ORIGINAL RESEARCH ARTICLE
published: 30 August 2011
doi: 10.3389/fgene.2011.00055

Gender-speciﬁc hierarchy in nuage localization of
PIWI-interacting RNA factors in Drosophila
Akihiro Nagao 1 † , Kaoru Sato 1 † , Kazumichi M. Nishida 1 , Haruhiko Siomi 1 and Mikiko C. Siomi 1,2 *12

Siomi Lab, Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan
Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Saitama, Japan

Edited by:
Muller Fabbri, The Ohio State
University, USA
Reviewed by:
Patrick Provost, Université Laval,
CanadaFrancesca Fanini, Istituto ScientiﬁcoRomagnolo per lo Studio e la Cura dei
Tumori, Italy
Michael Frohman, Stony Brook
University, USA*Correspondence:
Mikiko C. Siomi , Keio University
School of Medicine, 3 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan.
e-mail: siomim@z6.keio.jp†

Akihiro Nagao and Kaoru Sato havecontributed equally to this work.

PIWI-interacting RNAs (piRNAs) are germline-speciﬁc small non-coding RNAs that form
piRNA-induced silencing complexes (piRISCs) by associating with PIWI proteins, a subclade of the Argonaute proteins predominantly expressed in the germline. piRISCs protect
the integrity of the germline genome from invasive transposable DNA elements by silencing
them. Multiple piRNA biogenesis factors have been identiﬁed in Drosophila. The majorityof piRNA factors are localized in the nuage, electron-dense non-membranous cytoplasmic structures located in the perinuclear regions of germ cells. Thus, piRNA biogenesis
is thought to occur in the nuage in germ cells. Immunoﬂuorescence analyses of ovaries
from piRNA factor mutants have revealed a localization hierarchy of piRNA factors in female
nuage. However, whether this hierarchy is female-speciﬁc or can also be applied in malegonads remains undetermined. Here, we show by immunostaining of both ovaries andtestes from piRNA factor mutants that the molecular hierarchy of piRNA factors shows
gender-speciﬁcity, especially for Krimper (Krimp), a Tudor-domain-containing protein of
unknown function(s): Krimp is dispensable for PIWI protein Aubergine (Aub) nuage localization in ovaries but Krimp and Aub require each other for their proper nuage localizationin testes. This suggests that the functional requirement of Krimp in piRNA biogenesis may
be different in male and female gonads.
Keywords: nuage, piRNA, PIWI, Drosophila, germline

INTRODUCTIONPIWI-interacting RNAs (piRNAs) are germline-speciﬁc small
RNAs that function in RNA silencing. In Drosophila, piRNAs arise
mostly from transposable elements and associate with germlinespeciﬁc Argonaute proteins (PIWI proteins), Aubergine (Aub),Piwi, and Ago3, to guide PIWI proteins to their targets (Saito et al.,2006, 2009; Vagin et al., 2006; Aravin et al., 2007; Brennecke et al.,2007; Gunawardane et al., 2007; Nishida et al., 2007). Mutationsin the piwi genes lead to de-repression of transposable elements(Cox et al., 1998; Schmidt et al., 1999; Vagin et al., 2004; Kalmykova
et al., 2005; Klattenhoff et al., 2007; Li et al., 2009). Mutations in thepiRNA-producing genomic loci, the piRNA clusters, show a similar
phenotype (Brennecke et al., 2007). Thus, both PIWI and piRNAs
are necessary for transposon silencing in the germline. Throughthis mechanism, the PIWI–piRNA complex, also called the piRNAinduced silencing complex (piRISC), protects the integrity of thegermline genome from invasive transposable DNA elements andensures oogenesis and spermatogenesis.
PIWI-interacting RNAs are produced through two mechanisms, the primary processing pathway and the ampliﬁcation loop
(or the Ping-Pong cycle; Aravin et al., 2007; Brennecke et al., 2007;
Gunawardane et al., 2007; Li et al., 2009; Malone et al., 2009; Siomi
et al., 2011). Primary piRNAs are ﬁrst produced through the primary processing pathway; then, the majority of primary piRNAs
are ampliﬁed by the Ping-Pong cycle to maintain a high expression level of piRNAs in the germline. Maternally deposited piRNAs

