Department of Molecular Biology, Princeton University, Princeton, NJ
08544, USA
*
These authors contributed equally to this work
Present address: Department of Biology, DCMB Program, Duke University, Durham,
NC 27708, USA
Accepted 25 May 2001
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SUMMARY |
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Key words: Orb, Drosophila, DER signaling pathway, gurken, mRNA localization
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INTRODUCTION |
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The establishment of the dorsoventral (DV) axis of the Drosophila
egg and embryo also depends upon mRNA localization (see Nilson and Schupbach,
1999; Shulman and St Johnston,
1999
for reviews). Around
stage 7 of oogenesis, the microtubule network is reorganized, and the oocyte
nucleus moves from the posterior of the oocyte to the dorsal-anterior corner
(Theurkauf, 1994
). Shortly
thereafter, K(10) and squid (sqd) begin
concentrating gurken (grk) mRNA, which encodes a
transforming growth factor
(TGF
) homolog, in a cap just above
the oocyte nucleus (Neuman-Silberberg and Schupbach,
1993
; Gonzales-Reyes et al.,
1995; Serano et al., 1995
;
Saunders and Cohen, 1999
).
Translation of grk mRNA in stage 9 egg chambers results in the
localized production of Grk protein. Grk is secreted through the oocyte plasma
membrane and it signals dorsal fate to the overlying follicle cell epithelium
by interacting with the Drosophila epidermal growth factor receptor
(DER; Egfr FlyBase) (Price et al.,
1989
; Schejter and Shilo,
1989
). Mutations in either
grk or DER disrupt the DV signaling pathway, leading to the
production of eggs with a ventralized chorion that either lack or have fused
dorsal appendages. The mis-specification of follicle cell identity during
oogenesis also disrupts embryonic development (Roth and Schupbach,
1994
). Although the loss of
grk activity in the developing oocyte or DER in the follicle
cells results in the ventralization of the egg shell and embryo, mutations in
K(10) and sqd have the opposite effect on DV polarity,
giving a gain-of-function dorsalization of the egg chamber (Kelly,
1993
; Neuman-Silberberg and
Schupbach, 1993
; Serano et
al., 1995
). The dorsalized
phenotype arises because grk mRNA is distributed all along the
anterior margin of the oocyte. Translation of the mislocalized mRNA results in
the secretion of Grk protein around the entire circumference of the oocyte,
and it signals dorsal fate to follicle cells on both the dorsal and ventral
sides.
Another gene required for the establishment of polarity in the egg and
embryo is oo18 RNA binding (orb). orb functions at
multiple steps during oogenesis (Lantz et al.,
1994; Christerson and
McKearin, 1994
). In the
presumed null mutant, orb343, oogenesis arrests just
before the formation of the 16-cell cyst, and neither oocyte nor nurse cell
fates are determined. The orb303 mutation is slightly less
severe than orb343, and it arrests oogenesis after the
16-cell cyst is formed. There is also a much weaker hypomorphic allele,
orbmel, which was generated by the imprecise excision of a
P-element inserted into 5' UTR sequences in the second female-specific
exon (Christerson and McKearin,
1994
). Unlike the stronger
alleles, oogenesis is not irreversibly blocked in orbmel,
and homozygous mutant females lay eggs. Many of the eggs produced by
orbmel females have ventralized or lateralized chorions
that are indicative of a failure in the grk-DER signaling pathway
(Christerson and McKearin,
1994
; Roth and Schupbach,
1994
). When fertilized, the
embryos from orbmel mothers often show developmental
abnormalities expected from defects not only in DV patterning but also in
posterior patterning.
The morphological abnormalities in orbmel egg shells
and embryos can be attributed to defects in the execution of the mRNA
localization pathways responsible for axes determination. In the case of the
posterior pathway, the target for Orb function appears to be osk
mRNA. Instead of being localized in a cap at the posterior pole, osk
mRNA is distributed throughout much of the oocyte in stages 8-10
orbmel chambers (Christerson and McKearin,
1994). As Orb protein is
predicted to have RNA-binding activity (the C-terminal half of the
100
kDa female Orb protein has two 90 amino acid RNA recognition motif (RRM)
domains and a short 60 amino acid cysteine- and histidine-rich region that
resembles a zinc finger; Lantz et al.,
1992
) it might be expected to
play a direct role in the osk mRNA localization pathway. Indeed,
osk mRNA is found in an immunoprecipitable complex with Orb protein
in vivo (Chang et al., 1999
).
