Copyright Ó 2007 by the Genetics Society of America
DOI: 10.1534/genetics.107.071472
The Molecular Chaperone Hsp90 Is Required for mRNA Localization in
Drosophila melanogaster Embryos
Yan Song,*,1 Lanette Fee,* Tammy H. Lee* and Robin P. Wharton*,†,2
*Howard Hughes Medical Institute, Department of Molecular Genetics & Microbiology and †Department of Cell Biology,
Duke University Medical School, Durham, North Carolina, 27710
Manuscript received January 29, 2007
Accepted for publication May 25, 2007
ABSTRACT
Localization of maternal nanos mRNA to the posterior pole is essential for development of both the
abdominal segments and primordial germ cells in the Drosophila embryo. Unlike maternal mRNAs such as
bicoid and oskar that are localized by directed transport along microtubules, nanos is thought to be trapped as
it swirls past the posterior pole during cytoplasmic streaming. Anchoring of nanos depends on integrity of the
actin cytoskeleton and the pole plasm; other factors involved specifically in its localization have not been
described to date. Here we use genetic approaches to show that the Hsp90 chaperone (encoded by Hsp83 in
Drosophila) is a localization factor for two mRNAs, nanos and pgc. Other components of the pole plasm are
localized normally when Hsp90 function is partially compromised, suggesting a specific role for the
chaperone in localization of nanos and pgc mRNAs. Although the mechanism by which Hsp90 acts is unclear,
we find that levels of the LKB1 kinase are reduced in Hsp83 mutant egg chambers and that localization of pgc
(but not nos) is rescued upon overexpression of LKB1 in such mutants. These observations suggest that LKB1
is a primary Hsp90 target for pgc localization and that other Hsp90 partners mediate localization of nos.
S
UBCELLULAR localization of mRNA is an efficient
strategy for spatially restricting the encoded protein ( Johnstone and Lasko 2001; St. Johnston 2005).
This strategy is common in highly polarized cell types
such as neurons, as well as during the development of
oocytes or embryos when gene regulation is limited
to post-transcriptional mechanisms. Asymmetric RNA
localization prior to mitosis also serves to distinguish
daughter cells in Saccharomyces cerevisiae. In this organism, localization of ASH1 mRNA to buds just prior to
cytokinesis currently provides the most completely understood example of mRNA localization (Niessing et al.
2004; Gonsalvez et al. 2005).
Localization of maternal mRNAs drives much of the
patterning along the anteroposterior axis of the Drosophila embryo. The anterior determinant, bicoid (bcd)
mRNA, is localized during oogenesis as ribonucleoprotein (RNP) cargo associated with molecular motors that
traverse the microtubule cytoskeleton (Riechmann et al.
2002; Schnorrer et al. 2002; Snee et al. 2005; Weil et al.
2006). Posterior patterning is nucleated by the localization of oskar (osk) mRNA to the posterior pole of the
oocyte, also via directed movement along microtubules
1
Present address: Department of Pathology, Stanford University School
of Medicine, Stanford, CA.
2
Corresponding author: Howard Hughes Medical Institute, Department
of Molecular Genetics & Microbiology, Box 3657, Duke University
Medical School, Durham, NC 27710. E-mail: [email protected]
Genetics 176: 2213–2222 (August 2007)
(Cha et al. 2002; Braat et al. 2004; Huynh et al. 2004;
Yano et al. 2004). Localization of both bcd and osk mRNAs
is thought to occur via complex, multistep pathways
with many components.
One role of localized Osk is to direct the subsequent
localization of a fraction of the nanos (nos) mRNA late in
oogenesis (Bergsten and Gavis 1999). Localized nos
mRNA is the sole source of Nos protein in the early
embryo (Gavis and Lehmann 1992) where it plays a
number of key roles in development. Nos is required in
the somatic cytoplasm of the early embryo to repress
translation of maternal hunchback mRNA, thereby governing abdominal segmentation (Sonoda and Wharton
1999). Nos is also required in the primordial germ cells
that form at the posterior extreme of the embryo to delay proliferation, repress transcription, facilitate migration into the somatic gonad, and promote survival
(Kobayashi et al. 1996; Forbes and Lehmann 1998;
Asaoka-Taguchiet al. 1999; Schaner et al. 2003; Hayashi
et al. 2004; Kadyrova et al. 2007). The germ line functions of Nos appear to be conserved in many organisms
(Subramaniam and Seydoux 1999; Tsuda et al. 2003).
