Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027, USA
* Author for correspondence (e-mail: ddk1{at}colombia.edu)
Accepted 11 October 2005
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SUMMARY |
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Key words: Drosophila, Oogenesis, Polarity, Microtubules, Squid, PKA, hnRNP
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Introduction |
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The Drosophila oocyte develops within an egg chamber, which
consists of 15 nurse cells and an oocyte surrounded by an epithelial monolayer
of somatic follicle cells (Spradling,
1993). The oocyte and nurse cells are produced from a single
germline cystoblast, which arises when a germline stem cell divides. The
cystoblast undergoes four synchronous mitotic divisions to produce a germline
cyst of 16 cystocytes; each division ends with incomplete cytokinesis, leaving
the cystocytes interconnected by cytoplasmic bridges called ring canals. As
the cyst forms, MTs are organized by a specialized cytoskeletal structure
called the fusome, which spans the entire cyst. During the cystocyte
divisions, the fusome attaches to one pole of every mitotic spindle, ensuring
the stereotypical arrangement of cystocytes
(Grieder et al., 2000
).
Subsequently, the fusome directs the formation of an MT organizing center
(MTOC) within the oocyte, which enables cytoplasmic determinants to accumulate
within the ooplasm and maintain oocyte fate
(Huynh and St Johnston, 2004
).
The cyst then rearranges such that the oocyte is positioned posteriorly, and
somatic follicle cells encapsulate the cyst. At this stage, defined as stage
1, the MTOC is positioned at the anterior of the oocyte, and the MT plus-ends
extend through the ring canals into the nurse cells
(Theurkauf et al., 1992
). At
stage 2, the egg chamber buds off from the germarium, where it is formed, and
enters the vitellarium, where it will develop into a mature egg. The MTOC and
the oocyte nucleus move to the posterior of the oocyte
(Theurkauf et al., 1992
), and
the polarized MT network directs the localization of several cytoplasmic
factors that are synthesized in the nurse cells to the oocyte
(Koch and Spitzer, 1983
;
Pokrywka and Stephenson,
1995
). In this manner grk RNA becomes concentrated within
the oocyte and accumulates at its posterior cortex, where it is translated
during stages 2-6 (Neuman-Silberberg and
Schupbach, 1993
; Van Buskirk
and Schupbach, 1999
). The posteriorly localized Grk protein, a
TGF-
homolog, signals via the Epidermal growth factor receptor (EGFR)
to the follicle cells overlying the oocyte at the posterior of the egg
chamber, causing posterior cells to express different markers and behave
differently from the other follicle cells
(Gonzalez-Reyes et al., 1995
;
Gonzalez-Reyes and St Johnston,
1998
; Roth et al.,
1995
). At stage 7-8, there is a rearrangement of the germline MT
cytoskeleton such that the MT network emanating from the posterior MTOC is
replaced by MTs nucleated from multiple sites at the anterior cortex of the
oocyte and extending toward the posterior
(Theurkauf et al., 1992
). The
newly polarized MT cytoskeleton allows bcd RNA to be directed toward
the anterior of the oocyte in association with a minus-end directed motor
(Arn et al., 2003
;
Schnorrer et al., 2000
), and
osk RNA to be transported to the posterior by the plus-end directed
motor, kinesin (Brendza et al.,
2000
). MT rearrangement also allows the oocyte nucleus to move
from its early position near the posterior to the presumptive dorsal anterior
corner (Koch and Spitzer,
1983
; Swan et al.,
1999
). During stages 8-11, grk RNA accumulates in a cap
between the nucleus and the plasma membrane, and the resulting Grk protein
signals via the EGFR to specify dorsal cell fate in the overlying follicular
epithelium (Neuman-Silberberg and
Schupbach, 1993
;
Neuman-Silberberg and Schupbach,
1996
).
Mutations that alter Grk signaling during stages 8-11 disrupt DV patterning
of the follicle cells, leading to altered DV polarity in the eggshell and
embryo. For instance, loss of Squid (Sqd) activity in the germline leads to
the delocalization of grk RNA and Grk protein along the entire
anterior circumference of the oocyte, resulting in loss of ventral follicle
cell fates (Neuman-Silberberg and
Schupbach, 1993;
Neuman-Silberberg and Schupbach,
1996
). Mutations that prevent Grk signaling to the posterior
follicle cells during stages 2-6 disrupt AP patterning of the oocyte. For
example, mutations in grk itself lead to an aberrant MT rearrangement
at stages 7-8, altered localization of bcd and osk RNAs, and
failure of the oocyte nucleus to migrate to the anterior
(Gonzalez-Reyes et al., 1995
;
Roth et al., 1995
). This,
along with the discovery of similar AP phenotypes when specific gene
activities (e.g. Notch and Laminin A) are lost in the follicle cells alone
(Deng and Ruohola-Baker, 2000
;
Ruohola et al., 1991
), led to
the proposal that correctly specified posterior follicle cells must signal to
the oocyte at stages 6-7 to allow normal MT rearrangement. However, this
hypothetical signal has not been defined.
