphic alleles). All females used for
staining were grown at 18°C.
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Results |
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par-1 and exu are required for bicoid mRNA localisation
In wild type oocytes, two phases of bicoid mRNA localisation can
be distinguished by in situ hybridisation
(St Johnston et al., 1989;
Schnorrer et al., 2002
).
First, bicoid mRNA forms a ring at the anterior margin of stage 9-10a
oocytes (Fig. 2A,D). Next, at
stage 10b, the ring of bicoid mRNA evolves into a disk, such that the
mRNA is present along the entire anterior surface of the oocyte
(Fig. 2G). As a first step
towards the analysis of the role of Exu phosphorylation by Par-1, we
re-examined the distribution of bicoid mRNA in exu and
par-1 mutants. Consistent with previous reports
(Berleth et al., 1988
;
St Johnston et al., 1989
), we
found that in exu null mutants
(exuXL/exuVL)
(Marcey et al., 1991
)
bicoid mRNA localisation is severely affected and the anterior ring
does not form (Fig. 2B).
Interestingly however, bicoid mRNA localisation is not completely
abolished in exu mutants, as the mRNA still concentrates in the
anterior region of stage 9 oocytes (Fig.
2B,E). In addition, residual amounts of bicoid mRNA are
detected at the anterior cortex of disk stage oocytes
(Fig. 2H). Residual anterior
localisation of bicoid mRNA was also observed in to other
exu alleles (exuQR and
exuPJ), confirming that exu does not completely
abolish bicoid mRNA localisation to the anterior of the oocyte.
|
Exu and Par-1 colocalise with bicoid mRNA in the nurse cells
To further investigate the connection between Exu and Par-1, we compared
their distributions by detecting Exu as a GFP fusion protein and Par-1 with an
antibody. Both proteins have previously been detected in the cytoplasm of the
nurse cells (Wang and Hazelrigg,
1994; Theurkauf and Hazelrigg,
1998
; Shulman et al.,
2000
; Tomancak et al.,
2000
). Strikingly, the proteins show a near-identical localisation
in patches in the apical regions of the nurse cells
(Fig. 3A). It has previously
been shown that Exu-GFP colocalises with bicoid mRNA in the nurse
cells (Cha et al., 2001
). We
therefore tested whether bicoid mRNA is also present in these patches
in the nurse cells. bicoid mRNA is enriched in the apical patches,
although a substantial amount of the mRNA is also detected outside of the
patches (Fig. 3B). The
extensive colocalisation of Par-1 and Exu-GFP in these patches suggests
phosphorylation of Exu by Par-1 may occur at these sites. The presence of
bicoid mRNA in the apical patches supports our hypothesis that Exu
phosphorylation is involved in the localisation of bicoid mRNA.
|
After identifying Par-1 target sites in Exu in vitro, we generated
transgenes to express the mutant Exu proteins in ovaries under the control of
the exu promoter (Wang and
Hazelrigg, 1994). The transgenes were crossed into an exu
protein null background to assess the phosphorylation state of the mutant Exu
proteins by western blot analysis. As in the in vitro assay, a knockout of
site A causes only a slight decrease in Exu phosphorylation
(Fig. 4E, A6), while a knockout
of site B leads to a dramatic reduction
(Fig. 4E, B3). We observed the
same strong decrease in phosphorylation when only S457 of site B was mutated
as when both relevant serines in B were mutated
(Fig. 4E, B2 and B3) confirming
that this Serine is especially important for Exu phosphorylation. Most
importantly, upon mutation of all serines in A and B
(Exu
A+B), the protein migrates as a single band, indicating
absence of any phosphorylation (Fig.
4E, A6B3). Hence, the motifs mediating phosphorylation of Exu by
Par-1 in vitro also mediate Exu phosphorylation in vivo. Together with the
finding that Exu phosphorylation is dependent on Par-1 during oogenesis, these
data strongly suggest that Par-1 phosphorylates Exu on sites A and B.
Phosphorylation does not affect Exu localisation or mobility
Different aspects of Exu function have been revealed by analysis of an
Exu-GFP fusion protein, which, when expressed under the control of the
endogenous promoter, rescues the exu phenotype
(Wang and Hazelrigg, 1994).
Exu-GFP can be detected at the anterior of the oocyte and accumulates at the
posterior of stage 9-10 oocytes (Fig.
3D). Within the nurse cells, Exu-GFP forms particles that move
through the ring canals into the oocyte
(Theurkauf and Hazelrigg,
1998
). To examine whether one of these properties of Exu is
phosphorylation dependent, we expressed a mutant Exu-GFP, in which all serines
in site A and B are mutated in ovaries of exu null mutant females.