The role of Osk in nos mRNA localization is indirect;
Osk governs assembly of the pole plasm (specialized
cytoplasm that specifies germ line identity) via an
elaborate, genetically defined pathway in which recruitment of nos mRNA is one of the final steps (Ephrussi
and Lehmann 1992; Kim-Ha et al. 1993). Recruitment is
thought to involve trapping of nos RNPs as they swirl
past the posterior pole during cytoplasmic streaming
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Y. Song et al.
Figure 1.—A dominant modifier screen for nos
mRNA localization factors. Shown schematically
are the 39-UTRs of wild-type nos1 mRNA and a
mini-nos1 mRNA that lacks nt 185-849. The first
column shows a qualitative assessment of the relative efficiency of mRNA localization to the posterior pole (see Figure 2), the second column
shows the typical number of abdominal segments
in embryos where the sole source of Nos is the indicated mRNA, and the third column is a drawing
of the body plan after 24 hr of embryonic development. The rationale for the screen is that an EMSinduced mutation (*) would dominantly reduce
segmentation such that embryos do not hatch
(and thus remain within the vitelline membrane).
(Forrest and Gavis 2003; Serbus et al. 2005), rather
than the directed movement that underlies localization
of osk or ASH1 RNPs. The factors involved specifically in
localizing nos mRNA have yet to be identified.
In this report, we describe the results of a genetic
screen for nos mRNA localization factors that rely on
the abdomen-patterning role of Nos.
MATERIALS AND METHODS
Isolation and mapping of the 966 mutation: Homozygous
w1118 ; e nosBN males were mutagenized with EMS and mated
en masse with w1118 virgin females. Male progeny were individually crossed to generate P½mini-nos1, nosBN/e nosBN * females,
which were screened for the ability to give rise to hatching
progeny. The P½mini-nos1 transgene is described by Dahanukar
and Wharton (1996), where it is named nos1(DBX). Candidate mutant chromosomes (*) were recovered from sibling
males for further testing. Meiotic recombination with a rucuca
chromosome placed 966 between ru and h on the left arm of
the third chromosome. Fine mapping by P element-induced
male recombination further mapped 966 to a 57-kb interval
between P{SUPor-P}KG05210 and P{SUPor-P}KG00982. The
966 mutation was definitively identified by sequencing the
Hsp90 coding region amplified from genomic DNA extracted
from homozygous 966 larvae (identified by the absence of a
GFP-marked balancer chromosome).
Fly strains and reagents: The following strains were from
the Bloomington Stock Center: Hsp83 alleles scratch (08445),
E317K (e6D), S529F (e6A), and j5C2A; transformants bearing the
17.5 genomic Hsp83 rescue construct; flies with the P{SUPorP}KG05210, P{SUPor-P}KG00982, P{SUPor-P}KG03657, and
P{SUPor-P}KG07503 elements used in male recombination.
Flies with the maternal tubulin-GAL4 driver as well as the GFPLKB1 transgene (Huynh et al. 2001; Martin and St. Johnston
2003) were from D. St. Johnston; flies with the P{GAL4-arm.S}11
armadillo-GAL4 driver were from Bloomington. Antibodies
against various proteins were gifts of P. Macdonald (Hb), A.
Nakamura (Nos), D. St. Johnston (Stau), A. Ephrussi (Osk), and
K. Howard (Vas).
Immunohistochemistry: Egg chamber fixation and antigen
detection were performed as described (Palacios and St.
Johnston 2002). Primary antibodies were diluted as follows:
chicken anti-Vas 1:2000, rabbit anti-Osk 1:2000, rabbit antiStau 1:2000, rat anti-Hb (1:500), and rabbit anti-Nos 1:1000.
FITC-, rhodamine-, or Texas-red-conjugated secondary antibodies ( Jackson Laboratories) were used at 1:200. Nuclei were
stained with TOTO-3, oligreen, or TOPRO-3 (Molecular
Probes). Samples were mounted in Vectashield and imaged
on a Zeiss LSM510 confocal microscope. For embryo staining,
primary antibodies were diluted as follows: rat anti-Hb (1:500),
rabbit anti-Nos 1:1000, rabbit anti-Osk 1:2000. In situ hybridization was by standard methods using digoxigenin-labeled
dsDNA probes prepared from cDNA clones. The adducin-like/
hts probe was from clone N4 (Ding et al. 1993).
Western and Northern blots: Homozygous 966 mutant
larvae (e.g., non-Tubby) were identified shortly after hatching
and grown under noncrowded conditions. Samples from
whole larvae homogenized in SDS sample buffer were analyzed following transfer to Immobilon P by standard methods.