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also serve as primary piRNAs to induce the Ping-Pong cycle inembryos.
Primary piRNAs are mostly antisense to transposon mRNAs,
show a strong bias for uracil at the 5 end (1U bias) and are favorably loaded onto Aub and Piwi (Li et al., 2009; Malone et al., 2009).
Primary piRNAs in ovarian somatic cells require the female sterile (1) Yb (Yb) gene for their accumulation (Olivieri et al., 2010;
Saito et al., 2010; Qi et al., 2011). Yb is necessary for self-renewalof germline and somatic stem cells in ovaries (Szakmary et al.,2009). Germ cells do not express Yb and, therefore, Yb is unnecessary for primary piRNA production in germ cells. Zucchini (zuc)and armitage (armi) are necessary for primary piRNA production in both ovarian somas (Haase et al., 2010; Olivieri et al.,2010; Saito et al., 2010) and germ cells (Malone et al., 2009). Vasa,
maelstrom (mael), krimper (krimp), armi, and spindle-E (spn-E)
are necessary for primary piRNA accumulation in testes (Nagao
et al., 2010). Other genes necessary for primary piRNA production
remain elusive.
The Ping-Pong cycle performs piRNA ampliﬁcation andtransposon silencing, simultaneously; this is because transposon
mRNAs serve as substrates for the Aub–piRNA complexes. The
Aub-Slicer activity, the endonuclease activity required for cleaving the target mRNAs (Gunawardane et al., 2007; Nishida et al.,2007), determines the 5 end of mature piRNAs in the senseorientation. Mature piRNAs are then loaded onto Ago3. Thus,
Ago3-associating piRNAs are mostly in the sense orientation. The

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Ago3–piRNA complex, in turn, cleaves antisense transcripts oftransposons and the resultant piRNAs are loaded onto Aub. Other
factors required for the ampliﬁcation loop include Vasa, Tudor(Tud), Krimp, Spn-E, and Mael (Findley et al., 2003; Lim and Kai,
2007; Malone et al., 2009).
Tud is one of the Tud domain-containing proteins expressedin ﬂy gonads (Boswell and Mahowald, 1985; Thomson and Lasko,
2004; Arkov et al., 2006). The Tud domains in Tud speciﬁcallyinteract with the symmetrical dimethylarginines (sDMAs) in Aub
and Ago3 (Kirino et al., 2009; Nishida et al., 2009; Liu et al.,2010a,b; Siomi et al., 2010). The association between Tud andAub or Ago3 occurs simultaneously and the Tud–Aub–Ago3 complex contains piRNA intermediate-like RNA molecules but, in thecomplex, Aub and Ago3 are free from mature piRNAs. Thus, Tudmight serve as a platform for the ampliﬁcation loop (Nishida et al.,2009). Spn-E has a Tud domain (Gillespie and Berg, 1995; Ponting,1997); however, it does not seem to associate with Aub and Ago3
(Nishida et al., 2009). Krimp also contains a Tud domain (Lim andKai, 2007). Whether Krimp has the ability to associate with PIWI
members through their sDMAs remains undetermined.
Until now, the piRNA biogenesis factors that have been identiﬁed in Drosophila are localized in the cytoplasm and are preferentially accumulated in speciﬁc cytoplasmic perinuclear structures,
the nuage, and Yb bodies in germ and somatic cells, respectively.
Hence, both nuage and Yb bodies are considered to be the locations for piRNA biogenesis (Findley et al., 2003; Lim and Kai, 2007;
Olivieri et al., 2010; Saito et al., 2010; Qi et al., 2011; for review see
Siomi et al., 2011). Zuc is localized to mitochondria (Olivieri et al.,2010; Saito et al., 2010). How Zuc localization to mitochondria ismechanistically involved in piRNA biogenesis remains unknown.
Accumulated evidence has shown that in ovaries, nuage localization of the piRNA factors occurs in a molecular hierarchicalfashion. The current understanding is that Vasa, a DEAD-box RNA
helicase that is detected speciﬁcally in the germ cells, is the ﬁrst to
be located to the nuage (Findley et al., 2003): Vasa localization isfollowed by Spn-E, Aub, Ago3, and Mael. Whether this hierarchy
is female-speciﬁc or occurs also in males remains unexamined. To
address this question, we performed immunoﬂuorescence analyses
using monoclonal antibodies against individual piRNA factors andfound that the nuage localization hierarchy was gender-speciﬁc; anobvious difference was seen for Krimp. Krimp is placed between
Aub and Ago3 in ovaries, but is placed at the same level with Aub,but upstream of Ago3 in testes. These results suggest that the functional requirement of Krimp in piRNA biogenesis may be different
between male and female gonads.