First, we confirmed by western analysis that the mutant Exu-GFP is not
phosphorylated (Fig. 4E, A6
B3-GFP). Next, we analysed the distribution of mutant Exu-GFP protein in egg
chambers and found that neither its subcellular localisation in the oocyte nor
in the nurse cells is affected (Fig.
3C,E). Consistent with the finding that the overall distribution
of the protein is not altered, we could not detect any differences in the
mobility of unphosphorylated and phosphorylated Exu-GFP particles (data not
shown). Finally, we tested if Exu phosphorylation affects the ability of the
protein to colocalise with bicoid mRNA in the nurse cells. Notably,
bicoid mRNA is still enriched in the Exu-GFP containing apical
patches in the nurse cells (Fig.
3C). In conclusion, our Exu-GFP analysis suggests that
phosphorylation of Exu is neither required for its localisation, nor its
mobility, nor its ability to colocalise with bicoid mRNA.
Exu phosphorylation is required for anterior patterning of the embryo
A Bicoid protein gradient controls the hierarchy of segmentation genes at
beginning of embryogenesis (Driever,
1993). The mislocalisation of bicoid mRNA in embryos from
exu females disturbs this gradient, resulting in head defects
(Schüpbach and Wieschaus,
1986
; Frohnhöfer and
Nüsslein-Volhard, 1987
). To test whether phosphorylation of
Exu is required for anterior patterning of the embryo, we first attempted to
rescue the head defects of exu null mutant embryos using the
exu
A+B transgene, which
expresses unphosphorylated Exu. More than one-third of the embryos produced by
exuVL/exuXL;
exu
A+B females develop head defects
typical of exu mutants and fail to hatch (data not shown), indicating
that the Bicoid protein gradient is affected when Exu is unphosphorylated.
Next, we directly analysed the Bicoid gradient in embryos produced by
exu
A+B mothers, using an
anti-Bicoid antibody. We could detect no Bicoid gradient in approximately
one-third of the embryos (Fig.
5E). In the rest of the embryos, a gradient of Bicoid protein was
detected, however, its extent appears reduced compared with the wild type
(Fig. 5E). These results
suggest that the head defects observed in one third of the embryos produced by
exu
A+B mothers are caused by
the absence of a Bicoid gradient in the embryo at the beginning of
embryogenesis. The fact that two third of the embryos hatch indicates that, in
those embryos, the Bicoid gradient is sufficiently strong to support anterior
patterning.
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Different mechanisms mediate bicoid mRNA localisation during mid- and late oogenesis
Although exu-null and the phosphorylation defective
exuA+B mutants abolish
bicoid mRNA localisation during mid-oogenesis, the two mutants differ
in the strength of their embryonic patterning defects. Although we never
observed Bicoid protein at the anterior of the embryos from exu null
mutants, 73% of the progeny of the
exu
A+B mutants form a Bicoid
protein gradient (Fig. 5).
Though the extension of this gradient is reduced compared with the wild type,
its existence in a large proportion of the embryos reveals that
unphosphorylated Exu retains some activity. The fact that exu null
and exu
A+B mutants show
identical phenotypes until stage 10b suggests that the residual activity of
unphosphorylated Exu protein is required only during the later stages. We
therefore compared bicoid mRNA localisation in the two mutants after
mid-oogenesis. During late oogenesis,
exu
A+B oocytes start to
accumulate bicoid mRNA at the anterior cortex
(Fig. 7C), while in
exu null mutants only traces of bicoid mRNA are anteriorly
localised, and most of the mRNA is either uniformly distributed in the ooplasm
or cortically localised (Fig.
7B). The partial recovery in the
exu
A+B mutants shows that
bicoid mRNA localisation is less phosphorylation dependent during
late oogenesis. We conclude that during mid-oogenesis, bicoid mRNA is
localised by a mechanism that is strictly dependent on Exu phosphorylation,
and that during late oogenesis the mode of bicoid mRNA localisation
changes to a mechanism that is still dependent on Exu but less dependent on
its phosphorylation by Par-1. The fact that the amount of bicoid mRNA
that is localised during late oogenesis in the
exu
A+B mutants is sufficient for the
formation of a Bicoid protein gradient in 73% of the embryos shows that
mutants, in which localisation is abolished during the early stages of
bicoid mRNA localisation can recover in late oogenesis. Therefore,
the two mechanisms that mediate bicoid mRNA localisation during mid-
and late oogenesis are redundant, and defects that occur during mid-oogenesis
can be compensated in late oogenesis.