Hsp90 was detected with the 3E6-1.92 monoclonal antibody,
a gift from R. Tanguay (Carbajal et al. 1990), and ECL Plus
(Amersham). The loading control was a-tubulin, detected
with DM 1A monoclonal antibody (Sigma F-2168) and ECL
(Amersham). For Northern blots, 5-mg samples of total RNA
prepared from 0- to 4.5-hr embryos were analyzed by standard
methods using radio-labeled probes to detect smaug mRNA
(as a loading control) and mini-nos1 mRNA. Quantitation
was performed on a Typhoon phosphoimager.
RESULTS
A genetic screen for nos localization factors: To
identify factors involved in nos mRNA localization, we
chemically mutagenized flies and screened for dominant maternal-effect mutations that (further) compromise abdominal segmentation in a sensitized background.
The rationale for the screen is shown in Figure 1.
Inefficient localization and translation of a mini-nos1
mRNA generates only sufficient Nos activity to allow
development of 5–6 abdominal segments if the endogenous nos genes are mutant (Dahanukar and Wharton
1996). In such a background, we reasoned that a mutation in one of the two alleles encoding a localization
factor might compromise Nos activity sufficiently to preclude hatching (either because the allele is dominant
negative or the gene is haplo-insufficient). A similar
rationale has been used extensively in screens based on
changes in morphology of the Drosophila eye (e.g., Rebay
et al. 2000).
From a pilot screen of 6000 EMS-mutagenized third
chromosomes, we isolated a number of mutations that
Hsp90 As an mRNA Localization Factor
2215
Figure 2.—The 966 mutation dominantly interferes with localization of mini-nos1 mRNA. (A)
Northern blot of samples from 0 to 4.5 hr nosBN/
P½mini-nos1, nosBN (lane 1) and 966 nosBN/
P½mini-nos1, nosBN (lane 2) embryos to detect
smaug and mini-nos1 mRNAs. The mobility of molecular weight markers is indicated on the left. (B)
The distributions of mini-nos1 mRNA during embryonic stages 2 (early cleavage, first row) and 4
(syncytial blastoderm, second row), Hb protein
in nuclear cycle 10 (third row), and the resulting
embryonic body plan (darkfield micrographs of secreted cuticle in the fourth row) are shown. Here
and in all subsequent figures, embryos and egg
chambers are oriented anterior to the left and dorsal up. Note that the enzymatic reaction used to
detect hybridized digoxigenin-labeled probe was
nearly twice as long for the embryos in row 2 (vs.
row 1) due to the presence of reduced mRNA levels; control experiments suggest that an appreciable fraction of the signal in the somatic cytoplasm is due to background at the longer reaction time. In the cuticle photographs,
abdominal segments are marked by the presence of bands of large denticles that are white in darkfield; the eighth abdominal segment
is highlighted with an arrowhead on the left. The maternal genotypes are indicated above.
reduce or eliminate abdominal segmentation in the
sensitized background. One mutation proved to be an
allele of pumilio and another an allele of spindle-E; both
genes are known to play roles in posterior specification
(Lehmann and Nüsslein-Volhard 1987; Gillespie
and Berg 1995; Martin et al. 2003; Cook et al. 2004),
validating the premise of the screen. A third mutation,
966, is the subject of this report.
The 966 mutation appears to affect the localization
but not the synthesis or stability of mini-nos1 mRNA
(Figure 2). Both the analysis of Northern blots (Figure
2A) and examination of the unlocalized mini-nos1
mRNA in embryos hybridized with digoxigenin-labeled
probes (Figure 2B) support the idea that mini-nos1
mRNA stability is unaffected by the 966 mutation. In
contrast, localization of mini-nos1 mRNA is defective
from the beginning of embryonic development (stage
1) through formation of the syncytial blastoderm (stage
4) in embryos from heterozygous 966 mutant females
(Figure 2B). Because Nos protein is generated exclusively from translation of localized mRNA, the embryos
from 966 heterozygotes apparently have reduced Nos
activity, because Hunchback (Hb) accumulates in the
posterior and they subsequently fail to develop abdominal segments (Figure 2B). The 966 allele is homozygous
lethal and germ line clones are rudimentary, precluding
analysis of embryos derived from homozygous females.
We examined the distribution of other mRNAs in
embryos from heterozygous 966 mutant females (hereafter 966 mutant embryos) to determine whether the
defects in mini-nos1 mRNA localization are specific. As
shown in Figure 3, the localization of full-length nos1
Figure 3.—The 966 mutation does not significantly affect localization of other mRNAs in the
early embryo. The distributions of various mRNAs
(indicated on the left) are shown in stage 2 embryos. Insets on rows 1 and 2 show the posterior
of stage 4 embryos. The maternal genotypes are indicated above. Note that other experiments show
that the distributions of pgc, bcd, and add-like mRNAs
are unaffected by the nos genotype (not shown).