Although one function may be to help anchor localized mRNAs such as
osk, Orb also appears to play a central role in controlling the
translation of localized mRNAs. Orb is a member of the cytoplasmic
polyadenylation element binding protein (CPEB) family of translation
regulators (Hake and Richter,
1994
). During oogenesis, CPEB
proteins bind to the 3' UTRs of masked maternal mRNAs and control their
translation. Initially this interaction is thought to help repress the
translation of the masked mRNAs; however, at egg maturation, the CPEB proteins
activate translation by promoting polyadenylation (Fox et al.,
1989
; Paris and Richter,
1990
; Paris et al.,
1991
; Hake and Richter,
1994
; Sheets et al.,
1994
; Barkoff et al.,
1998
; Minshall et al.,
1999
). Although a role in
repressing osk translation has not yet been demonstrated, Orb is
required for the translation of osk mRNA that has been transported to
the posterior pole (Markussen et al.,
1995
; Chang et al.,
1999
). In addition to
interacting with and promoting the translation of osk mRNA, Orb
autoregulates its own expression by localizing orb mRNA in the oocyte
and then activating the translation of the localized message. Both
localization and translational regulation seem to be mediated through
sequences in the orb mRNA 3' UTR (Tan et al.,
2001
).
orb is also required for mRNA localization in the DV polarity
pathway. Previous studies have shown that the localization of mRNA for two
genes in this pathway, K(10) and grk, is affected in
orb mutant ovaries (Christerson and McKearin,
1994; Lantz et al.,
1994
; Roth and Schupbach,
1994
). The distribution of
grk mRNA in vitellogenic orbmel chambers shows
similarities to that seen in K(10) and sqd mutant ovaries:
instead of being concentrated in a crescent at the dorsal anterior corner of
the oocyte, grk mRNA is distributed around the anterior margin of the
oocyte. (While the mislocalized grk mRNA in K(10) and
sqd mutants remains tightly associated with the oocyte cortex, giving
a ring in cross sections, grk mRNA in orbmel egg
chambers is more diffusely distributed.) The fact that the distribution of
grk mRNA in orbmel is similar to that in
K(10) or sqd is surprising as the DV defects in
orbmel more closely resemble those in grk or
DER mutants. In the studies reported here, we have examined the role
of the orb gene in this grk-DER signaling pathway, as well
as earlier in oogenesis, when the grk-DER pathway functions in
anteroposterior (AP) polarity.
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MATERIALS AND METHODS |
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Immunoprecipitations and PCR assays |
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Immunocytochemistry and western analysis
Ovaries were dissected in phosphate-buffered saline (PBS), fixed and
stained for Orb, Grk and K(10) proteins (essentially as described by Lantz et
al., 1994). The Orb antibody
is a mouse monoclonal, while the Grk and K(10) antibodies are from rats.
Imaging was by laser scanning confocal microscopy (Krypton-Argon Laser, BioRad
MRC 600 or a Zeiss Confocal). Western analysis was as described previously
(Lantz et al., 1994
).
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RESULTS |
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While high levels of Grk are found concentrated in a cap above the oocyte
nucleus in wild-type chambers, there are severe abnormalities in Grk
expression in most orbmel chambers (see
Fig. 1). In many of the mutant
chambers, there is no detectable Grk protein. In other chambers, a small
amount of Grk can be seen in the region just above the oocyte nucleus;
however, the level of Grk protein is greatly reduced compared with that seen
in wild-type chambers. These findings indicate that orb is required
in the DV signaling pathway for both the localization of grk mRNA and
the proper expression of Grk. Similar results have been reported elsewhere
(Neuman-Silberberg and Schupbach,
1996).
orb is required for grk expression at the posterior
of the oocyte in pre-vitellogenic stages
In addition to its role in DV polarity, the grk-DER signaling
pathway also functions earliemr in oogenesis in the establishment of A-P
polarity (Gonzales-Reyes et al., 1995; Roth et al.,
1995). This process begins in
region 3 of the germarium, which contains incompletely budded stage 1
chambers, and continues during the pre-vitellogenic stages. Grk protein
translated from grk mRNA localized at the posterior of the oocyte
signals the posterior-most follicle cells, specifying posterior identity (see
Fig. 2). As high levels of Orb
are concentrated in the posterior cortical cytoplasm of the oocyte in stage 1
and older pre-vitellogenic chambers, it seemed possible that orb
might also function in the AP grk-DER signaling pathway.
|
The pre-vitellogenic stages of oogenesis in the orbmel
mutant appear to be completely normal, and the pattern of Orb protein
accumulation before stages 7-8 is indistinguishable from wild type. For this
reason, we did not expect, nor did we observe, any abnormalities in Grk
expression during the early stages of oogenesis in orbmel
ovaries (not shown; Chang et al.,
1999). However, defects in
early Grk expression are seen in the two stronger mutants
orb303 and orb303.