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Discussion |
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A role for Exu phosphorylation in recruiting anterior targeting factors to bicoid mRNA
Exu protein is an essential mediator of bicoid mRNA localisation.
In this study we have shown that Par-1 kinase phosphorylates Exu, and that
this phosphorylation is necessary for anterior localisation of bicoid
mRNA during mid-oogenesis. We have also shown that Exu phosphorylation does
not affect Exu localisation, its ability to form mobile particles, or its
colocalisation with bicoid mRNA. How then might Par-1 phosphorylation
enable Exu to mediate bicoid mRNA localisation? Experiments in which
fluorescently labelled bicoid mRNA was microinjected into living egg
chambers have revealed that Exu is required in the nurse cells for anterior
localisation of bicoid mRNA within the oocyte. These experiments have
led to a model whereby Exu associates in the nurse cells with bicoid
mRNA and mediates the recruitment of additional nurse cell factors required
for targeting of bicoid mRNA to the anterior of the oocyte
(Cha et al., 2001). Our finding
that mutation of Exu phosphorylation sites results in a phenotype that is,
during mid-oogenesis, indistinguishable from that of exu-null mutants
suggest that Exu phosphorylation is involved in the recruitment of these
anterior-targeting factors in the nurse cells. Phosphorylation might increase
the binding affinity of Exu for these nurse cell factors, promoting their
association with bicoid mRNA. The colocalisation of Exu-GFP, Par-1
and bicoid mRNA in patches in the nurse cells suggests that this is
where the bicoid RNP complexes assemble.
Cortical bicoid mRNA localisation precedes its anterior transport in the oocyte
The consequences of exu and par-1 mutations on
bicoid mRNA localisation are distinct. Although loss of exu
function results in diffuse bicoid mRNA distribution in the ooplasm,
a reduction in par-1 function causes cortical localisation of the
mRNA. We have generated an Exu protein that localises bicoid mRNA
independent of phosphorylation by Par-1 and rescues exu mutants, but
that is unable to rescue bicoid mRNA localisation in par-1
mutants. Therefore, the cortical mislocalisation of bicoid mRNA in
par-1 mutant oocytes is independent of Exu function. What might be
the other function of Par-1 in localisation of bicoid mRNA? The fact
that bicoid localisation requires the microtubule cytoskeleton
(Pokrywka and Stephenson,
1991), together with the report that oocyte microtubules are
improperly polarised in par-1 mutants
(Shulman et al., 2000
),
suggests that cortical localisation in the mutants is caused by a microtubule
defect. It has been proposed that microtubules of different qualities may
nucleate from different regions of the oocyte cortex
(Cha et al., 2001
). A simple
explanation for the aberrant localisation of bicoid mRNA in
par-1 oocytes would be that the subset of microtubules nucleating
from the anterior corners of the oocyte and serving as tracks for anterior
transport of bicoid mRNA are not restricted to the anterior corners,
but spread along the cortex, resulting in the lateral cortical localisation of
bicoid mRNA. However, this model is not supported by our genetic
epistasis experiments, which indicate that the exu independent
function of par-1 acts at a step upstream of exu in
bicoid mRNA localisation. Therefore, we favour a different model, in
which in wild-type oocytes bicoid mRNA first localises cortically
preceding its targeted transport along microtubules. In this model, most of
the bicoid mRNA entering the oocyte moves in a nonpolar fashion,
either passively or by active transport, to the oocyte cortex. Only after this
cortical localisation does the targeted transport of bicoid mRNA to
the anterior corners of the oocyte commence. In par-1 mutants, the
improperly organised microtubule cytoskeleton prevents release of the mRNA
from the cortex to the (anterior-targeting) microtubules and the mRNA remains
cortically localised. In exu mutants, the polarity of the
microtubules is normal and bicoid mRNA is released from the cortex.
However, its targeted transport to the anterior is impaired and the mRNA is
diffusely distributed in the ooplasm.
A trapping mechanism for bicoid mRNA localisation in late oogenesis
The requirement for Exu phosphorylation in bicoid mRNA
localisation decreases during the later stages of oogenesis. This is revealed
by the partial recovery of bicoid mRNA localisation in exu
mutants that abolish phosphorylation. These mutants are indistinguishable from
exu-null mutants through stage 10b of oogenesis, but during early
embryogenesis two-thirds of the mutants localise enough bicoid mRNA
at the anterior to support formation of a Bicoid protein. This indicates that
the mechanism of bicoid mRNA localisation changes after stage 10b of
oogenesis, from an early phase that is strictly dependent on Exu
phosphorylation, to a late phase that is less dependent on phosphorylation.