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Y. Song et al.
mRNA is essentially normal in early 966 mutant embryos, although the pole cells in slightly older embryos
appear to retain somewhat reduced levels of mRNA. In
contrast, localization of pgc mRNA to the posterior or
bicoid and adducin-like mRNAs to the anterior is indistinguishable in wild-type and 966 mutant embryos.
We next wished to determine whether the 966 gene
product acts late in the posterior pathway (when nos
mRNA is localized) or early, perhaps acting indirectly to
govern oocyte polarity and accumulation of Osk, for
example. Three observations suggest that the 966 gene
product acts downstream of Osk. First, Osk accumulation is normal in 966 mutant embryos (Figure 4A). Second, Osk activity appears normal in 966 mutant embryos
by the criterion that they have a similar number of pole
cells as wild-type embryos (data not shown). The formation of pole cells is known to be a sensitive indicator of
Osk activity (Ephrussi and Lehmann 1992; Smith et al.
1992). Third, we find that the 966 mutation interferes
with localization of mini-nos1 mRNA to the anterior of
osk-bcd embryos (Figure 4B). In osk-bcd embryos, a chimeric mRNA (bearing the protein-coding region of osk
and the localization signals of bcd) generates Osk activity
at the anterior of the embryo independent of the upstream factors that regulate localization and translation
of native osk mRNA. This ectopic Osk directs efficient
localization and translation of full-length nos1 mRNA,
resulting in the suppression of head and thoracic segments, which are replaced by a mirror symmetric duplication of abdominal segments (Ephrussi and Lehmann
1992). We find that mini-nos1 mRNA is inefficiently localized to the anterior of osk-bcd embryos where it apparently is translated into only enough Nos to suppress
head segmentation (Figure 4B). If, in addition, the females are heterozygous for 966, then recruitment of
mini-nos1 mRNA to the anterior is almost eliminated
and the suppression of anterior development by ectopic
Nos is largely relieved (Figure 4B).
Taken together, the results described above suggest
that the 966 mutation alters the function of a factor that
is required downstream of Osk to localize mini-nos1
mRNA.
Hsp90 requirement for mRNA localization: We
mapped the 966 mutation (primarily scoring the associated homozygous lethality) using deficiencies, meiotic
recombination, and P element-induced male recombination. In the course of these experiments, we discovered that 966 is semilethal in trans to the scratch (stc)
allele (Yue et al. 1999) of Hsp83, which encodes the
highly conserved Hsp90 chaperone. Two additional
lines of evidence demonstrate that 966 is indeed an
allele of Hsp83. First, the Hsp83 gene on the 966 chromosome bears a single nucleotide substitution that
results in an alanine to aspartate substitution at a highly
conserved residue (133) in the N-terminal ATPase
domain (Figure 5A). Second, two independently isolated Hsp83 alleles that encode missense forms of the
Figure 4.—The 966 gene product acts downstream of Oskar in the posterior pathway to localize mini-nos1 mRNA. (A)
The distribution of Osk in stage 2 embryos, maternal genotypes as indicated. (B) The distribution of mini-nos1 mRNA
in stage 2 embryos and anterior cuticle (phase-contrast micrographs) of mature embryos from females of the indicated genotype. Note that virtually the entire head skeleton is absent
in the embryo on the left, with only a trace of sclerotized cuticle in evidence (arrowhead). With the exception of minor
defects in the dorsal bridge (arrowhead), the head skeleton
(arrowhead) in the embryo on the right is wild type.
Hsp90 protein (E317K and S592F) (Cutforth and
Rubin 1994; van der Straten et al. 1997) suppress
abdominal segmentation in the mini-nos1 background
in a manner similar to 966 (Figure 5A). Thus, Hsp90
function is required for normal localization of mini-nos1
mRNA.
We next asked whether the A133D allele acts in a
dominant negative or haplo-insufficient manner to
modify mini-nos1-dependent segmentation. The E317K
and S592F alleles were identified in screens for dominant modifiers of kinase-dependent eye phenotypes;
since they also modify mini-nos1 activity, we suspected
that the A133D mutant and the other missense mutants
act as dominant negatives for mini-nos1 localization.
Consistent with such an idea, a presumptive null allele
of Hsp83, j5C2A, which bears a P-element in the first
(nonprotein-coding) exon, does not dominantly modify the mini-nos1 segmentation phenotype. Moreover,
rescue of the segmentation phenotype caused by the
A133D allele is inefficient. A single wild-type Hsp83
transgene rescues very poorly, yielding embryos with 1 to
2 abdominal segments; two copies of the wild-type Hsp83
transgene are required for full rescue (Figure 5B).