In the presumed protein null, orb343 (Lantz et al.,
1994), little or no Grk can be
detected (Fig. 2). A different
result is obtained for orb303. orb303
expresses only one of the two female Orb isoforms and this protein exhibits an
abnormal pattern of accumulation (Lantz et al.,
1994
). In wild-type ovaries,
Orb protein begins concentrating in the presumptive oocyte in region 2 of the
germarium, which contains newly formed 16-cell cysts. Unlike wild type, the
mutant Orb303 protein does not properly localize to the presumptive
oocyte and instead is distributed at abnormally high levels in all 16 germ
cells (Fig. 2). The level of
Orb303 protein remains elevated when the aberrant cysts exit the
germarium; however, as these pseudo-egg chambers `mature', the mutant protein
begins to disappear. As can be seen in Fig.
2, Grk expression is prematurely induced in
orb303 ovaries. Newly formed 16-cell cysts in region 2 of
the germarium not only have elevated amounts of the mutant Orb protein but
also have abnormally high levels of Grk. Moreover, instead of being localized
in only a single germ cell (the presumptive oocyte), as in wild type, Grk is
distributed throughout the cyst and can also be detected in the surrounding
somatic tissue. This can be seen most readily in pseudo-egg chambers that have
just budded off from the germarium. As Grk is thought to help polarize the
early egg chamber, the uniform distribution of Grk protein in these early
mutant cysts could help explain why the germ cells in mutant cysts do not
undergo the rearrangements that position the oocyte at the posterior. In older
pseudo-egg chambers that have little Orb protein, Grk also disappears. These
findings indicate that wild-type orb function is required for the
localized expression of Grk protein during early oogenesis.
Orb is required for K(10) protein expression
In wild-type stage 8-10 egg chambers, K(10) mRNA is concentrated
along the anterior margin of the oocyte. While K(10) mRNA also
accumulates along the anterior margin of orbmel
vitellogenic chambers, it is not as tightly concentrated as in wild type and
spreads towards the center of the oocyte. To test whether orb is also
required for the expression of K(10) protein, we stained wild-type and
orbmel ovaries with antibodies against K(10) and Orb
proteins (Fig. 3). In wild-type
ovaries, K(10) protein can first be detected in the oocyte in previtellogenic
stages. By the time Grk is first expressed at the dorsal anterior corner of
the oocyte in stage 8-9 chambers, high levels of K(10) are found concentrated
in the oocyte nucleus. The pattern of K(10) protein expression in
previtellogenic orbmel chambers appears to be normal. In
contrast, in most stage 8-10 orbmel chambers, little or
only low levels of K(10) are observed in the oocyte nucleus
(Fig. 3). We also examined
K(10) expression in orb303 ovaries. Low levels of K(10)
could be detected in cysts that had high levels of Orb303 protein
(not shown). These findings indicate that orb is required for the
proper expression of K(10) protein.
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As shown in Fig. 4,
K(10) mRNA seems to be associated with Orb protein in ovary extracts.
The expected smear of K(10) 3' UTR-specific amplification products is
observed in total RNA and in the Orb immunoprecipitate, while these
amplification products are absent in the Dorsal immunoprecipitate.
Unexpectedly, the results for grk were the same as for nos;
we were unable to detect 3' UTR sequences from grk mRNA in the
Orb immunoprecipitates (see Fig.
3). One possible explanation for this result could be that the
bound mRNAs are partially hydrolyzed during the immunoprecipitation procedure.
For example, in the case of 5 kb orb mRNA, we found sequences
from the
1.0 kb 3' UTR in Orb immunoprecipitates, while sequences
upstream of the 3' UTR in the protein-coding region were not detected
(Tan et al., 2001
). Though
grk mRNA is only about
1.5 kb in length, it is possible that we
failed to detect grk in the immunoprecipitates because Orb complexes are
associated with sequences close to the 5' end of grk mRNA
rather than with the 3' UTR. To test this possibility, we reverse
transcribed and PCR amplified with primers complementary to sequences in the
grk 5' UTR. While appropriate amplification products were
observed in the total RNA control, we did not detect amplification products in
the Orb immunoprecipitates (data not shown).
