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First published online 15 March 2006
doi: 10.1242/dev.02318
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B kinase family, is required for mRNA localization during oogenesis
1 Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue,
New York, NY 10021, USA.
2 Biochemistry, Cell and Molecular Biology Program, Weill Graduate School of
Medical Sciences, Cornell University, 445 East 69th Street, New York, NY
10021, USA.
* Author for correspondence (e-mail: k-anderson{at}ski.mskcc.org)
Accepted 9 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B kinases (IKKs) regulate
the activity of Rel/NF-B transcription factors by targeting their
inhibitory partner proteins, IBs, for degradation. We identified
mutations in ik2, the gene that encodes one of two
Drosophila IKKs, and found that the gene is essential for viability.
During oogenesis, ik2 is required in an NF-B-independent
process that is essential for the localization of oskar and
gurken mRNAs; as a result, females that lack ik2 in the
germline produce embryos that are both bicaudal and ventralized. The abnormal
RNA localization in ik2 mutant oocytes can be attributed to defects
in the organization of microtubule minus-ends. In addition, both mutant
oocytes and mutant escaper adults have abnormalities in the organization of
the actin cytoskeleton. These data suggest that this IB kinase has an
NF-B-independent role in mRNA localization and helps to link
microtubule minus-ends to the oocyte cortex, a novel function of the IKK
family.
Key words: ik2, IB kinases, mRNA localization, Oogenesis, Dynein-based transport, oskar, gurken
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B kinase (IKK) family are known for their
roles in innate immune response signaling pathways in both mammals and
Drosophila (Ghosh and Karin,
2002
; Peters and Maniatis,
2001; Silverman and Maniatis,
2001). Mammalian IKKs all have roles in immune responses, but have
a variety of targets. IKK
and IKKß were identified in a protein
complex that phosphorylates IB and targets it for degradation, thereby
allowing the nuclear localization and activation of NF-B transcription
factors (DiDonato et al.,
1997; Mercurio et al.,
1997; Zandi et al.,
1997). Gene targeting experiments in the mouse demonstrated that
IKKß, but not IKK, is required for NF-B activation by
pro-inflammatory stimuli through receptors such as TLR4
(Li et al., 1999a;
Li et al., 1999b;
Tanaka et al., 1999).
IKK activates the Rel/p52 transcription factor, because it activates
proteolytic processing of the p100 precursor of p52 in an
IB-independent process (Senftleben
et al., 2001). IKK
and TANK binding kinase 1 (TBK1) are
required to phosphorylate and activate the transcription factor Interferon
regulatory factor 3 (IRF3) in response to viral infection
(Fitzgerald et al., 2003;
McWhirter et al., 2004;
Sharma et al., 2003). In
addition to these immune response functions, IKK has an
NF-B-independent role in epidermal differentiation and limb development
(Hu et al., 1999;
Hu et al., 2001;
Sil et al., 2004;
Takeda et al., 1999).
Dorsoventral patterning of the Drosophila embryo relies on the
activation of Dorsal, a Rel-family transcription factor, by a signaling
pathway that is homologous to mammalian TLR pathways
(Anderson, 2000). In response
to activation of the receptor Toll, Cactus (the Drosophila IB)
is degraded, which allows Dorsal to move to embryonic nuclei and activate
genes, such as twist, that are required for specification of ventral
cell types. Phosphorylation of Cactus is required for its degradation
(Fernandez et al., 2001), but
the responsible kinase has not been identified. The Drosophila genome
encodes two members of the IKK family. DmIkkß (ird5 -
FlyBase) is essential for the response to bacterial infection
(Lu et al., 2001).
DmIkkß is required for proteolytic processing and activation of
Relish, a p100-like Rel/ankyrin-repeat protein, like the role of mammalian
IKK in the activation of p100. The function of the second
Drosophila protein kinase of the IKK family, ik2
(IB kinase-like 2), has not been characterized, but it was a
good candidate to control the phosphorylation and degradation of Cactus.
To test whether ik2 encodes a Cactus kinase, we characterized the phenotypes caused by loss of ik2 function. Here, we present data showing that Ik2 is essential for dorsoventral and anteroposterior embryonic patterning of the Drosophila embryo. However, Ik2 does not act as a Cactus kinase, but exerts its effects on embryonic patterning through the localization of specific mRNAs during oogenesis. The data indicate that Drosophila Ik2 regulates RNA localization through regulation of the cytoskeleton and define a novel function for this protein family.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
consists of five alleles and were renamed accordingly:
Ea36 (ik21), Ea41
(ik22), Ea46
(ik23), Ea47
(ik24), Ea66
(ik25). The following mutations in the kinase domain were
identified from sequenced ik2 genomic DNA: ik21
(G250D), ik22 (N69I), ik23 (G19D),
ik24 (G109A), ik25 (D160N).
ik2alice was recovered in a maternal effect genetic screen
(Luschnig et al., 2004), and
also had a point mutation in the kinase domain (F297I).
Fly stocks and genetic analyses
The X chromosome FLP recombinase stock P[ry+,
hsFLP], the second chromosome FRT stock P[w+ FRT
40A] (Chou and Perrimon,
1996), Df(2L)KetelRX32
(Erdelyi et al., 1997),
BicD1 and BicD2
(Mohler and Wieschaus, 1986),
and the tubulin-Gal4 and daughterless-Gal4 drivers were obtained from the
Bloomington Stock Center. The recombinant chromosomes ik21
P[w+ FRT 40A] and ik2alice
P[w+ FRT 40A] were provided by F. Schnorrer and C.