Finally, we find that the A133D mutant protein accumulates to wild-type levels in extracts prepared from
larvae 1 day before they die (Figure 5B). Taken
together, these results suggest that the A133D missense
protein dominantly interferes with wild-type Hsp90 with
respect to the mini-nos1 segmentation phenotype.
To further investigate the role of Hsp90 in localization of maternal mRNAs in general and full-length nos1
Hsp90 As an mRNA Localization Factor
2217
Figure 5.—966 is a dominant
negative allele of Hsp83. (A) At
the top is a schematic representation of the domains of Hsp90
and the positions of missense substitutions encoded by the 966 allele and the two other alleles
used in these studies. Abdominal
segmentation is visualized in darkfield photographs of cuticle secreted by embryos from females
heterozygous for the indicated
Hsp83 allele (except in row 1,
which is from an Hsp831/Hsp831
female). The females here and in
B are also P½mini-nos1, nosBN/nosBN,
such that the sole source of Nos
in embryos is from the mini- nos1
transgene. (B) The A133D protein
acts in a dominant negative fashion.
Cuticle of embryos from females
heterozygous for the indicated
Hsp83 allele, as in A. Embryos in
rows 2 and 3 are from females that
also bear one and two extra copies of an Hsp831 transgene, respectively. Note that one copy of the transgene does not efficiently rescue
the lethality of Hsp83A133D/Hsp83stc animals, consistent with the idea that this allele, like other missense alleles of Hsp83 (Cutforth and
Rubin 1994; van der Straten et al. 1997), is dominant-negative. Western blot of extracts (each containing 5 mg of protein) prepared
from w1118 (lane 1), nosBN/TM3 (lane 2), and 966 nosBN/966 nosBN (lane 3) larvae 72–74 hr after egg laying. Following transfer, the
membrane was cut in half and probed separately with antibodies against Hsp90 and a-tubulin.
mRNA in particular, it was necessary to bypass the
requirement of Hsp90 function for viability. A comprehensive study of Hsp83 alleles had shown that several
trans-heterozygous allelic combinations are viable and
fertile (Yue et al. 1999). We reinvestigated the issue and
found that Hsp83stc/Hsp83E317K females are reasonably
healthy and produce large numbers of eggs, of which
1–2% develop through late embryonic stages. We therefore used this genetic background to investigate the
consequences of impairing maternal Hsp90 function
on mRNA localization.
As shown in Figure 6, localization of full-length nos1
mRNA is defective in 90–95% of embryos when maternal Hsp90 function is compromised. The phenotype is
heterogeneous and includes nearly normal localization
of a reduced amount of nos RNA to a crescent along the
posterior cortex, localization of a ‘‘ball’’ of nos mRNA
only partially in contact with the posterior cortex,
diffuse localization of a cloud near the posterior, and
no detectable localization. These defects in mRNA
localization have predictable effects on subsequent
development: essentially all Hsp83stc/Hsp83E317K embryos
have reduced levels of Nos protein (Figure 6). Very few
of these embryos cellularize, presumably due to some
other requirement(s) for maternal Hsp90 function. But
the 1–2% of embryos that eventually secrete cuticle have
on average 3 abdominal segments rather than the
normal complement of 8 (Figure 6). Thus, maternal
Hsp90 is critical for posterior localization of nos1 mRNA.
We next wished to determine whether localization of
other maternal mRNAs also relies on Hsp90. Accord-
ingly, we examined the distributions of polar granule
component (pgc), germcell-less (gcl), CyclinB (CycB) (Lehner
and O’Farrell 1990; Jongens et al. 1992; Nakamura
et al. 1996), and bcd mRNAs in Hsp83stc/Hsp83E317K embryos. Of these, only pgc mRNA localization is abnormal
(Figure 6 and not shown). Localization of pgc mRNA is
affected to a greater extent than is localization of nos
mRNA. Because Hsp90 activity is only partially ablated in
the Hsp83stc/Hsp83E317K egg chambers, we do not know
whether the posterior localization of gcl and CycB mRNAs
and the anterior localization of bcd mRNA rely on lower
Hsp90 activity or whether they are Hsp90 independent.
However, the significant observation is that proper deployment of only a subset of the late-localizing posterior
mRNAs (nos and pgc) requires normal Hsp90 activity.
Hsp90 is thought to interact with hundreds of proteins in most cells (Millson et al. 2005; Zhao et al. 2005)
and Hsp83 mutants are highly pleiotropic (Yue et al.