Is orbmel epistatic to K(10)?
If the reduction in K(10) protein expression were the only effect of the
orbmel mutation on the grk-DER DV signaling
pathway, it would be expected to result in dorsalized egg chambers. However,
the fact that orbmel produces ventralized rather than
dorsalized eggs can be explained by the finding that Grk protein expression is
also substantially reduced in orbmel vitellogenic
chambers. As grk mRNA is mislocalized in a K(10)-like
pattern in orbmel egg chambers, it would be reasonable to
suppose that the orb-dependent activation of Grk protein expression
is downstream of the K(10)-dependent step in the grk-DER
pathway. In this case, orbmel should be epistatic to
K(10) that is, the double mutant should have essentially the
same defects in DV polarity as observed in orbmel alone.
To test this prediction, we generated females homozygous for both the
orbmel and K(10) mutations, and examined their
eggs. The results of this analysis are presented in
Table 1. Contrary to our
expectations, the vast majority of the eggs produced by the double mutant
females have the dorsalized phenotype of K(10), rather than the
ventralized phenotype of orbmel.
|
Grk protein is upregulated in the K(10); orbmel
double mutant
If K(10) is epistatic to orbmel, then the
pattern of Grk protein expression in the double mutant ovaries should resemble
that observed in K(10) not orbmel mutant ovaries.
In the experiment shown in Fig.
5, we stained K(10), orbmel and K(10);
orbmel ovaries with antibodies against Grk protein. We also
monitored Orb protein expression in these different genetic backgrounds. The
K(10) mutant differs from wild type in that Grk protein is expressed
all along the anterior margin of the oocyte in stage 8-10 chambers, instead of
being restricted to the dorsal anterior corner. As expected from the egg shell
phenotype of the double mutant, we found that Grk protein expression in
K(10);orbmel egg chambers resembles that seen in
K(10) not that of orbmel: high levels of protein
are observed all along the anterior margin of the oocyte (see
Fig. 5).
Orb protein expression in orbmel is upregulated
by the K(10) mutation
These findings indicate that the K(10) mutation suppresses the
orbmel defect in the translation of grk mRNA, and
consequently Grk protein expression. A possible mechanism is suggested by a
comparison of the Orb expression pattern in K(10); orbmel
and orbmel mutant ovaries. While little Orb is detected in
most vitellogenic orbmel chambers, the level of Orb
protein in stage 7-10 K(10);orbmel chambers is close to
that seen in wild type (compare Orb antibody staining in the different
chambers shown in Fig. 5). As
the defect in grk mRNA translation in orbmel is
thought to be a consequence of the greatly reduced levels of Orb protein in
vitellogenic mutant chambers, it is reasonable to think that the increase seen
in the double mutant would be sufficient to restore Grk expression.
To provide further evidence that the level of Orb protein accumulation is close to that of wild type in the double mutant, we probed western blots of ovary extracts. The results are shown in Fig. 6. Whereas the amount of Orb protein in orbmel ovary extracts is greatly diminished (estimated to be about 1/20th that in wild type), the level of protein in the double mutant is much closer to wild type (estimated to be about 1/3rd the level in wild type). The increased accumulation of Orb protein seen in both whole mounts and western blots is consistent with the idea that K(10) negatively regulates Orb expression.
|
We also examined Orb protein expression in K(10) mutant ovaries that are wild type for the orb gene. However, we were unable to detect any obvious difference in the amount of protein. As Orb is already present in substantial quantities in wild-type chambers, there could be other, K(10)-independent mechanisms that limit accumulation above a certain level. Alternatively, it is possible that the effects of the K(10) mutation on Orb protein expression are specific for the orbmel allele. The orb mRNA expressed by orbmel lacks sequences from the 5' UTR and this might make this message especially sensitive to the repressive activity of the K(10) protein.
To further test the effects of K(10) on orb, we took
advantage of the dominant negative activity of a transgene, hsp83:Lac-Z
orb 3' UTR, which constitutively expresses a transcript that
contains lacZ-coding sequences fused to the orb 3'
UTR. Previous studies (Tan et al.,
2001) have shown that this
transgene RNA interferes with orb autoregulation by competing with
the endogenous mRNA for orb function. Because Orb activity is
required for its own synthesis, this competition downregulates Orb expression.