Protein kinase A (PKA; PKA-C1 - FlyBase) was identified as a potential
transducer of the posterior follicle cell signal because it is required in the
germline for normal MT polarity and RNA localization in stage 8-9 oocytes
(Lane and Kalderon, 1994). In
this study, we characterized the role of PKA further and identified a mutation
in sqd as a weak modifier of the PKA mutant phenotype. Sqd
is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family
of RNA-binding proteins, and its role in establishing DV polarity has been
well characterized (Kelley,
1993
; Matunis et al.,
1994
; Neuman-Silberberg and
Schupbach, 1993
;
Neuman-Silberberg and Schupbach,
1996
). We found that loss of Sqd function produces a highly
penetrant defect in MT organization along the AP axis at stages 8-9, as well
as aberrant localization of osk RNA and other molecules that are
normally localized posteriorly. A less penetrant defect in MT polarity, which
affects Grk localization, is also evident before stage 6 in sqd
mutants. Despite this earlier defect, sqd mutants display normal
specification of posterior follicle cells in response to the early Grk signal.
Thus, Sqd is essential for the establishment of MT polarity in both early and
mid-oogenesis.
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Materials and methods |
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RNA in-situ hybridization
Ovaries were hand-dissected from ten females fattened on yeast paste.
Ovaries were teased open with forceps and fixed for 9 minutes in a biphasic
1:6 mixture of 6% formaldehyde:heptane. Ovaries were washed in PBS + 0.1%
Tween-20 (PBT), permeabilized for 1 hour in PBT + 1% Triton-X 100, dehydrated
into methanol, and stored overnight at -20°C in 100% methanol. After
rehydration, ovaries were treated for 10 minutes with 50 µg/ml proteinase K
and for 2 minutes with 2 mg/ml glycine. Ovaries were post-fixed for 15 minutes
in 4% formaldehyde and washed 5x5 minutes in PBT. Subsequent steps were
according to Tautz and Pfeifle with slight adjustments. Digoxigenin-labeled
riboprobes were hybridized at 1:50 overnight at 55°C. Hybridization buffer
was adjusted to pH 5. Alkaline-phosphatase-conjugated anti-digoxigenin
antibody was used at 1:2000 (Roche). Ovaries were mounted in 60% glycerol in
PBS.
Riboprobes were synthesized from cDNAs subcloned into a Bluescript vector (bcd) or a pNB40 vector (osk) using Ampliscribe transcription kit (Epicentre) and DIG RNA Labeling Mix (Roche). Following the transcription reaction, probes were hydrolyzed in carbonate buffer (2x: 120 mmol/l Na2CO3, 80 mmol/l NaHCO3, pH 10.2) for 20 minutes at 65°C. Hydrolysis was stopped with one volume 0.2 mol/l NaOAc (pH 6), and the probes were precipitated at -20°C with 0.1 volume 4 mol/l LiCl, 20 µg/ml tRNA, and 2.5 volumes chilled 100% ethanol. Probes were stored at -20°C in 200 µl hybridization buffer.
Antibody staining
Ovaries were dissected and fixed as for RNA in-situ hybridization. After
washing in PBT, ovaries were blocked for 1 hour in PBT + 1% Triton X-100, 1%
BSA. Primary antibodies were diluted in PBT + 0.1% BSA and incubated overnight
at 4°C. Anti-Grk (mouse, 1:30), anti-Orb (mouse, 1:30) and
anti-Nintra (mouse, 1:1000) were from the Developmental Studies
Hybridoma Bank. Anti-ß-galactosidase antibodies were from Promega (mouse,
1:1000) and Cappel (rabbit, 1:4000). Anti-PKC was from Santa Cruz
Biotech (rabbit, 1:1000). Anti-Dhc (mouse, 1:100), anti-Baz (rabbit, 1:1000),
anti-Sqd (mouse, 1:10) and anti-Osk (rabbit, 1:3000) were gifts from T. Hays,
A. Wodarz, T. Schupbach and A. Ephrussi respectively.
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Tubulin staining
Ovaries were dissected as for RNA in-situ hybridization and fixed for 10
minutes in 100% methanol at -20°C. After rehydration into PBT, ovaries
were blocked as for antibody staining. FITC-conjugated anti--tubulin
antibody (Sigma) was incubated at 1:250 in PBT + 0.1% BSA overnight at
4°C. Ovaries were washed 3x10 minutes in PBT + 0.1% BSA, incubated
for 1 hour at room temperature with Alexa 488-conjugated secondary antibodies
at 1:500 (Molecular Probes), and mounted in Aquapolymount or Fluoromount G.