1999). We therefore considered whether the defects in
nos and pgc localization might be quite indirectly caused
by earlier defects in the assembly of pole plasm components at the posterior of the egg chamber. To this
end, we examined the localization of upstream components of the posterior pathway in Hsp83stc/Hsp83E317K egg
chambers where Hsp90 activity is compromised.
Reduction of Hsp90 activity has a relatively specific
effect on the posterior localization of nos and pgc mRNAs.
The initial localization of three key factors that act upstream of nos is essentially normal in Hsp83stc/Hsp83E317K
egg chambers (Figure 7). These include: (1) Stau protein
½an effective proxy for osk mRNA (Martin et al. 2003),
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Y. Song et al.
Figure 6.—Hsp90 is required for localization of nos and pgc
mRNAs. The distributions of various mRNAs and proteins
(except in row 3, which shows cuticle secreted by mature embryos) in stage 2 embryos from wild-type (w1118) and Hsp83stc/
Hsp83E317K females. Many of the Hsp83stc/Hsp83E317K embryos
have terminal defects not readily visible in the photograph,
consistent with previous reports that Hsp90 is a cofactor of
the Raf kinase (van der Straten et al. 1997), which acts
downstream of Torso to specify terminal identity in the embryo. The embryos in row 5 were hybridized simultaneously
with probes to bcd (the prominent signal at the anterior)
and pgc (the low-level unlocalized signal as well as the posterior cap that is present in wild type but not Hsp83 mutant embryos). Note that many Hsp83 mutant embryos have an
abnormal shape, with elongated anterior ends to which the
vitelline membrane and micropyle frequently remain attached (particularly evident in rows 4 and 5).
(2) Osk protein, and (3) Vasa protein. Since accumulation of Osk is acutely interdependent with localization
of osk mRNA, Vasa, and the Par-1 kinase (Breitwieser
et al. 1996; Riechmann et al. 2002; Benton and St.
Johnston 2003; Johnstone and Lasko 2004), the data
presented in Figure 7 suggest that pole plasm assembly
is essentially normal until the late recruitment of nos and
pgc. Oogenesis appears grossly normal in the Hsp83stc/
Hsp83E317K background, which suggests that polarization
of the microtubule cytoskeleton is likely to be normal.
This idea is further supported by the normal initial localization of Stau to the posterior and the maintenance
of bcd mRNA at the anterior, both dependent critically
on microtubule integrity (Pokrywka and Stephenson
1991; Brendza et al. 2000; Weil et al. 2006). Not only
is the initial formation of pole plasm normal, but also its
integrity is maintained in Hsp83stc/Hsp83E317K embryos,
based on the normal posterior localization of Osk and
Vasa (Figure 7). Distribution of the latter protein was
detected in double-staining experiments, which show
that Hsp83stc/Hsp83E317K embryos with very little or no
detectable Nos have a normal crescent of Vasa at the
posterior pole. Thus, both the initial formation and
maintenance of the pole plasm appear unperturbed in
Hsp83stc/Hsp83E317K flies.
Taken together, the observations outlined above lead
us to conclude that, despite its general pleiotropy,
Hsp90 plays a relatively specific role for the localization
of nos and pgc mRNAs to the posterior of the embryo.
Rescue of pgc mRNA localization in Hsp83 mutant
embryos by overexpression of LKB1: Hsp90 is a molecular chaperone that activates and stabilizes a wide variety
of client regulatory and signaling proteins (Pearl and
Prodromou 2001); a priori, it seemed unlikely that Hsp90
interacts directly with nos and pgc mRNAs. Therefore, one
approach to further understanding its role in mRNA
localization would be to identify molecules whose activity is dependent on Hsp90. To date, none of the other
genetic modifiers identified in the screen that yielded
the 966 allele of Hsp83 encodes an obvious Hsp90 client.
Therefore, we turned to a candidate gene approach, focusing on protein kinases previously implicated in various aspects of posterior patterning. Two such proteins are
Par-1 and LKB1, which are required for polarization of
the oocyte microtubule cytoskeleton and the proper deposition of osk mRNA at the posterior (Shulman et al.
2000; Tomancak et al. 2000; Martin and St. Johnston
2003; Doerflinger et al. 2006).