The downregulation of Orb protein disrupts the grk-DER signaling
pathway, giving ventralized eggs. A single copy of the transgene has only very
modest phenotypic effects, inducing DV polarity defects in a few percent of
the eggs. However, when the hsp83:lacZ orb 3' UTR transgene is
introduced into females heterozygous for orb343, about 20%
of the eggs have DV defects. If K(10) functions as a negative
regulator of the wild-type orb gene, then a K(10) mutation
might act as a suppressor, reducing the frequency of DV defects in eggs from
hsp83:lacZ orb 3' UTR, orb343/+ females. As
can be seen in the second part of Table
1, K(10) is a weak suppressor. When hsp83:lacZ
orb 3' UTR, orb343/+ females have only a single
wild type K(10) gene, the frequency of DV defects is reduced.
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DISCUSSION |
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How does orb function in regulating translation and localization?
Orb homologs in other organisms, the CPEB proteins, interact with elements in
the 3' UTRs of masked mRNAs, and activate their translation by a
mechanism that is thought to involve polyA addition (see Richter,
1999 for a review). As the
translational function of the CPEB proteins is conserved in animals as diverse
as clams and mice, it would be reasonable to suppose that the role of the
orb gene in the Drosophila grk-DER signaling pathway also
involves translational activation. Accordingly, the defects in the expression
of both Grk and K(10) proteins would arise because wild type orb
activity is required to properly regulate the translation of grk and
K(10) mRNAs. In the case of K(10), it seems possible that
Orb protein might act directly on the mRNA. First, K(10) mRNA is
associated with Orb protein in an immunoprecipitable complex (see above) and
second, K(10) mRNA is mislocalized in orb mutant ovaries
(Christerson and McKearin,
1994
; Lantz et al.,
1994
). For grk, the
situation appears to be more complicated and will be considered further.
As translational activation by CPEB proteins in other systems has been tied
to polyadenylation, an obvious question is whether the polyA tails of
K(10) mRNA are affected in orb mutants. Unfortunately,
experiments aimed at testing this point have been inconclusive. Using the
anchored-dT RT-PCR procedure of Salles et al. (Salles et al.,
1994), we found that
K(10) mRNA isolated from the strong loss-of-function orb
mutant, orb343, had shorter poly(A) tails than wild type
(J. S. C., unpublished). However, we cannot excluded the possibility that the
short poly (A) tails in this mutant arise because K(10) mRNA is
targeted for deadenylation in the absence of translation. For
orbmel, the average poly(A) length appeared, at most, to
be only marginally shorter than wild type. Of course, as K(10) protein is
expressed normally in pre-vitellogenic stages in this mutant, the presence of
mRNAs with extended poly(A) tails is not altogether surprising. Further
studies will be required to determine whether the mechanism used to promote
the translation of K(10) mRNA depends upon polyA addition as is
thought to be the case in other organisms.
In contrast to K(10), grk mRNA was not found in Orb
immunoprecipitates. Although there are many reasons why an Orb
protein:grk mRNA complex might not be detected, this result forces us
to consider the possibility that orb acts on grk only
indirectly. In this case, we would have to propose other mechanisms to account
for the defects in both the localization and translation of grk mRNA
that are observed in orb mutants. It seems possible that the
mislocalization of grk mRNA in orbmel could
arise, at least in part, because the expression of K(10) protein is greatly
reduced in stage 8-10 orbmel chambers. However, as the
localization defects in orbmel are more severe than those
seen in K(10), orb may regulate some other factor in addition to
K(10) that helps direct the proper localization of grk mRNA. An
obvious candidate is sqd. Although we did not detect any alterations
in Sqd protein expression in orbmel chambers, it should be
noted that only one of the three Sqd isoforms seems to be involved in
grk mRNA localization (Norvell et al.,
1999). Consequently, any
effects on the expression of this specific isoform could be obscured by the
other isoforms.