For partial MT depolymerization, ovaries were rocked at room temperature in 20
µg/ml colchicine + 0.1% DMSO in modified Robb's medium
(Theurkauf et al., 1992
) for
10 minutes and rinsed in PBT before methanol fixation.
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Results |
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Germline clones of a PKA-C1 null allele, H2, showed over
90% penetrance of mid-oogenesis AP polarity defects, as detected by a
kinesin-ß-galactosidase (kin-ß-gal) protein that marks the plus-ends
of MTs and a GFP-Staufen (GFP-Stau) (St
Johnston et al., 1991) protein that generally co-localizes with
osk RNA (Fig. 1E,G).
Such polarity defects can be a consequence of failed posterior follicle cell
specification, as in grk and cornichon (cni)
mutants, for example (Gonzalez-Reyes et
al., 1995
; Roth et al.,
1995
). However, the expression of the posterior follicle cell
marker pointed998/12 (pnt) was normal in 89% of
egg chambers containing PKA-C1H2 germline clones
(n=187; Fig. 1C).
pnt998/12 expression was absent from posteriorly
positioned follicle cells only when those cells also lacked PKA activity,
consistent with previously noted cell-autonomous effects. Similarly,
expression of anterior markers in posterior follicle cells surrounding
PKA mutant germline clones was never observed for the centripetal
cell marker BB127 (Fig.
1D) and only very rarely for the general anterior marker
L53b and the border cell marker 5A7 (5% each, data not
shown). The rare ectopic expression of L53b and 5A7 at the
posterior is probably due to loss of PKA in the follicle cells and cannot
plausibly be responsible for the >90% incidence of AP polarity phenotypes
in PKA null germline clones. Thus, loss of PKA in the germline
disrupted oocyte polarity without substantially affecting established markers
of posterior follicle cell identity. These results support the hypothesis that
PKA affects the oocyte MT network in mid-oogenesis in response to a normal
posterior follicle cell signal.
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Even though mC* efficiently rescued oocyte polarity and nurse cell fusion defects in PKA-C1H2 germline clones, those eggs did not develop normally. Fewer than 4% hatched even if zygotically rescued by a wild-type paternal PKA-C1 allele, and most were either unfertilized or arrested development very early (see Table S1A in the supplementary material). Of the embryos that hatched, roughly half developed to adulthood if zygotically rescued, but all died before third instar in the absence of zygotic rescue. mC* was also unable to rescue animals zygotically null for PKA-C1 (but derived from heterozygous parents) beyond second instar (see Table S1B in the supplementary material). Thus, there are PKA functions in oogenesis or early embryogenesis, as well as during larval development, that are not adequately substituted by mC*.
LKB-1 does not rescue PKA mutant oocytes
Drosophila homologs of the Caenorhabditis elegans partition
defective (par) genes are involved in organizing the germline MT
cytoskeleton at mid-oogenesis (Pellettieri
and Seydoux, 2002). Germline mutation of the Drosophila
par-4 homolog, lkb1, can produce a mid-oogenesis phenotype
similar to that of PKA mutants. As PKA can phosphorylate LKB1 at a
specific site in vitro, and as rescue activity of an LKB1 transgene is reduced
if the PKA site is substituted with an alanine (GFP-LKB1S535A) and
enhanced if substituted with a glutamate residue (GFP-LKB1S535E),
it has been suggested that LKB1 is a key target of PKA in oocyte polarity
development (Martin and St Johnston,
2003
). If the sole role of PKA in AP polarity were to
phosphorylate LKB1, then expressing the LKB1 transgene carrying the
phosphorylation-mimicking mutation in the PKA site should bypass the need for
PKA. However, PKA-C1H2 germline clones that overexpress
GFP-LKB1S535E displayed 100% kin-ß-gal mislocalization
(n=19; Fig. 1J),
whereas wild-type oocytes overexpressing the transgene did not exhibit any AP
polarity defects (Fig. 1I).
Thus, LKB1 does not seem to be a major target for PKA function in oocyte
polarity.