Nüsslein-Volhard (Tübingen). The following stocks were also used:
Tau-GFP line 2.1 (Micklem et al.,
1997); Kinesin-ß-galactosidase insertion line KZ503
(Clark et al., 1994); and
Nod-ß-galactosidase insertion line NZ143.2
(Clark et al., 1997). To
induce expression of FLP recombinase, flies were mated for 24 hours, and
second instar larvae were heat shocked in a 37°C water bath for two hours
on two consecutive days.
Eggshell and cuticle preparations
To visualize the chorion under the microscope, eggs were washed with 0.7%
NaCl and 0.1% Triton X-100, and mounted in Hoyer's medium
(Van der Meer, 1977). For
cuticle preparations, embryos were collected on apple juice agar plates,
washed with 0.7% NaCl and 0.1% Triton X-100, and bleached to remove the
chorion. Embryos were fixed for 1 hour at 65°C in 1:4 glycerol:acetic acid
and mounted in Hoyer's medium.
Ovarian and embryonic in situ hybridization
Whole-mount ovaries were hybridized with digoxigenin-labeled grk,
bcd and osk RNA probes, all described previously
(Berleth et al., 1988;
Ephrussi et al., 1991;
Neuman-Silberberg and Schüpbach,
1993). Ovaries were fixed and stained according to Suter and
Steward (Suter and Steward,
1991). Embryonic fixation and hybridization were performed as
described previously (Tautz and Pfeifle,
1989). Fluorescent ovarian in situ hybridization to detect
osk mRNA was performed as described previously
(Cha et al., 2002).
Immunohistochemistry
For Twist staining of embryos, 0- to 2-hour embryos were collected, aged 2
hours at 25°C, and fixed with 4% paraformaldehyde. Embryos were rehydrated
with 1xPBS and incubated in 0.3% BSA in PBST (1xPBS with 0.3%
Triton X-100) for 30 minutes. After overnight incubation at 4°C with
primary antibody (in 0.3% BSA in PBST) and washing with PBST, the samples were
incubated for 1 hour with biotin goat anti-rabbit secondary antibody (Jackson
ImmunoResearch) and signal was amplified using the Vector Elite ABC kit
(Vector Laboratories). Rabbit anti-Twist (1:5000) was kindly provided by
Siegfried Roth (Cologne).
Ovaries from 24- to 48-hour-old females were dissected and fixed as
previously described (Verheyen and Cooley,
1994a). Antibodies were used at the following concentrations:
mouse monoclonal P1H4 anti-dynein heavy chain, 1:500
(McGrail and Hays, 1997);
anti-ß-galactosidase monoclonal antibody, 1:2000 (Promega). For Grk
antisera (1:10), ovaries were fixed and stained as described previously
(Queenan et al., 1999). For
visualization of actin, ovaries were incubated with either FITC-phalloidin or
rhodamine-phalloidin (Molecular Probes) for 2 hours. Images were captured
using a Leica TCS SP2 confocal microscope system and Leica Confocal Services
software (version 2.61).
Electron microscopy
Fly heads were fixed with 4% formaldehyde overnight, then dehydrated with a
graded ethanol series. Samples were critical-point dried in CO2,
then sputter coated with 30 nm of gold palladium and examined with a scanning
electron microscope.
UAS-ik2 rescue construct
An ik2 genomic DNA fragment from 95 bp before the first codon to
350 bp following the stop codon was cloned from Oregon R genomic DNA using the
primers 5'-GCTCTAGAGTCACAATCGAGAAGGCGCTT-3' and
5'-GCTCTAGAGCTCAATGGCGTCGAG-3', which incorporated an
XbaI site on both the 5' and 3' ends. The resulting 3.1
kb fragment was inserted into the XbaI site of the UASp vector to
drive maternal expression (Rørth,
1998). The rescue plasmid was injected into yw embryos
and transgenic lines were selected for expression of the white
gene.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TANK binding kinase 1 (TBK1), which
are 60-61% identical to Ik2 in the kinase domain and 51% identical across the
entire protein; by contrast, Ik2 is only 28% identical to the other
Drosophila IKK, DmIkkß. A saturation mutagenesis experiment had
identified lethal complementation groups in polytene chromosome region 38E
(Kozlova et al., 1998), the
region that includes the ik2 gene. We identified missense mutations
in the ik2 kinase domain in all five alleles of the l(2)38Ea
complementation group. A sixth allele, ik2alice,
identified in a genetic mosaic screen for maternal effect mutations
(Luschnig et al., 2004), had a
missense mutation in the C-terminal end of the kinase domain. All six
ik2 alleles caused recessive lethality, and the majority of the
mutants died as first instar larvae. At low temperature and in uncrowded
culture conditions, rare escaper adults (<1%) were observed, but they died
shortly after eclosion.
Loss of ik2 in the female germline results in bicaudal and ventralized embryos
If ik2 encoded the Cactus kinase, embryos produced by females that
lack ik2 would not be able to degrade Cactus, so Dorsal would not
enter embryonic nuclei to activate genes required for ventral cell fate
specification and the embryos would be dorsalized. Because ik2
mutations were lethal, we used FRT/FLP recombination combined with the
ovoD dominant female-sterile mutation to generate mutant
clones in the female germline (Chou and
Perrimon, 1992). More than 95% of the embryos laid by
ik2alice and ik21 mutant females did
not hatch; however, larval cuticle preparations showed that none of the
embryos were dorsalized. Instead, the majority of embryos produced by
ik2alice and ik21 mutants had a
bicaudal phenotype, ranging from headless embryos
(Fig. 1B) to embryos with a
duplicated abdomen in place of the head and thorax
(Fig. 1C). In addition to this
anteroposterior patterning defect, a large number of embryos from both
ik2alice (Fig.
2B) and ik21
(Fig. 2C) germline clones had
expanded ventral cuticular structures, the opposite of the expected phenotype.