We must also explain why grk mRNA is not properly translated in
orb mutant ovaries. Orb protein could be required for the expression
of factors that activate translation of grk mRNA. In
orb303 this factor(s) could be prematurely produced
throughout the cyst, leading to the very high levels of unlocalized Grk seen
in this mutant. As K(10) and sqd do not seem to function in
the localization or translation of grk mRNA at the posterior of the
oocyte in pre-vitellogenic stages, the orb regulatory target(s) early
in oogenesis could be different from that used later in DV signaling. Another
possibility is that orb regulates the expression of a signal(s) that
coordinates the activation of grk mRNA translation with other events
in oogenesis. This function is suggested by the fact that CPEB activity in
other organisms helps govern progression through oogenesis (Sheets et al.,
1995; De Moor and Richter
1999
; Barkoff et al.,
2000
; Groisman et al.,
2000
) and by the finding that
grk expression in the DV pathway is sensitive to check points that
monitor progression through meiosis (Gonzalez-Reyes et al.,
1997
; Ghabrial and Schupbach,
1999
). In this case, signals
crucial for translation of grk mRNA might not be produced in the
absence of orb activity.
Role of K(10) in Orb protein expression
The epsitatic relationship between orbmel and
K(10) is rather surprising. As orb is required for the
localization and translation of grk mRNA, we expected that
orbmel would be epistatic to K(10). However,
contrary to this expectation, eggs produced by
K(10);orbmel double mutant females have the dorsalized egg
shell phenotype that is characteristic of K(10) mutations, rather
than the ventralized phenotype of orbmel. This result
implies that the loss of K(10) function rescues the
orbmel defect in grk mRNA translation (but not
the localization defect). Interestingly, a similar epistatic relationship is
found for K(10) and mutations in the spindle (spn) genes
(Gonzalez-Reyes et al., 1997).
Mutants in the spn genes resemble orb in that grk
mRNA is mislocalized in a K(10)-like pattern but is not properly
translated, giving ventralized eggs. Moreover, the defects in grk
mRNA translation in spn mutants can also be rescued by mutations in
K(10) and double mutant females produce dorsalized eggs. To explain
these findings, Gonzalez et al. have postulated that the function of the
spn genes is to alleviate K(10)-dependent repression of
grk mRNA translation (Gonzalez-Reyes et al.,
1997
).
Although orb could have a similar role in alleviating K(10)-dependent repression of grk, an alternative (or additional) explanation for the epistatic relationship between orbmel and K(10) is that K(10) negatively regulates Orb protein expression. This possibility is suggested by the finding that the amount of Orb protein in vitellogenic chambers from the double mutant is close to that seen at equivalent stages in wild-type ovaries. The restoration of near wild-type levels of Orb protein in these orbmel;K(10) chambers would in turn be expected to produce a concomitant increase in Grk expression, giving the observed gain-of-function phenotype.
Complicating our conclusion that K(10) negatively regulates Orb expression is the finding that K(10) protein does not properly accumulate in the oocyte nucleus of vitellogenic orbmel chambers. One might have expected that this reduction in the level of K(10) protein would alleviate the K(10)-dependent repression of Orb protein expression, leading to an increased accumulation of Orb protein in the orbmel mutant and a dorsalized (not ventralized) DV phenotype. However, it does not. One explanation for this paradox is that orbmel is wild type for K(10), whereas this is not the case in the double mutant. In addition, there are no apparent defects in K(10) expression in pre-vitellogenic orbmel chambers. It is possible that there is sufficient residual K(10) protein remaining at later stages to effectively repress orb (see examples in Fig. 3), or that K(10) repression of orb is linked to a process that occurs before the time when the accumulation of K(10) protein drops below some critical threshold value in the orbmel chambers. In this context, it is interesting to note that the most severe defects in both orb mRNA localization and Orb protein expression in orbmel occur after the reorganization of the cytoskeleton and the concomitant movement of the oocyte nucleus from the posterior to the anterior of the oocyte. This marks a shift in the localization of orb mRNA and the site of Orb protein synthesis from the posterior of the oocyte to the anterior. As the expression of K(10) protein before this time is normal in orbmel ovaries, its possible that K(10) repression may be somehow linked to this spatial transition in orb regulation.
Although the K(10) mutation had quite dramatic effects on Orb expression in orbmel ovaries, there were no obvious changes in Orb expression in K(10) mutant ovaries that are wild type for orb. It seems possible that there may be some special features of the orbmel mutation that make it especially sensitive to K(10) repression. However, our genetic interaction experiments suggest that K(10) also negatively regulates expression of the wild-type orb gene. An important unanswered question is the mechanism of regulation. Here, there is a problem of compartmentalization. For example, as orb mRNA is thought to be synthesized in nurse cells, K(10) protein is unlikely to influence transcription. Even effects on the localization/translation of orb mRNA must be indirect. Further studies will clearly be required to understand how K(10) regulates orb expression.
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ACKNOWLEDGMENTS |
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