MATERIALS AND METHODSDROSOPHILA STRAINSThe yellow white (y w) and Oregon-R strains were used as
wild-type (wt ) strains. The tud allele used was tud1 bw sp/CyO
l(2)DTS5131 (Drosophila Genetic Resource Center stock number: 106505). The aub alleles used were aubHN2 cn bw/CyO
and aubQC42 cn bw/CyO. aubHN2 cn bw/CyO and aubQC42 cn
bw/CyO were crossed to yield aub heterozygous mutant ﬂies;
aubQC42 /aubHN2 (Nishida et al., 2007). The ago3 alleles usedwere ago3t2 /TM6B Tb and ago3t3 /TM6B Tb. ago3t2 /TM6B Tb
and ago3t3 /TM6B Tb were crossed to yield ago3 heterozygous

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piRNA factors in male nuage

mutant ﬂies; ago3t2 /ago3t3 (Li et al., 2009). The krimp allele used
was w1118 ; PBac{WH}krimpf06583 /CyO (Bloomington Drosophila
Stock Center; 18990). w1118 ; PBac{WH}krimpf06583 /CyO andw1118 ; Df(2R)BSC309/CyO (Bloomington Drosophila Stock Center; 23692) were crossed to yield krimp transheterozygote mutant
ﬂies; krimpf06583 /Df. Transheterozygotes krimpf06583 /Df exhibitedmale and female sterility and an extent of loss in krimp perinuclear staining similar to that exhibited by homozygous krimpf06583(Lim and Kai, 2007, and data not shown). Hence, krimpf06583 wasused as a loss-of-function allele to characterize krimp phenotypes
in this study. All stocks were maintained at 25˚C.
PRODUCTION OF ANTIBODIES AND WESTERN BLOTTING

An anti-Tudor monoclonal antibody was raised speciﬁcally against
the C-terminus of the protein. A 327 amino acid fragment at the Cterminal end of Tudor (amino acid 2189 to the end) was fused withglutathione S-transferase (GST) and used to immunize mice. An
anti-Krimp monoclonal antibody was raised speciﬁcally against
the N-terminus of the protein. A 200 amino acid fragment at the
N-terminal end of Krimp was fused with GST and used to immunize mice. Anti-Tudor and anti-Krimp monoclonal antibodies
were produced essentially as described previously (Ishizuka et al.,2002) and puriﬁed from culture supernatant of hybridoma cells
under standard procedures using Thiophilic-Superﬂow resin (BDBiosciences). Western blot analysis was performed as described
previously (Miyoshi et al., 2005). Anti-Aub and anti-Ago3 antibodies were used as described previously (Nishida et al., 2007,2009).
IMMUNOHISTOCHEMISTRY

Testes and ovaries were stained with the various antibodies as previously described (Nishida et al., 2007; Nagao et al., 2010). An
anti-Aub antibody was directly labeled using the HiLyte Fluor 555
Labeling Kit-NH2 (Dojindo Molecular Technologies). Anti-Ago3(1:5,000 dilution; Nagao et al., 2010), anti-Tud (1:250 dilution;
Nishida et al., 2009), and anti-Krimp (1:250 dilution) antibodies
were used as primary antibodies. Alexa 488-conjugated or Alexa
546-conjugated anti-mouse IgG (Molecular Probes; 1:1,000 dilution) were used as secondary antibodies. DNA was stained with
DAPI. All images were collected using a confocal microscope (Zeiss
LSM5 EXCITER). All immunohistochemical analyses were performed at least three times and representative images are shownin the ﬁgures.