Some embryos were both ventralized and bicaudal, with expanded ventral
denticle bands and filzkörper (a posterior structure) in both the tail
and the anterior of the embryo. Both ik2 alleles produced bicaudal
and ventralized embryos, but 89% (n=190) of the embryos produced by
ik2alice mutant females were bicaudal with no apparent
dorsoventral abnormalities, whereas only 47% (n=110) of embryos
produced by ik21 mutant females were bicaudal, and the
remainder of the embryos appeared to be too ventralized to score for ectopic
posterior cuticular structures. We observed a similar range of phenotypes in
embryos produced by ik22, ik23 and
ik25 females with mutant germline clones.
The ik2 bicaudal phenotype is the result of ectopic localization of oskar mRNA during oogenesis
The anteroposterior pattern of the embryo depends on the localization of
maternal mRNAs. The bicoid (bcd) mRNA is localized to the
anterior end of the egg, and specifies anterior cell fates including the head
and thorax (Berleth et al.,
1988). nanos mRNA is localized at the posterior pole of
the egg, a process that depends on prior posterior localization of
oskar (osk) mRNA, and specifies the pattern of the abdomen
(Nüsslein-Volhard et al.,
1987; Ephrussi et al.,
1991).
|
;
Suter and Steward, 1991;
Wharton and Struhl, 1989;
Schüpbach and Wieschaus,
1991; Mahone et al.,
1995; Castagnetti and Ephrussi,
2003). Bicaudal embryos produced by BicC and
BicD females have ectopic anterior nanos and oskar
mRNA, leading to duplicated posterior structures
(Mahone et al., 1995;
Ephrussi et al., 1991). We
examined the localization of these key patterning mRNAs in embryos produced by
ik2 germline clones to determine whether the cause of the
ik2 bicaudal phenotype was similar to that of the BicC and
BicD mutants.
In wild-type embryos prior to gastrulation, bcd mRNA is localized
to the anterior pole (Fig. 1D)
(Berleth et al., 1988) and
osk mRNA is found exclusively at the posterior pole
(Fig. 1H)
(Ephrussi et al., 1991).
bcd mRNA was localized normally in all ik2alice
(Fig. 1E) and
ik21 (Fig.
1F) mutant embryos examined, similar to
BicD1/BicD2 mutants
(Fig. 1G). By contrast,
oskar mRNA was present at both the anterior and posterior poles in
100% of the embryos produced by ik2alice
(Fig. 1I) and
ik21 (Fig.
1J) mutant germline clone females, and 100% of the embryos
produced by BicD1/BicD2 mutant females
(Fig. 1K). Thus even though a
bicaudal phenotype could not be detected in all cuticle preparations of the
ventralized class of embryos, all embryos produced by
ik2alice and ik21 mutant females had
anteriorly localized oskar mRNA. While gain-of-function BicC
and BicD alleles produce bicaudal embryos at high frequency,
ik2 is the first locus to be described where loss of function causes
a completely penetrant bicaudal phenotype.
|
). We
found ectopic nanos mRNA at the anterior pole of all
ik2alice (Fig.
1M) and ik21
(Fig. 1N) mutants, which
indicated that oskar localized at the anterior of ik2
embryos was sufficient to mislocalize nanos to the anterior pole.
The bcd and osk mRNAs are transcribed in the nurse cells,
transported into the oocyte and targeted to the anterior and posterior ends of
the oocyte during stages 9-10 of oogenesis
(Schnorrer et al., 2000;
St Johnston et al., 1989;
Ephrussi et al., 1991). In
BicD mutant females, osk mRNA is transported efficiently
from the nurse cells into the oocyte, but accumulates at both the anterior and
posterior poles of the oocyte (Ephrussi et
al., 1991). We examined osk mRNA in both
BicD1/BicD2 (data not shown) and ik2
mutant oocytes using a probe conjugated directly to fluorescein. At a stage
when osk mRNA was localized tightly to the posterior pole of
wild-type oocytes (Fig. 3A),
osk mRNA was present in all regions of the ik2 mutant oocyte
cortex (Fig. 3B,C) and was
enriched at both the anterior and posterior poles.
While osk mRNA is being transported to the posterior pole of
wild-type oocytes, the message is translationally repressed, ensuring that the
protein is only active once it reaches its correct subcellular location
(Kim-Ha et al., 1995;
Markussen et al., 1995;
Rongo et al., 1995). Females
that carry mutations in BicC produce bicaudal embryos as a result of
ectopic anterior osk mRNA and premature translation of Osk at ectopic
sites in the oocyte (Mahone et al.,
1995; Saffman et al.,
1998). In ik2 oocytes, Osk protein was localized
exclusively at the posterior of the oocyte at stage 9 and was not detectable
at earlier stages (data not shown). Thus the ectopic localization of
osk mRNA in ik2 mutant oocytes, and not premature
translation, is the cause of the ik2 bicaudal phenotype.
The bcd mRNA is transported from the nurse cells to the oocyte and
then restricted to the anterior of the developing oocyte by stages 8-9 of
oogenesis (Berleth et al.,
1988; St Johnston et al.,
1989). The bcd transcript was tightly localized to the
anterior cortex of the oocyte in most ik2 egg chambers. However, in
approximately 20% of ik21 mutant egg chambers the
expression domain of bcd mRNA was expanded toward the posterior of
the oocyte (data not shown), a pattern that has been seen when dynein-mediated
RNA transport is disrupted (Duncan and
Warrior, 2002; Januschke et
al., 2002; Schnorrer et al.,
2000).
|
).
Establishment of the dorsoventral pattern of the embryo is dependent on two
sequential signaling pathways, the Gurken/Egf receptor pathway, which acts
during oogenesis, and the embryonic Toll-Dorsal signaling pathway
(Morisato and Anderson, 1995).