Two lines of evidence suggest that LKB1 is a significant Hsp90 client for the localization of pgc mRNA. First,
the level of GFP-LKB1 is significantly reduced when
Hsp90 activity is compromised in essentially all Hsp83stc/
Hsp83E317K egg chambers (Figure 8A). In contrast, no
consistent effect of reducing Hsp90 activity is seen on
levels of GFP-Par-1 (not shown). Second, overexpression of LKB1 in Hsp83stc/Hsp83E317K females significantly
rescues the localization of pgc mRNA (Figure 8B). For
this experiment, we used the armadillo-GAL4 driver to
achieve low-level overexpression of GFP-LKB1 in the ovaries, as previously described (Martin and St. Johnston
2003). The rescue of pgc localization does not appear to
be due to an indirect elevation of posterior Osk levels,
which are normal during both oogenesis and early embryogenesis (Figure 8B); these observations are consistent
with the previous finding that up to 10-fold overexpression of LKB1 has no significant effect on localization of the
pole plasm component Stau (Martin and St. Johnston
2003). In contrast to the rescue of pgc, no significant rescue of nos localization was observed (Figure 8B). Similar
negative results were obtained using nos-GAL4-VP16 to
drive higher level expression of LKB1 in the germ line
(Van Doren et al. 1998) (not shown). Taken together,
these observations suggest that, for localization of pgc
mRNA, a major function of Hsp90 is to stabilize LKB1.
Presumably other Hsp90 partners or targets mediate localization of nos mRNA.
Hsp90 As an mRNA Localization Factor
2219
Figure 7.—Assembly and maintenance of the
pole plasm in Hsp83 mutants. The distributions
of various proteins in stage 10B egg chambers
or stage 2 embryos from wild-type (w1118) and
Hsp83stc/Hsp83E317K females. Nuclei in rows 1–3
are in blue, green, and red, respectively. Note
that the initial accumulation of Osk at the posterior is slightly defective in some stage Hsp83 mutant egg chambers at stage 7–8; however, by stage
9–10, the level of posterior Osk in all mutant egg
chambers is indistinguishable from that in wild
type. Also note that the level of Osk in many
Hsp83 mutant embryos is higher for reasons we
do not understand. As a result, the protein is detectable further toward the anterior than in wildtype embryos. In row 5, distributions of Vasa
(red) and Nos (green) at the posterior of stage
2 embryos are shown, with both channels overlaid
in the third image for each genotype.
DISCUSSION
The specific role that maternal Hsp90 plays in
localization of a subset of mRNAs to the pole plasm is
somewhat surprising, given the number of proteins that
are thought to require the activity of this chaperone.
Although ubiquitously distributed, Hsp90 is enriched
in the testes and ovaries and the male germ line is particularly sensitive to a reduction in Hsp90 activity (Yue
et al. 1999). The experiments reported here define a role
for maternal Hsp90 in the localization of nos and pgc
mRNAs. We do not yet know the mechanism by which
Hsp90 acts. However, the apparent integrity of the pole
plasm in Hsp83stc/Hsp83E317K ovaries and embryos (Figures 6 and 7) and the rescue of pgc localization upon
overexpression of a single kinase (Figure 8) are consistent with the idea that the defects in pgc (and perhaps
also nos) localization arise from the reduction in activity
of a few discrete Hsp90 clients. Hsp83 mRNA is itself
concentrated in the pole plasm (Ding et al. 1993) and
Hsp90 is found at particularly high levels in the germ
line precursors of other organisms (Vanmuylder et al.
2002; Inoue et al. 2003), which may reflect a conserved
role in mRNA localization.
We do not know whether Hsp90 acts directly or
indirectly to stabilize LKB1. Mammalian LKB1 binds
directly to Hsp90 and Cdc37, a cochaperone for kinase
clients (Boudeau et al. 2003). However, we have not
observed a direct interaction between Drosophila Hsp90
and LKB1, either by co-immunoprecipitation or in yeast
interaction experiments in which the DNA-binding
domain was fused to the Hsp90 C terminus to avoid
interfering with dimerization, as described (Millson
et al. 2005). We do not currently know whether Hsp90
binding to LKB1 is ephemeral (and thus difficult to
detect) or whether Hsp90 acts indirectly to stabilize
LKB1.
How might LKB1 act to localize pgc mRNA? Despite
its conserved role in regulation of cellular polarity (see
Alessi et al. 2006 and references therein), no LKB1
substrate that plays a direct role in mRNA localization
has been described, to our knowledge. Loss of LKB1
function in germ line clones of presumptive null alleles
prevents the reorganization of the oocyte microtubule
network at stage 7 that is required for posterior localization of osk mRNA and affects epithelial polarity in
the ovarian follicle cells (Martin and St. Johnston
2003). LKB1 colocalizes with cortical actin in the oocyte,
integrity of which is required for anchoring of pole
plasm components and nos mRNA (Lantz et al. 1999;