A P-element insertion in sqd causes an AP patterning defect in the oocyte
In an effort to identify factors involved in transducing the mid-oogenesis
signal with PKA, we performed a dominant enhancer screen in a PKA
hypomorphic background. We found that the P-element allele l(3)j4B4
dominantly enhanced the kin-ß-gal mislocalization phenotype of the
PKA hypomorph from less than 10% penetrance to 29%. In the absence of
any PKA mutation, l(3)j4B4 homozygous germline clones made
from either of two independent recombinant chromosomes showed over 90%
kin-ß-gal mislocalization in stage 9 (n=58;
Fig. 2A) and close to 100%
GFP-Stau mislocalization in stages 9 and 10 (n=106;
Fig. 2C). Like PKA
germline clones, l(3)j4B4 germline clones showed no defect in
movement of the oocyte nucleus to the anterior cortex (data not shown). To
verify that the P-element insertion was the cause of the phenotype, we
generated a revertant line in which the P-element had been mobilized and
excised completely out of the genome. The revertant line was homozygous viable
and showed 93% normal GFP-Stau localization (n=149; data not shown),
a penetrance that is well within the range of GFP-Stau localization observed
for various controls. The l(3)j4B4 P-element is inserted into the
first intron of the sqd gene
(http://flybase.org/).
Thus, a P-element insertion in sqd is responsible for a strong defect
in AP patterning in the oocyte.
sqd alleles cause AP and DV patterning defects
To test whether l(3)j4B4 behaves like other alleles of
sqd, we examined l(3)j4B4 germline clones for classic
sqd phenotypes. DV defects, including eggshells with dorsal appendage
material around their entire anterior circumference, specification of dorsal
fate in all the follicle cells surrounding the anterior of the oocyte, and Grk
protein accumulation along the entire anterior cortex of stage 9-10 oocytes,
were observed for l(3)j4B4 germline clones (see Fig. S1A-D in the
supplementary material). Furthermore, in both l(3)j4B4 germline
clones and sqd1 mutant ovaries, the nurse cell chromosomes
failed to disperse completely beyond stage 6 (see Fig. S1F in the
supplementary material) (Goodrich et al.,
2004), in contrast to wild-type oocytes, in which the nurse cell
chromosomes remain polytene only for the early stages of oogenesis (see Fig.
S1E in the supplementary material, arrow). Finally, no Sqd protein was
detected in the nurse cells or oocyte in l(3)j4B4 germline clones
(see Fig. S1H in the supplementary material). Thus, l(3)j4B4 mutants
clearly are deficient for Sqd activity, displaying all the previously
described oogenesis phenotypes characteristic of sqd mutants. We
conclude that l(3)j4B4 is indeed an allele of sqd, and we
now refer to it as sqdj4B4.
Because sqdj4B4 shows a highly penetrant defect in AP
patterning during oogenesis, we examined whether other sqd alleles
also exhibit a similar phenotype. We found GFP-Stau to be substantially
mislocalized in stage 9-10 oocytes of escaper females from all strong
sqd allelic combinations tested, including
sqdj4B4/sqdix50,
sqdj4B4/sqdix77, sqdj4B4/Df,
sqdix77/Df, as well as in females containing
sqdix77 germline clones
(Table 1A). For all these
genotypes, we often observed a small amount of GFP-Stau in the correct
location at the posterior of the oocyte, in addition to the ectopic GFP-Stau
in the center of the oocyte, and the incidence of oocytes with normal
localization was consistently higher in later stages. We also observed
GFP-Stau mislocalization in homozygotes of the weak, viable allele
sqd1 and in sqdj4B4/sqd1
trans-heterozygotes (Table 1A).
In these genotypes, GFP-Stau rarely accumulated as a `cloud' in the center of
the oocyte as it does in sqdj4B4 germline clones; rather
it seemed to aggregate in `flecks' in the ooplasm or in ectopic locations
along the lateral cortex (Fig.
2D). Additionally, all sqd alleles showed aberrant
GFP-Stau aggregation surrounding the nuclei of the nurse cells
(Fig. 2J). A SqdS transgene
that expresses one of the three described Sqd splice variants
(Norvell et al., 1999) rescued
the partial lethality of sqdj4B4 in combination with other
strong sqd alleles and also restored normal GFP-Stau localization in
those females (Table 1A,B).
Both the lethality complementation tests and the GFP-Stau mislocalization data
suggest that sqdj4B4 is a stronger allele than the
previously described allele sqdix77, which is the result
of a small deletion in the 5' UTR
(Kelley, 1993
)
(Table 1). Germline clones of
the probable null allele, sqdix50
(Kelley, 1993
), did not
produce vitellogenic egg chambers. We conclude from these data that all
sqd alleles disrupt AP polarity during oogenesis to a degree
commensurate with their allelic strength.