Loss of the Tgf ligand Gurken (Grk) or its receptor, the
Drosophila Egf receptor (Egfr) causes ventralization of both the
embryo and the surrounding eggshell
(Schüpbach, 1987). The
eggshell, which is made by the somatic follicle cells that surround the
developing oocyte, has a clear dorsoventral polarity, marked by a pair of
dorsal appendages at a dorsoanterior position on the eggshell
(Fig. 2D). The majority of eggs
produced by ik21 germline clones had a single, fused
dorsal appendage (Fig. 2E), and
some lacked dorsal appendages altogether
(Fig. 2F), such as grk
or Egfr mutants. Because both the embryos and the eggshells produced
by ik2 mutants were ventralized, like grk or Egfr
mutants, it seemed likely that ik2 affected the Grk/Egfr pathway.
By stage 9 of oogenesis, grk mRNA is localized as a cap around the
oocyte nucleus, in the dorsoanterior corner of the oocyte
(Fig. 3D)
(Neuman-Silberberg and Schüpbach,
1993). Grk protein is translated adjacent to the oocyte and
signals to the Egfr on nearby follicle cells to instruct those cells to adopt
a dorsal cell fate, which sets up the dorsoventral axis of the eggshell and
embryo. In the majority of the ik2alice oocytes
(Fig. 3E) and in all
ik21 oocytes (Fig.
3F), grk mRNA was localized to the anterior margin of the
oocyte but was never concentrated dorsally. The pipe gene, which is
required to activate the Toll ligand, is expressed only in ventral follicle
cells, as the result of repression by Egfr signaling in dorsal follicle cells
(Sen et al., 1998). During
stages 9-10 of oogenesis, pipe is expressed in ventral follicle
cells, corresponding to the future ventral side of the embryo where the
Toll-Dorsal signaling pathway will be activated
(Fig. 3G). By contrast,
pipe mRNA was expressed in both ventral and dorsal follicle cells in
the majority of the ik21 egg chambers analyzed
(Fig. 3H,I). Thus, the absence
of a source of grk mRNA at the dorsoanterior corner of ik2
mutant oocytes prevents the specification of dorsal follicle cells and causes
ventralization of the eggshell and embryo.
To confirm that the anteroposterior and dorsoventral defects observed in
ik2 mutants are the result of a genetic requirement for ik2
in oogenesis only, we also performed an epistasis analysis of pipe.
Females that are homozygous for recessive mutations in pipe produce
dorsalized embryos (Fig. 4C),
as the Toll ligand is never activated and ventral cell fates are never
specified (Sen et al., 1998).
We examined the embryos produced by mothers that carry germline clones of
ik21 in a
pipe1/pipe1 and a
pipe1/pipe2 mutant background. A
hundred percent of the embryos examined were dorsalized in a manner similar to
pipe mutants (Fig.
4D), suggesting that ik2 affects dorsoventral patterning
at a step upstream of the Toll-Dorsal pathway.
Abnormal minus-end directed microtubule transport in ik2 oocytes
Localization of mRNAs to the correct position within the oocyte depends on
microtubules and microtubule motors
(Brendza et al., 2000;
MacDougall et al., 2003;
Pokrywka and Stephenson, 1991;
Pokrywka and Stephenson,
1995). Although recent re-examinations of the oocyte microtubule
cytoskeleton have demonstrated that the bulk oocyte microtubules are non-polar
(Cha et al., 2001;
Cha et al., 2002), it has been
proposed that subsets of microtubules are used by microtubule motors to
localize mRNAs to specific subcellular regions
(MacDougall et al., 2003;
Januschke et al., 2006).
Localization of osk mRNA to the posterior pole depends on
kinesin-based transport, whereas grk mRNA localization to the
anterior dorsal corner depends on dynein-based transport. We therefore
evaluated the organization of the microtubule cytoskeleton and microtubule
motors in ik2 mutants.
|
), which
decorates all microtubules, revealed that the microtubule cytoskeleton is
abnormal in ik2 mutants. In wild-type stage 9 oocytes
(Fig. 5A) fixed to preserve the
microtubule cytoskeleton, Tau-GFP highlighted the non-polar microtubules in
the cytoplasm, but was excluded from the oocyte nucleus. In stage 9
ik2 mutant egg chambers (Fig.
5B), we observed staining throughout the ooplasm, but there were
abnormal aggregates of Tau-GFP at the circumference of the nucleus, which
suggested that a subpopulation of microtubules was disrupted in the
mutant.
After stage 7 of oogenesis, the plus-ends of microtubules are concentrated
at the posterior end of the oocyte, and kinesin, the plus-end directed motor,
is enriched at the posterior. In wild-type stage 9 egg chambers, the
Kinesin Heavy Chain-ß-galactosidase fusion
(KHC-ß-lacZ) transgene was localized to the posterior pole of
the oocyte (Fig. 5C)
(Clark et al., 1994). At stage
8-9, ik2alice (Fig.
5D) and ik21 (data not shown) mutant egg
chambers were indistinguishable from wild type. This indicates that
microtubule plus-ends are correctly concentrated at the posterior of the
oocyte in ik2 mutants at the time when the kinesin motor is actively
transporting osk mRNA to the posterior pole.
During mid-oogenesis, Dynein heavy chain (Dhc) accumulates at the posterior
of the oocyte (Fig. 5E)
(McGrail and Hays, 1997),
where it presumably is stored to allow repeated rounds of minus end-directed
transport. In ik2alice, ik21 and
BicD1/BicD2 mutant egg chambers, Dhc was
distributed along the lateral cortex of the oocyte
(Fig. 5F and data not shown)
and was sometimes enriched in the dorsal and ventral corners of the anterior
margin.