RESULTS AND DISCUSSION
To ensure that we had a constant supply of anti-Tud and antiKrimp antibodies for this study, we produced Tud and Krimp
monoclonal antibodies. We used recombinant Tud and Krimp
peptide fragments expressed in and puriﬁed from E. coli to immunize mice. Western blotting of total ovary and cultured Schneider
2 (S2) lysates showed that the antibodies reacted with proteins of
the expected sizes of Tud and Krimp (Figures 1A,B, respectively).
The Tud band in S2 lysate migrated slightly faster than that inovary lysate (Figure 1A). This may reﬂect post-translational modiﬁcation of Tud protein, although clear evidence for this is stillrequired. We also performed western blotting on tud and krimp
mutant ovaries (Figures 1A,B). The bands corresponding to Tud

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FIGURE 1 | Specificity of anti-Tud and anti-Krimp antibodies. (A) Western
blotting was performed on Schneider 2 (S2) and total ovary lysates using ananti-Tud monoclonal antibody raised in our laboratory. A band of the size
predicted for Tud was detected in both lysates. In tud mutant (tud1 /tud1 )ovaries, the bands corresponding to Tud disappeared, indicating that theantibody was speciﬁc to the antigen. Tubulin was used as a loading control.
(B) Western blotting was performed on S2 and total ovary lysates using ananti-Krimp monoclonal antibody. A band of the size predicted for Krimp was
detected. In krimp mutant (krimpf06583 /Df ) ovaries, the bands corresponding to
Krimp disappeared, indicating that the antibody was speciﬁc to Krimp.

and Krimp were not present in the mutants, indicating a highspeciﬁcity of the antibodies for the individual antigens.
We previously examined the subcellular localization of Tud in
aub mutant ovaries using an anti-Tud antibody that was a kind
gift from A. Nakamura (CDB-RIKEN, Japan; Nishida et al., 2009).
Here, we used the newly produced anti-Tud antibody to repeat
this immunoﬂuorescence study on both control and aub mutant
ovaries. Very little Tud was localized to the nuage when Aub was not
expressed (Figure 2A), conﬁrming the previous observation that
Aub is necessary for Tud nuage localization (Nishida et al., 2009).
Tud localization has not been previously examined in ago3 mutant
ovaries. We immunostained for Tud in ago3 mutant ovaries anddetected Tud in the nuage (Figure 2A), suggesting that Ago3 is dispensable for Tud nuage localization. Thus, the nuage localizationof Aub, but not of Ago3, is necessary for Tud nuage localization.
Aub localization to nuage is not affected by loss of ago3 expression(Figure 2A), although Ago3 localization to nuage is affected byloss of aub expression (Figure 2A). Hence, for nuage localization,
Ago3 requires Aub but Aub does not require Ago3.
We then examined the subcellular localization of Tud in aub
and ago3 mutant testes. The Tud signal was detected in the nuage
in ago3 mutant testes (Figure 2B) the same as in control. In aub
mutant testes, however, the Tud signal was hardly detected in
the nuage; instead, it appeared to be in the nucleus in particular in spermatogonia cells (Figure 2B). These results suggest that
Aub controls the Tud nuage localization even in testes. Aub was
localized to the nuage in ago3 testes (Figure 2B), conﬁrming that
Aub nuage localization does not require Ago3 in both female andmale gonads. Ago3 is not localized to nuage in aub mutant testes(Figure 2B). Thus, Ago3 nuage localization requires Aub in bothmales and females.

We then investigated how the subcellular localization of Aub
and Ago3 was affected by loss of tud function. In ovaries, Aub was
detected only slightly in the nuage in tud mutant ﬂies (Figure 3A),
as has been reported previously (Nishida et al., 2009). In contrast, Ago3 was localized to the nuage, the same as in the control.
These results suggest that under conditions where tud function isabsent, Ago3 ﬁnds an alternative way to be localized to the nuage
in a Tud-independent manner. Another possible interpretation isthat a small amount of Aub in the nuage is sufﬁcient for Ago3
to be localized to the nuage. Also in testes, Ago3 localization wasbarely affected by loss of tud function, whereas Aub localization
was drastically changed (Figure 3B); Aub was sparsely localized in
the nuage and instead was dispersed in the cytosol. These results
indicate that the requirement and dispensability of Tud for nuage
localization of Aub and Ago3, respectively, are similar in male andfemales.
We also examined how loss of krimp affects the localizationof Aub and Ago3 in the germline. In krimp mutant ovaries, Ago3
lost its ability to localize to the nuage, although Aub was accumulated in the nuage the same as in the control (Figure 4A). Thus,
Krimp is necessary for nuage localization of Ago3, but not of Aub.
Tud nuage localization was maintained even after krimp expression was lost (Figure 4A). In contrast, in krimp mutant testes,
Aub, Ago3, and Tud nuage localization was abolished (Figure 4B).
The requirement of Krimp for Aub and Tud nuage localization isdifferent in ovaries and testes.
An earlier study showed that Krimp is localized to the nuage inovaries (Lim and Kai, 2007). In the present study, we conﬁrmed thislocalization (Figure 5A) using our newly produced anti-Krimp
antibody (Figure 1B). We then examined by immunostaining ofovaries and testes how Krimp nuage localization is affected by loss