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|
We therefore examined MT distribution directly with -tubulin
antibody. In wild-type stage 8 and 9 oocytes,
-tubulin staining is
higher at the anterior than at the posterior
(Fig. 3A), because MTs are
nucleated primarily at the anterior cortex
(Theurkauf et al., 1992
) and
are excluded from the posterior pole (Cha
et al., 2002
). In sqdj4B4 germline clones, we
detected MTs at uniform density all around the oocyte cortex, indicating that
in these mutants the asymmetrical mid-oogenesis MT array does not form
correctly (Fig. 3B).
We did not find any combination of sqd alleles that shows a
significantly greater penetrance of mislocalization for osk RNA than
for kin-ß-gal. In the viable trans-heterozygote,
sqdj4B4/sqd1, kin-ß-gal was improperly
localized at 65% penetrance, either in the center of the oocyte or diffusing
away from the posterior pole (n=165;
Fig. 2B). This penetrance is
comparable to the 65% GFP-Stau mislocalization and 74% osk
mislocalization observed in oocytes of this genotype
(Table 1A and
Fig. 2D,F), implying that the
mislocalization of osk RNA in this and other sqd allelic
combinations results principally from aberrant MT organization. This is in
contrast to, but not mutually exclusive with, the suggestion that Sqd guides
osk RNA to the posterior by a direct interaction
(Norvell et al., 2005). Our
observation that GFP-Stau particles cluster aberrantly around the periphery of
nurse cell nuclei in sqd mutants supports the idea that Sqd affects
the behavior of complexes likely to include osk RNA, despite the fact
that we did not observe any specific effect of Sqd on osk RNA
localization independent of the MTs.
Cytoplasmic streaming normally occurs at stages 10b-12 and depends on
reorganization of the MT cytoskeleton after stage 10a
(Theurkauf, 1994;
Theurkauf et al., 1992
). In
cappuccino (capu) mutants, mislocalization of osk
RNA is the result of premature cytoplasmic streaming during stages 8-10a
(Manseau et al., 1996
). In
sqdj4B4 germline clones, we observed normal streaming at
stage 10b but not earlier (data not shown), indicating that the MT
arrangements that govern streaming do not depend on Sqd function.
In a subset of polarity mutants, including PKA, bcd RNA localizes
ectopically to the oocyte posterior at stages 8-10; it has been suggested that
this reflects localization to an MTOC aberrantly persisting at the oocyte
posterior after stage 7 (Lane and
Kalderon, 1994). We did not detect ectopic bcd at the
posterior of sqdj4B4 mutant oocytes (n=40,
Fig. 3H), even though it was
readily observed in PKA-C1H2 germline clones (31%,
n=26, data not shown), as well as in another polarity mutant,
mago nashi (mago; Fig.
3G).
Partial MT depolymerization in sqd and PKA mutants
To further investigate the nature of the MT defects in sqd and
PKA mutants, we examined MTs with -tubulin antibody following
partial depolymerization with colchicine. In wild-type stage 8 and 9 oocytes,
MT stubs emanate mostly from the anterior cortex following partial
depolymerization (Fig. 3C),
reflecting the presence of an anterior MTOC after stage 7
(Theurkauf et al., 1992
). By
contrast, partial MT depolymerization in sqdj4B4 germline
clones revealed MTs emanating from the entire oocyte cortex at stages 8 and 9
(Fig. 3D,F). Likewise, in
PKA-C1H2 germline clones, MT stubs could be detected all
around the oocyte cortex following partial depolymerization at stages 8-10.
Additionally, however, a strong focus of MTs sometimes could be detected at
the posterior pole of PKA-C1H2 germline clones
(Fig. 3E), whereas such
staining was never seen in sqdj4B4 germline clones. This
suggests that the whole cortex, including the posterior pole, nucleates MTs in
both sqd and PKA mutants but that only PKA mutants
retain a discrete posterior MTOC. The cortical MTs that persist at the
posterior pole in sqd mutants presumably do not represent a true
organizing center, as they are not capable of supporting ectopic posterior
bcd RNA, in contrast to those of the PKA mutant.