Forrest and Gavis 2003). It is therefore attractive to
speculate that LKB1 might act at the cortex, where actin
and microtubule filaments meet, phosphorylating a
currently unknown substrate to promote the trapping
of pgc-containing RNPs. Our results suggest that the
level of LKB1 in Hsp83stc/Hsp83E317K flies is insufficient
for pgc localization but sufficient for viability as well as
proper polarization of microtubules during oogenesis
and localization of osk. According to this idea, LKB1
2220
Y. Song et al.
Figure 8.—Overexpression of
the LKB1 kinase rescues pgc
mRNA localization in Hsp83 mutant embryos. (A) The distribution of GFP-LKB1 in sibling wild
type (Hsp83/1) or Hsp83stc/
Hsp83E317K stage 10B egg chambers. (B) The distributions of
pgc mRNA, Osk, and nos mRNA
are shown in stage 2 embryos or
stage 10B egg chambers for three
maternal genotypes: wild type
(w1118), Hsp83stc/Hsp83E317K, and
armadillo-GAL4/UAS-GFP-LKB1 ;
Hsp83stc/Hsp83E317K .
hypomorphs might exhibit many of the defects we
observe in flies with reduced Hsp90 function.
Two other kinases have been implicated in mRNA
localization in Drosophila, but as is the case for the role
of LKB1 in pgc localization, the critical direct substrate(s) for each have yet to be identified. Protein
kinase A (PKA) is required for the microtubule reorganization described above that leads to posterior
localization of osk mRNA (Lane and Kalderon 1994).
Although PKA has been shown to phosphorylate LKB1
at residue 535 in vitro (Martin and St. Johnston 2003),
overexpression of LKB1 bearing a phosphomimetic
S535E substitution does not rescue the microtubule defects in PKA mutant ovaries, suggesting that some other
protein is the major target for PKA during microtubule
reorganization (Steinhauer and Kalderon 2005). A
second kinase, IkB kinase-like2 (Ik2), and its binding
partner, Spindle-F (Spn-F), have recently been shown to
regulate both microtubule and actin filament distributions in the female germ line (Abdu et al. 2006; Shapiro
and Anderson 2006). The authors proposed that Ik2/
Spn-F facilitates the connection of a subset of microtubules to cortical actin, although the mechanism of their
action is unknown. For each of these kinases, LKB1,
PKA, and Ik2, further biochemical and genetic experiments will be required to determine how they act to
localize mRNA.
The differential effects we observe on localization of
nos, pgc, and CycB (Figures 6 and 8) suggest that each
mRNA is localized by a somewhat different mechanism.
CycB mRNA localization is normal in Hsp83stc/Hsp83E317K
embryos and thus appears to be relatively Hsp90 independent; pgc mRNA localization requires normal
levels of Hsp90 activity, primarily to stabilize LKB1; and
nos mRNA localization requires normal levels of Hsp90
activity, presumably to stabilize or activate other (currently unknown) factors. Studies of the polar granule
component Tudor support the idea that different mechanisms underlie localization of nos, pgc, and gcl mRNAs,
each of which is concentrated at the posterior pole late
in oogenesis (Thomson and Lasko 2004; Arkov et al.
2006). Among this class of mRNAs, only nos has been
studied in detail (Forrest and Gavis 2003). Similar
detailed studies of pgc, gcl, and CycB localization might
reveal some of the mechanistic differences.
The genetic screen we employed to identify Hsp90
appears to constitute a promising approach for the
identification of additional nos mRNA localization factors.
One key aspect of the screen was reliance on the identification of dominant modifier mutations. Such mutations
may be relatively easy to isolate in the case of Hsp83, as
the encoded protein is a multidomain dimer that forms
large complexes with other factors, including its cochaperones (Pearl and Prodromou 2001). Nevertheless,
Hsp90 As an mRNA Localization Factor
we are optimistic that a scaled-up version of the screen
that surveys the entire genome and characterization of
resulting mutants will yield additional components of
the nos mRNA localization machinery.
We especially thank Daniel St. Johnston for his generosity in
supplying strains and reagents; Anne Ephrussi, Akira Nakamura, Paul
Lasko, Paul Macdonald, and Ken Howard for reagents; Joe Heitman,
Chris Nicchitta, and Danny Lew for comments on the manuscript and
suggestions; Sandy Curlee and Estelle Tsalik for assistance in the office
and lab, respectively; and Jian Chen for media preparation. We are also
indebted to the Bloomington Stock Center. Y. S. was a Predoctoral
Fellow of the HHMI and Robin Wharton is an Investigator of the
Howard Hughes Medical Institute.
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Communicating editor: W. M. Gelbart