Although the plus-ends of the microtubules appeared normal in ik2
mutants, the position of the minus-ends was not. In most cells, microtubule
minus-ends are anchored in a microtubule organizing centre (MTOC) at the
centrosome, whereas the minus-ends in the Drosophila oocyte are
distributed along the anterior and lateral cortex, and are enriched in the
area around the oocyte nucleus (Theurkauf
et al., 1992; Cha et al.,
2002). A transgene that encodes a fusion protein of the motor-like
domain of Nod, a kinesin-related protein, with the coiled-coil domain of KHC
and ß-galactosidase has been used in previous studies as a marker of
microtubule minus-ends (Clark et al.,
1997). Although Nod preferentially binds microtubule plus-ends in
vivo (Matthies et al., 2001;
Cui et al., 2005), the
Nod-ß-gal transgene reliably localizes to the oocyte microtubule
minus-ends, probably as a result of sequences outside of the Nod motor-like
domain. At stage 8-10 in wild type, Nod-ß-gal is localized to the
anterior margin of the oocyte and is enriched in the dorsoanterior corner
adjacent to the oocyte nucleus (Fig.
5G). Nod-ß-gal was not detected at the anterior of stage 8-9
ik21 oocytes (Fig.
5H), and instead was present at low levels at the posterior and
lateral cortex; at later stages, Nod-ß-gal could not be detected in
ik21 oocytes (Fig.
5J). Thus, microtubule minus-ends are not properly distributed at
the anterior of ik2 mutant oocytes during mid-oogenesis.
ik2 mutations also disrupt the actin cytoskeleton
In addition to the microtubule abnormalities in ik2 mutant
oocytes, all of the rare ik2 escaper adults had abnormal bristles
with a morphology that suggested defects in the actin cytoskeleton
(Fig. 6). Bristles are
constructed with rings of membrane-attached, cross-linked actin bundles
(Tilney et al., 1995;
Tilney et al., 1996). At high
magnifications, the actin footprints along the length of the bristle shaft in
ik2 escaper adult eyes (Fig.
6F) appeared less organized than those in wild-type bristles
(Fig. 6E). Loss of activity of
specific actin-associated proteins, including Profilin (chickadee)
(Verheyen and Cooley, 1994b)
and the ß-subunit of capping protein
(Hopmann et al., 1996), causes
abnormal bristles similar to those seen in ik2 mutants. All of the
ik2 allelic combinations analyzed, as well as the alleles in trans to
the deficiency Df(2L)KetelRX32, which removes the
ik2 genomic region, displayed similar bristle phenotypes. Expression
of a UASp-ik2 transgene under the direction of either the ubiquitous
tubulin-Gal4 or daughterless-Gal4 driver rescued both the
viability and bristle abnormalities of ik2 allelic combinations
completely (data not shown), which confirmed that these phenotypes are caused
by loss of ik2.
|
|
), and we observed that Grk protein colocalized with the
ectopic actin in affected ik2 egg chambers
(Fig. 7H). Thus, both the actin
cytoskeleton and the anchoring of microtubule minus-ends are disrupted in
ik2 mutants.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B kinase in the Drosophila embryo. The embryonic
ventralization caused by loss of ik2 is the opposite of the phenotype
predicted for a Cactus kinase, and all effects of ik2 on the
dorsoventral pattern of the embryo can be explained by a loss of activity of
the Grk/Egfr pathway during oogenesis. Embryos that lack maternal activity of
both Drosophila IKKs, Ik2 and DmIkkß, are ventralized and are
indistinguishable from ik2 single mutants (data not shown), which
rules out the possibility that the Drosophila IKKs act in both the
Grk/Egfr and Toll pathways, and indicates that an unidentified kinase of
another family is required to target Cactus for degradation. Additional
experiments will be required to test whether ik2 plays other roles in
the immune response.
We found that instead of playing a role in Cactus degradation,
Drosophila ik2 is required for the localization of specific mRNAs
during oogenesis. Both the actin and microtubule cytoskeletons are disrupted
in ik2 mutants, and defects in microtubule-based transport are
sufficient to account for the defects in mRNA localization seen in
ik2 mutants. Because Drosophila ik2 is specifically required
for organization of the oocyte cytoskeleton, our results raise the possibility
that some of the NF-B-independent roles of the mammalian IKKs may act
through the cytoskeleton.
The embryonic patterning defects caused by the loss of ik2 function are due to the failure to transport all osk mRNA to the posterior pole of ik2 mutant oocytes, which leads to bicaudal embryos, and failure to localize grk mRNA to the dorsal anterior of the oocyte, which leads to ventralized embryos. Loss of ik2 has a milder effect on bcd mRNA localization; bcd is correctly localized in most oocytes, but is not tightly restricted to the anterior pole in a minority of cases.
Many lines of evidence indicate that osk localization to the
posterior pole depends on kinesin and gurken localization to the
dorsoanterior corner depends on dynein
(Brendza et al., 2000;
Cha et al., 2002;
MacDougall et al., 2003).
However, the kinesin and dynein motors in the oocyte are interdependent. For
example, posterior localization of dynein
(Fig. 5) and the anterodorsal
localization of gurken are both disrupted in Khc mutants
(Brendza et al., 2002), and
hypomorphic Dhc mutants have a reduced amount of
Khc-ß-gal at the posterior pole
(McGrail and Hays, 1997). Both
motor systems are at least partially functional in ik2 mutants: most
oskar is localized to the posterior pole of the oocyte (a
kinesin-dependent process) and grk mRNA is localized anteriorly (a
dynein-dependent process).