Stage 10b is the stage at which ooplasmic streaming commences, providing a
possible mechanism for localisation of bicoid mRNA in mutants in
which Exu phosphorylation cannot occur. Before stage 10b, anterior targeting
of bicoid mRNA could be mediated solely by directed transport of
bicoid mRNA complexes along microtubules, a process that is strictly
dependent on Exu phosphorylation. After stage 10b, this directed transport
might be complemented or replaced by a passive trapping mechanism, which has
also been postulated for the localisation of oskar and nanos
mRNAs during late oogenesis (Glotzer et
al., 1997; Forrest and Gavis,
2003
). This mechanism relies on the movements generated by
ooplasmic streaming, which could bring bicoid mRNA complexes into
contact with the anterior cortex of the oocyte, where the mRNA could be
trapped by localised anchoring molecules. This change in the mechanism of
bicoid mRNA localisation would occur at the time of assembly of the
anterior MTOC that is essential in the late phase of bicoid mRNA
localisation (Schnorrer et al.,
2002
), suggesting that the MTOC might be involved in the trapping
mechanism. Such a trapping mechanism would be differentially affected in
Exu-null mutants and in mutants that specifically abolish Exu phosphorylation.
It is possible that Exu provides bicoid mRNA not only with factors
required for anterior targeting, but also with factors required for anchoring
of bicoid mRNA. Unphosphorylated Exu might be inactive in recruiting
the factors for anterior targeting, but be competent for binding of factors
required for anchoring.
A model for bicoid mRNA localisation
In summary, our data supplement the models for bicoid mRNA
localisation previously presented by Cha et al.
(Cha et al., 2001) and
Schnorrer et al. (Schnorrer et al.,
2002
) in the following way: in the first phase of bicoid
mRNA localisation, the mRNA is transported to the anterior corners of the
oocyte, resulting in a ring-like distribution. This targeted transport
requires the formation of RNP complexes that contain bicoid mRNA and
specific anterior-targeting factors that allow the RNPs to identify those
microtubules that nucleate from the anterior corners of the oocyte. Assembly
of this complex takes place in the nurse cells and requires the
phosphorylation of Exu by Par-1. Upon entry of the complex into the oocyte, a
specific proportion of the RNP complexes encounter the microtubules that
nucleate from the anterior corners, and these complexes are directly
transported to their final destination. However, a large proportion of the
complexes does not find these microtubules directly, and moves first to the
oocyte cortex. The transfer of these cortically localised complexes to
microtubules nucleating from the anterior corners solely requires a properly
polarised microtubule network. Only at this stage can the nurse cell factors
assembled on the mRNA act to transport the cortically localised complexes to
the anterior corners of the oocyte. During the second phase of bicoid
mRNA localisation, the ring-shaped distribution changes to a disc-shaped
distribution and a MTOC forms at the anterior of the oocyte. The third phase
of bicoid mRNA localisation begins after the onset of ooplasmic
streaming. In this late phase, the mechanism of bicoid mRNA
localisation changes from targeted transport to passive trapping, mediated by
ooplasmic streaming, and the mRNA is anchored at the anterior margin. The
generation of exu mutants that abolish phosphorylation allows us to
distinguish between the early and the late mechanisms of bicoid mRNA
localisation, as the two mechanisms differ in their sensitivity to Exu
phosphorylation.
Par-1 establishes polarity in the oocyte by different mechanisms
We have previously shown that Par-1 controls posterior patterning by
phosphorylating Oskar (Riechmann et al.,
2002). Here, we have shown that Par-1 regulates anterior
patterning by phosphorylating Exu. Although Oskar is an intrinsically unstable
protein whose stability is increased by Par-1 phosphorylation, Par-1
phosphorylation does not affect Exu stability
(Fig. 1B,
Fig. 3C,E; data not shown) but
does affect its ability to mediate bicoid mRNA localisation. Thus,
Par-1 uses at least two different mechanisms to generate polarity within the
same cell. Interestingly, these two Par-1 substrates, Oskar and Exu, are
unique to Diptera, showing that during evolution Par-1 gained fly-specific
mediators of cell polarisation as substrates. Par-1 is therefore flexible in
the mechanisms and in the targets by which it mediates cell polarisation. This
is in striking contrast to the PDZ-containing proteins Par-3 and Par-6, which
appear to establish polarity by the assembly of a conserved protein
complex.
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ACKNOWLEDGMENTS |
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Footnotes |
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