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To test whether early Grk signaling suffices to specify posterior follicle cells in sqdj4B4 germline clones, we examined two established markers of follicle cell fate. Expression of the posterior follicle cell marker, pntrm254, was normal in egg chambers where the germline was mutant for sqd (Fig. 6A). Additionally, we did not see ectopic expression of the anterior centripetal cell marker, BB127, in posterior follicle cells surrounding sqd mutant oocytes (Fig. 6B). Thus, the early grk signal, which is required for pntrm254 expression, is transmitted appropriately from sqd mutant oocytes, and we conclude that the mid-oogenesis phenotype of sqd oocytes is not the result of an earlier defect in grk signaling or in posterior follicle cell specification.
Although the anterior and posterior follicle cells are specified correctly in egg chambers containing sqdj4B4 germline clones, the migration of the anterior border and centripetal cells in those egg chambers was defective at high penetrance (see Fig. S2F in the supplementary material).
sqd mutants display defects in early oocyte polarity
The mislocalization of Grk protein in early sqd mutant oocytes led
us to question whether early sqd oocytes display other polarity
defects. Like Grk, Orb protein and osk RNA localize in a cap at the
posterior of wild-type oocytes in stages 2-6
(Fig. 4C and data not shown).
As was found for Grk, Orb was diffusely localized in the ooplasm of early
sqd mutants, at variable penetrance ranging from 12-52%
(n=339; Fig. 4D); the
penetrance increased with the age of the females, as was the case for various
other defects (see below). osk RNA was also mislocalized in a manner
similar to Orb and Grk in a subset of early oocytes (data not shown).
Likewise, stage 2-6 sqdj4B4 oocytes often had an even
distribution of MTs throughout their cytoplasm
(Fig. 4F), in contrast to
wild-type oocytes, which have a strong focus of tubulin staining at the
posterior, indicative of the posterior MTOC
(Fig. 4E). Thus, sqd
mutants display abnormal distributions of germline MTs in both early and
mid-oogenesis.
sqd mutants also display defects in oocyte specification and cystocyte mitosis
Ovarioles containing sqdj4B4 germline clones exhibited
a number of additional phenotypes likely to originate in the germarium. These
increased in penetrance over time, presumably reflecting progressive depletion
of perduring Sqd gene products below functional thresholds in the germarium.
Thus, for animals with germline clones induced during the third larval instar,
the penetrance of these phenotypes collectively increased from 17% of early
egg chambers in 1-3-day-old females (n=127; 6-8 days after clone
induction) to 89% in 10-12-day-old females (n=280; 15-17 days after
clone induction).
The most prominent of these phenotypes was the absence of stalks separating adjacent egg chambers (see Fig. S2B in the supplementary material). Also, many egg chambers contained aberrant numbers of germ cells, from as few as two (see Fig. S2C in the supplementary material) to well in excess of 16. Furthermore, the number of germ cells in adjacent egg chambers generally did not add up to 16, nor were excess germ cells present in multiples of 16. Cysts with 16 nurse cells and no oocyte were also observed (data not shown), although more commonly, cysts with no oocyte also contained abnormal numbers of germ cells (see Fig. S2B in the supplementary material). Additionally, in egg chambers containing the proper complement of nurse cells and an oocyte, the oocyte was sometimes mispositioned within the cyst, failing to reside at the posterior (see Fig. S2D in the supplementary material). In 10-12-day-old females, a number of egg chambers beyond stage 6 were seen to degenerate. The scoring of defects in AP polarity was always performed using young females and only included egg chambers with 16 germ cells and a normally positioned oocyte.
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sqd does not affect polarity in somatic cells
To determine whether Sqd affects polarity of somatic cells in addition to
its role in polarizing the oocyte, we examined the localization of several
markers of apical polarity in sqdj4B4 follicle cell
clones. We did not detect any effect on the localization of the apical
proteins Notch (N), Bazooka (baz), or Atypical protein kinase C (aPKC) in
sqd mutant follicle cell clones, even as long as 15 days after clone
induction (Fig. 6C).
Additionally, we did not detect any defects in planar cell polarity in the
trichome bristles of the wing blade, either in sqdj4B4
clones or in the wings of sqdj4B4/Df escaper
flies (Fig. 6D), and no planar
polarity or other defects were apparent in sqdj4B4
homozygous eye clones (data not shown). Thus, Sqd acts specifically in the
female germline to organize MTs and establish polarity.
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Discussion |
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Loss of PKA in the germline did not affect MT polarity in early oogenesis, as judged by Orb localization in stages 2-6 (data not shown), but, similarly to sqd mutants, it disrupted MT polarity in mid-oogenesis without discernibly altering follicle cell fate. However, despite several similarities, we observed a significant difference in the MT organization of sqd and PKA mutants at stages 8-10, as discussed in greater detail below.