Several lines of evidence suggest that the RNA localization defects seen in
ik2 oocytes are associated with defects in a subset of
dynein-mediated, minus-end-directed transport processes. The movement of
grk mRNA to the dorsoanterior corner of the oocyte depends on two
sequential dynein-based movements: grk mRNA moves first to the
anterior of the oocyte along microtubules with plus-ends at the posterior pole
and minus-ends at the anterior, and then moves dorsally on microtubules with
minus-ends that form a cage around the oocyte nucleus
(MacDougall et al., 2003). The
dorsal movement of grk mRNA is specifically blocked in ik2
mutants, which would be consistent with a failure in this dynein-based
movement. Restriction of bcd mRNA to the anterior margin of the
oocyte, which is disrupted in some ik2 oocytes, depends on the
swallow gene product, which binds dynein light chain
(Schnorrer et al., 2000).
Overexpression of dynamitin disrupts dynein function and causes changes to the
localization of grk and bcd mRNA that are similar to the
phenotype of ik2 oocytes (Duncan
and Warrior, 2002; Januschke
et al., 2002). In addition, BicD mutations produce a
maternal effect phenotype similar to that of ik2. BicD is part of a
protein complex with dynein light chain in early oocytes, neuroblasts and the
early embryo (Bullock and Ish-Horowicz,
2001; Hughes et al.,
2004; Navarro et al.,
2004), and has been proposed to link cargo to microtubules in both
Drosophila and mammalian cells
(Hoogenraad et al., 2003;
Matanis et al., 2002).
Although this evidence links ik2 to a dynein transport system, the most penetrant phenotype in ik2 mutants is osk mislocalization and subsequent production of bicaudal embryos, a kinesin-dependent process. However, loss of ik2 function, like the BicD gain-of-function mutations, does not eliminate kinesin function, because the majority of osk mRNA accumulates at the posterior pole. Because the kinesin and dynein motors in the oocyte are interdependent, osk mRNA mislocalization could be caused by a decreased kinesin activity that is secondary to dynein disruption.
In addition to defects in minus-end-directed transport, the organization of the microtubules is also perturbed in ik2 oocytes (Fig. 5). The plus-ends of microtubules are localized correctly to the posterior pole of the oocyte. However, there are abnormal aggregates of microtubules around the oocyte nucleus, where a population of microtubule minus-ends is normally anchored, and the microtubule minus-end marker, Nod-ß-gal, is not localized at the anterior of the oocyte. These defects suggest that abnormal organization of microtubule minus-ends during mid-oogenesis could be the basis of the defect in minus-end-directed transport.
The adult bristles and ovaries of ik2 mutants also displayed
abnormalities in the actin cytoskeleton. The bristle defects are nearly
identical to those caused by mutations in actin-associated proteins
(Hopmann et al., 1996;
Verheyen and Cooley, 1994b),
or to bristles that were treated with F-actin-inhibitors
(Tilney et al., 2000).
Bristles contain a central core of microtubules, but mutations in the dynein
heavy chain gene Dhc64C or treatment with drugs that disrupt
microtubule dynamics do not cause bristle phenotypes like the thick, branched
bristles seen in ik2 mutants
(Gepner et al., 1996;
Tilney et al., 2000) (data not
shown). The actin cytoskeleton of the oocyte is also disrupted in ik2
mutants, with ectopic sites of actin polymerization in the ooplasm
(Fig. 7). These actin defects
are distinct from those caused by mutations that affect nurse cell ring canal
actin (Hudson and Cooley,
2002), which suggests that actin organization is not globally
disrupted in ik2 mutants and that the actin defects are restricted to
the oocyte cortex.
Recent data have defined two sets of microtubules in the oocyte that are
both nucleated from minus-ends at the centrosome associated with the oocyte
nucleus; one set remains associated with the oocyte nucleus, whereas the
remaining microtubules shift their minus-ends from the oocyte to the cortex
(Januschke et al., 2006). It
was suggested that translocation of the minus-ends of the latter set of
microtubules to the cortex could depend on actin and motor proteins
(Januschke et al., 2006). Our
data suggest that anchoring of microtubule minus-ends to the oocyte cortex
depends upon an Ik2-dependent interaction of microtubule minus-ends with the
F-actin network, analogous to the interaction of microtubule plus-ends with
the actin cytoskeleton through microtubule tip proteins
(Gundersen et al., 2004).
The phenotypes of ik2 in the ovary and adult bristles are very
similar to those caused by mutations in spn-F
(Abdu et al., 2006). Like
ik2 mutations, null mutations in spn-F affect the
localization of osk and grk mRNAs during oogenesis, and
cause bicaudal and ventralized embryos. spn-F mutant oocytes have
ectopic sites of F-actin polymerization, and spn-F bristles are
similar to ik2 mutant bristles. Ik2 and Spn-F have been shown to
interact in a yeast two-hybrid screen
(Giot et al., 2003), which
suggests that these proteins can form a complex. Spn-F associates specifically
with microtubule minus-ends (Abdu et al.,
2006). We therefore propose that Ik2 and Spn-F act together to
regulate interactions between the minus-ends of microtubules and the
actin-rich cortex.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abdu, U., Bar, D. and Schüpbach, T.
(2006). spn-F encodes a novel protein that affects
oocyte patterning and bristle morphology in Drosophila.Development 133,1477
-1484.
Anderson, K. V. (2000). Toll signaling pathways in the innate immune response. Curr. Opin. Immunol. 12, 13-19.[CrossRef][Medline]
Berleth, T., Burri, M., Thoma, G., Bopp, D., Richstein, S., Frigerio, G., Noll, M. and Nusslein-Volhard, C. (1988). The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 7,1749 -1756.[Medline]
Brendza, R. P., Serbus, L. R., Duffy, J. B. and Saxton, W.
M. (2000). A function for kinesin I in the posterior
transport of oskar mRNA and Staufen protein.
Science 289,2120
-2122.
Brendza, R. P., Serbus, L. R., Saxton, W. M. and Duffy, J. B. (2002). Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes. Curr. Biol. 12,1541 -1545.[CrossRef][Medline]
Bullock, S. L. and Ish-Horowicz, D. (2001). Conserved signals and machinery for RNA transport in Drosophila oogenesis and embryogenesis. Nature 414,611 -616.[CrossRef][Medline]
Castagnetti, S. and Ephrussi, A. (2003). Orb
and a long poly(A) tail are required for efficient oskar translation
at the posterior pole of the Drosophila oocyte.
Development 130,835
-843.
Cha, B. J., Koppetsch, B. S. and Theurkauf, W. E. (2001). In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway. Cell 106,35 -46.[CrossRef][Medline]
Cha, B. J., Serbus, L. R., Koppetsch, B. S. and Theurkauf, W. E. (2002). Kinesin I-dependent cortical exclusion restricts pole plasm to the oocyte posterior. Nat. Cell Biol. 4, 592-598.[Medline]
Chou, T. B. and Perrimon, N. (1992). Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131,643 -653.[Abstract]
Chou, T. B. and Perrimon, N. (1996). The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144,1673 -1679.[Abstract]
Clark, I., Giniger, E., Ruohola-Baker, H., Jan, L. Y. and Jan, Y. N. (1994). Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr. Biol. 4,289 -300.[CrossRef][Medline]
Clark, I. E., Jan, L. Y. and Jan, Y. N. (1997). Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle. Development 124,461 -470.[Abstract]
Cui, W., Sproul, L. R., Gustafson, S. M., Matthies, H. J. G.,
Gilbert, S. P. and Hawley, R. S. (2005). Drosophila
Nod protein binds preferentially to the plus ends of microtubules and promotes
microtubule polymerization in vivo. Mol. Biol. Cell
16,5400
-5409.
DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E. and
Karin, M. (1997). A cytokine responsive IB kinase that
activates the transcription factor NF-B. Nature
388,548
-554.[CrossRef][Medline]
Duncan, J. E. and Warrior, R. (2002). The cytoplasmic dynein and kinesin motors have interdependent roles in patterning the Drosophila oocyte. Curr. Biol. 12,1982 -1991.[CrossRef][Medline]
Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37-50.[CrossRef][Medline]
Erdelyi, M., Mathe, E. and Szabad, J. (1997). Genetic and developmental analysis of mutant Ketel alleles that identify the Drosophila importin-beta homologue. Acta Biol. Hung. 48,323 -338.[Medline]
Fernandez, N. Q., Grosshans, J., Goltz, J. S. and Stein, D.
(2001). Separable and redundant regulatory determinants in Cactus
mediate its dorsal group dependent degradation.
Development 128,2963
-2974.
Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C.,
Latz, E., Golenbock, D. T., Coyle, A. J., Liao, S. M. and Maniatis, T.
(2003). IKK and TBK1 are essential components of the IRF3
signaling pathway. Nat. Immunol.
4, 491-496.[CrossRef][Medline]
Gepner, J., Li, M., Ludmann, S., Kortas, C., Boylan, K., Iyadurai, S. J., McGrail, M. and Hays, T. S. (1996). Cytoplasmic dynein function is essential in Drosophila melanogaster.Genetics 142,865 -878.[Abstract]
Ghosh, S. and Karin, M. (2002). Missing pieces
in the NF-B puzzle. Cell
109,S81
-S96.[CrossRef][Medline]
Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B.,
Li, Y., Hao, Y. L., Ooi, C. E., Godwin, B., Vitols, E. et al.
(2003). A protein interaction map of Drosophila melanogaster.Science 302,1727
-1736.
Gundersen, G. G., Gomes, E. R. and Wen, Y. (2004). Cortical control of microtubule stability and polarization. Curr. Opin. Cell Biol. 16,106 -112.[CrossRef][Medline]
Hoogenraad, C. C., Wulf, P., Schiefermeier, N., Stepanova, T., Galjart, N., Small, J. V., Grosveld, F., de Zeeuw, C. I. and Akhmanova, A. (2003). Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport. EMBO J. 22,6004 -6015.[CrossRef][Medline]
Hopmann, R., Cooper, J. A. and Miller, K. G.
(1996). Actin organization, bristle morphology, and viability are
affected by actin capping protein mutations in Drosophila. J. Cell
Biol. 133,1293
-1305.
Hu, Y., Baud, V., Delhase, M., Zhang, P., Deerinck, T.,
Ellisman, M., Johnson, R. and Karin, M. (1999). Abnormal
morphogenesis but intact IKK activation in mice lacking the IKK subunit
of IB kinase. Science
284,316
-320.
Hu, Y., Baud, V., Oga, T., Kim, K. I., Yoshida, K. and Karin,
M. (2001). IKK controls formation of the epidermis
independently of NF-B. Nature
410,710
-714.[CrossRef][Medline]
Hudson, A. M. and Cooley, L. (2002). Understanding the function of actin-binding proteins through genetic analysis of Drosophila oogenesis. Ann. Rev. Genet. 36,455 -488.[CrossRef][Medline]
Hughes, J. R., Bullock, S. L. and Ish-Horowicz, D. (2004). Inscuteable mRNA localization is dynein-dependent and regulates apicobasal polarity and spindle length in Drosophila neuroblasts. Curr. Biol. 14,1950 -1956.[CrossRef][Medline]
Januschke, J., Gervais, L., Dass, S., Kaltschmidt, J. A., Lopez-Schier, H., St Johnston, D., Brand, A. H., Roth, S. and Guichet, A. (2002). Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation. Curr. Biol. 12,1971 -1981.[CrossRef][Medline]
Januschke, J., Gervais, L., Gillent, L., Keryer, G., Bornens, M.
and Guichet, A. (2006). The centrosome-nucleus complex and
microtubule organization in the Drosophila oocyte.
Development 133,129
-139.