MT organization in polarity mutants
In grk and cni mutants, where the posterior follicle
cells do not differentiate properly, both the subsequent RNA localization
defects and the failure of the oocyte nucleus to migrate from the posterior to
the anterior have been attributed to defects in MT reorganization resulting
from loss of a posterior follicle cell signal
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995
). In
PKA and sqd mutant oocytes, the anterior migration of the
oocyte nucleus, which depends on MT function
(Koch and Spitzer, 1983
), was
unaffected, despite the accompanying MT defects and the highly penetrant
mislocalization of osk RNA and other posterior factors. Thus, it
appears that either discrete aspects of the MT organization, which direct
nucleus migration, are spared in PKA and sqd mutants or the
overall disruption of MT organization by loss of PKA or Sqd is simply less
severe than that caused by loss of posterior follicle cell fate.
The normal organization of MTs in stage 8-10 oocytes is not entirely clear.
In addition to MTs nucleated at the anterior cortex, MTs have been proposed to
emanate from all cortical positions, with the exception of the posterior pole
(Cha et al., 2001;
Cha et al., 2002
). This
assertion is based on the observations that components associated with MT
minus-ends, such as
-tubulin and the centrosome component Centrosomin
(Cnn) (Cha et al., 2002
), can
be seen along the entire oocyte cortex, and that injected bcd RNA
localizes to the lateral cortices as well as the anterior, but not to the
posterior pole (Cha et al.,
2001
). Hence, normal posterior localization of osk RNA
may require the clearing of MTs nucleated both from a discrete posterior MTOC
established before stage 6 and from dispersed cortical sites established after
stage 7.
Staining with -tubulin antibody following partial MT
depolymerization revealed MT stubs emanating mostly from the anterior in
wild-type oocytes, whereas PKA and sqd mutant oocytes
retained short MTs around the entire oocyte cortex, including the posterior
pole. Some PKA mutant oocytes also showed an elevated posterior
concentration of MTs not seen in sqd mutant oocytes. Thus, it appears
that the primary MT defect in sqd mutants is the failure to eliminate
cortical sites of MT nucleation beyond stage 7, whereas PKA mutants
additionally retain a posterior MTOC beyond stage 6. This hypothesis can
explain why ectopic bcd RNA localizes at the posterior of
PKA mutant oocytes but not sqd mutant oocytes. It should,
however, be noted that since classical MTOC components, such as
-tubulin, are present along the entire oocyte cortex at stages 9-10
even in wild-type oocytes (Cha et al.,
2002
) (J, Steinhauer, unpublished data), the inference of a
discrete posterior MTOC from partial MT depolymerization experiments cannot be
confirmed directly.
sqd mutants display polarity defects in early oogenesis
In a proportion of sqd mutant stage 2-6 oocytes, Grk, Orb,
osk RNA and MTs were distributed evenly throughout the ooplasm rather
than localizing in a cap at the oocyte posterior. Although these defects did
not appear to cause the subsequent AP defects by preventing posterior follicle
cell specification, we cannot rule out the possibility that the early and late
polarity phenotypes are causally related in some other way. For instance, it
is possible that a molecule(s) required at the posterior of the oocyte for the
MT reorganization at stages 7-8 is improperly localized by stage 6 in
sqd mutants, as are Grk, Orb and osk RNA. If MT
rearrangements were very sensitive to the localized concentration of this
hypothetical regulator, an early polarity defect of apparently low penetrance
could be translated into a much more penetrant polarity phenotype at
mid-oogenesis.
sqd is not the only mutant to cause polarity defects in both early
and mid-oogenesis. For example, defects in early polarity are caused by
mutations in Armitage (Armi), a component of the RNA silencing machinery, and
these defects were proposed to be the cause of a mid-oogenesis AP polarity
phenotype (Cook et al., 2004).
However, we found that pnt998/12 expression was not
disrupted in armi1 homozygotes (J. Steinhauer,
unpublished). Weak par-1 alleles also affect mid-oogenesis polarity
without affecting posterior follicle cell fate
(Shulman et al., 2000
),
whereas strong alleles disrupt early polarity severely, causing oocyte
identity to be lost (Cox et al.,
2001
; Huynh et al.,
2001
). Thus, for several mutations, including sqd, armi
and par-1, it is unclear whether MT organization is disrupted
independently at two distinct phases of development or whether there is a
causal connection between the early and later polarity phenotypes that is not
evident as a failure in posterior follicle cell specification. In either case,
a single molecular target might account for both the early and mid-oogenesis
phenotypes.