Kim-Ha, J., Kerr, K. and Macdonald, P. M. (1995). Translational regulation of oskar mRNA by Bruno, an ovarian RNA-binding protein, is essential. Cell 81,403 -412.[CrossRef][Medline]
Kozlova, T., Pokholkova, G. V., Tzertzinis, G., Sutherland, J.
D., Zhimulev, I. F. and Kafatos, F. C. (1998).
Drosophila hormone receptor 38 functions in metamorphosis: a role in
adult cuticle formation. Genetics
149,1465
-1475.
Li, Q., Van Antwerp, D., Mercurio, F., Lee, K. F. and Verma, I.
M. (1999a). Severe liver degeneration in mice lacking the
IB kinase 2 gene. Science
284,321
-325.
Li, Z. W., Chu, W., Hu, Y., Delhase, M., Deerinck, T., Ellisman,
M., Johnson, R. and Karin, M. (1999b). The IKKß subunit
of IB kinase (IKK) is essential for nuclear factor B activation
and prevention of apoptosis. J. Exp. Med.
189,1839
-1845.
Lu, Y., Wu, L. P. and Anderson, K. V. (2001).
The antibacterial arm of the Drosophila innate immune response
requires an IB kinase. Genes Dev.
15,104
-110.
Luschnig, S., Moussian, B., Krauss, J., Desjeux, I., Perkovic,
J. and Nusslein-Volhard, C. (2004). An F1 genetic screen for
maternal-effect mutations affecting embryonic pattern formation in
Drosophila melanogaster. Genetics
167,325
-342.
MacDougall, N., Clark, A., MacDougall, E. and Davis, I. (2003). Drosophila gurken (TGFalpha) mRNA localizes as particles that move within the oocyte in two dynein-dependent steps. Dev. Cell 4,307 -319.[CrossRef][Medline]
Mahone, M., Saffman, E. E. and Lasko, P. F. (1995). Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1. EMBO J. 14,2043 -2055.[Medline]
Markussen, F. H., Michon, A. M., Breitwieser, W. and Ephrussi, A. (1995). Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly. Development 121,3723 -3732.[Abstract]
Matanis, T., Akhmanova, A., Wulf, P., Del Nery, E., Weide, T., Stepanova, T., Galjart, N., Grosveld, F., Goud, B., De Zeeuw, C. I. et al. (2002). Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex. Nat. Cell Biol. 4,986 -992.[CrossRef][Medline]
Matthies, H. J. G., Baskin, R. J. and Hawley, R. S.
(2001). Orphan kinesin NOD lacks motile properties but does
possess a microtubule-stimulated ATPase activity. Mol. Biol.
Cell 12,4000
-4012.
McGrail, M. and Hays, T. S. (1997). The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila.Development 124,2409 -2419.[Abstract]
McWhirter, S. M., Fitzgerald, K. A., Rosains, J., Rowe, D. C.,
Golenbock, D. T. and Maniatis, T. (2004). IFN-regulatory
factor 3-dependent gene expression is defective in Tbk1-deficient mouse
embryonic fibroblasts. Proc. Natl. Acad. Sci. USA
101,233
-238.
Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett,
B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A. et al.
(1997). IKK-1 and IKK-2: cytokine-activated IB kinases
essential for NF-B activation. Science
278,860
-866.
Micklem, D. R., Dasgupta, R., Elliott, H., Gergely, F., Davidson, C., Brand, A., Gonzalez-Reyes, A. and St Johnston, D. (1997). The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr. Biol. 7, 468-478.[CrossRef][Medline]
Mohler, J. and Wieschaus, E. F. (1986).
Dominant maternal-effect mutations of Drosophila melanogaster causing
the production of double-abdomen embryos. Genetics
112,803
-822.
Morisato, D. and Anderson, K. V. (1995). Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. Annu. Rev. Genet. 29,371 -399.[Medline]
Navarro, C., Puthalakath, H., Adams, J. M., Strasser, A. and Lehmann, R. (2004). Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate. Nat. Cell Biol. 6,427 -435.[CrossRef][Medline]
Neuman-Silberberg, F. S. and Schüpbach, T.
(1993). The Drosophila dorsoventral patterning gene
gurken produces a dorsally localized RNA and encodes a
TGF-like protein. Cell
75,165
-174.[CrossRef][Medline]
Neuman-Silberberg, F. S. and Schüpbach, T.
(1996). The Drosophila TGF-like protein Gurken:
expression and cellular localization during Drosophila oogenesis.
Mech. Dev. 59,105
-113.[CrossRef][Medline]
Nüsslein-Volhard, C., Frohnhofer, H. G. and Lehmann, R.
(1987). Determination of anteroposterior polarity in
Drosophila. Science 238,1675
-1681.
Peters, R. T. and Maniatis, T. (2001). A new
family of IKK-related kinases may function as IB kinase kinases.
Biochim. Biophys. Acta
1471,M57
-M62.[Medline]
Pokrywka, N. J. and Stephenson, E. C. (1991). Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis. Development 113, 55-66.[Abstract]
Pokrywka, N. J. and Stephenson, E. C. (1995). Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev. Biol. 167,363 -370.[CrossRef][Medline]
Queenan, A. M., Barcelo, G., Van Buskirk, C. and Schüpbach, T. (1999). The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89, 35-42.[CrossRef][Medline]
Rongo, C., Gavis, E. R. and Lehmann, R. (1995). Localization of oskar RNA regulates oskar translation and requires Oskar protein. Development 121,2737 -2746.[Abstract]
Rørth, P. (1998). Gal4 in the Drosophila female germline. Mech. Dev. 78,113 -118.[CrossRef][Medline]
Saffman, E. E., Styhler, S., Rother, K., Li, W., Richard, S. and
Lasko, P. (1998). Premature translation of oskar in
oocytes lacking the RNA-binding protein Bicaudal-C. Mol. Cell.
Biol. 18,4855
-4862.