Additional early sqd phenotypes
Several additional phenotypes became prevalent in older
sqdj4B4 germline clones, rising to very high penetrances
after 2 weeks. Among the varied late onset sqd phenotypes, the oocyte
sometimes was mispositioned within the egg chamber, even in those egg chambers
containing the normal complement of nurse cells to oocyte. This phenotype can
arise in several ways, including as a result of delayed oocyte specification
(Gonzalez-Reyes et al., 1997).
A role for Sqd in oocyte specification is supported by the presence of cysts
with 16 nurse cells and no oocyte in these older ovarioles. In other cases,
cysts with fewer than 16 germ cells were observed, implicating Sqd in
cystocyte mitosis. Both oocyte specification and the normal cystocyte
divisions depend on specific arrangements of the MT cytoskeleton in the
germarium (Huynh and St Johnston,
2004
). Thus, it is likely that some of these late onset
sqd phenotypes, like the polarity phenotypes, are caused by a defect
in regulating MT dynamics.
osk translation in polarity mutants
In sqdj4B4 germline clones, we noticed the accumulation
of Osk protein in the cytoplasm of stage 9-10 oocytes. A similar observation
was reported for mutations in another hnRNP A/B family member, Hrp48 (Hrb27C -
FlyBase) (Yano et al., 2004).
Normally, Osk protein accrues only at the posterior cortex, and translation of
osk RNA is presumably repressed elsewhere
(Kim-Ha et al., 1995
).
Therefore, loss of sqd may cause de-repression of osk
translation. However, we did not see ectopic osk translation in stage
6-8 sqd mutant oocytes, detected either with Osk antibody or with an
osk translation reporter, in contrast to the premature expression
observed with the osk translation reporter in hrp48 mutants
(Yano et al., 2004
) or with
similar reporters lacking specific repressor elements
(Gunkel et al., 1998
;
Kim-Ha et al., 1995
). Thus,
although we do not discount the idea that sqd is directly involved in
translational regulation of osk, we propose an alternative hypothesis
for the ectopic Osk protein accumulation in sqd mutants. As most of
the posterior components that we examined were mislocalized to the center of
sqdj4B4 oocytes, we believe that the primary AP defect in
sqd mutants is that the MT plus-ends are focused incorrectly at the
center of the oocyte. Hence, all the necessary components for osk
translation may be localized together, and we hypothesize that the
osk translation machinery is assembled and activated in the middle of
the sqd mutants as it normally is at the posterior of wild-type
oocytes at stage 9.
We also detected a low penetrance of ectopic Osk protein in PKA
mutants. The scenario outlined above could be true for PKA mutants as
well. Regardless of the mechanism, it is clear from this result that PKA is
not absolutely required for Osk translation, although it may enhance
osk translation, as previously suggested
(Yoshida et al., 2004).
Targets of PKA and Sqd
Although sqd was identified in a screen for modifiers of
PKA in oocyte polarity, retesting with various alleles indicated that
there is not a strong genetic interaction between the two loci (data not
shown). Both Sqd and PKA act in mid-oogenesis to reorganize the oocyte MTs in
response to a normal posterior follicle cell signal, but specific MT defects
differ between the two mutants, as discussed above. Thus, they probably have
different targets and mechanisms in this complex process.
The hnRNP Sqd is an RNA-binding protein. Another hnRNP of the same family,
Hrp48, is also required for MT reorganization at mid-oogenesis
(Yano et al., 2004). Sqd and
Hrp48 bind each other in vitro, cooperate in grk RNA localization
(Goodrich et al., 2004
) and
have similar localization patterns throughout oogenesis
(Matunis et al., 1994
;
Yano et al., 2004
) (see Fig.
S1G in the supplementary material). Thus, one might expect these two proteins
to act together in MT reorganization. Although we were unable to detect a
strong genetic interaction between sqd and hrp48 in AP
polarity (data not shown), we speculate that they are collectively necessary
for the localization and translation of one or a small number of specific RNA
molecules required for MT repolarization at mid-oogenesis.
hnRNPs normally participate in the processing of many RNAs
(Dreyfuss et al., 2002), but
their generic functions may be partially redundant, so that, for example, in
sqd mutants, continued cell viability is not impaired despite the
presence of a strong AP polarity defect. Our ability to induce large,
persistent somatic cell clones for sqdj4B4 without causing
any polarity or other phenotypes supports this idea. Follicle cell polarity
was also normal in PKA mutant clones (data not shown). Thus, the
disruptions in MT polarity that we observe for both PKA and
sqd mutants represent specialized functions of these proteins in
germline cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5515/DC1
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ACKNOWLEDGMENTS |
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