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FIGURE 2 | Tud subcellular localization in aub and ago3 mutant ovaries
and testes. (A) Ovaries: the control (a), and aub (aubHN2 /aubQC42 ) (b), andago3 (ago3t2 /ago3t3 ) (c) mutant ovaries were stained with anti-Tud antibody;the control (d), and aub (aubHN2 /aubQC42 ) (e) ago3 (ago3t2 /ago3t3 ) (f) mutant
ovaries were stained with anti-Aub antibody; and the control (g), and aub(aubHN2 /aubQC42 ) (h), and ago3 (ago3t2 /ago3t3 ) (i) mutant ovaries were stainedwith anti-Ago3 antibody. Scale bars in all ovary images indicate 10 μm. (B)Testes: the control (a), and aub (aubHN2 /aubQC42 ) (b), and ago3 (ago3t2 /ago3t3 )Frontiers in Genetics | Non-Coding RNA

piRNA factors in male nuage(c) mutant testes were stained with anti-Tud antibody. Enlarged images of theregions that are boxed with white dotted lines in (B) (a–c) are shown in (B)(d–f), respectively. The control (g), and aub (aubHN2 /aubQC42 ) (h), and ago3
(ago3t2 /ago3t3 ) (i) mutant testes were stained with anti-Aub antibody; and thecontrol (j); and aub (aubHN2 /aubQC42 ) (k); and ago3 (ago3t2 /ago3t3 ) (l) mutant
testes were stained with anti-Ago3 antibody. Scale bars in all testis images
indicate 20 μm. All immunohistochemical analyses were performed at least
three times and representative images are shown.

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FIGURE 3 | Loss of tud function affects the subcellular localization of
Aub and Ago3 in ovaries and testes similarly. (A) Ovaries: tudheterozygous and homozygous mutant ovaries were immunostained with
anti-Tud, anti-Aub, and anti-Ago3 antibodies [(a): tud hetero/anti-Tud; (b):tud homo/anti-Tud; (c): tud hetero/anti-Aub; (d): tud homo/anti-Aub; (e): tudhetero/anti-Ago3; (f): tud homo/anti-Ago3]. Scale bars in all ovary images
indicate 10 μm. (B) Testes: tud mutant and control testes were
immunostained with anti-Aub and anti-Ago3 antibodies [(a): tudhetero/anti-Aub; (b): enlarged image of the region boxed with a white line in(a); (c): tud homo/anti-Aub; (d): enlarged image of indicated region in (c);(e): tud hetero/anti-Ago3; (f): enlarged image of indicated region in (e); (g):tud homo/anti-Ago3; (h): enlarged image of indicated region in (g)]. Scalebars in all testis images indicate 20 μm. All immunohistochemical analyses
were performed at least three times and representative images are shown.

of other piRNA factors. We found that loss of ago3 and tud expression in ovaries did not affect Krimp localization (Figure 5A). However, in aub mutant ovaries, Krimp signals were hardly detectedin the nuage; instead, Krimp accumulated to unknown cytoplasmic structures (Figure 5A). Thus, Aub, but not Ago3 or Tud, isrequired for Krimp nuage localization. Why Krimp accumulates
in cytoplasmic structures remains unknown. The Krimp signalof immunostaining was lower in aub and tud mutant testes thanthat in the control, while Krimp expression was unchanged byloss of ago3 expression (Figure 5B). Western blotting conﬁrmed
a severe reduction in the expression level of Krimp in aub andwww.frontiersin.org

FIGURE 4 | Loss of krimp function affects the subcellular localizationof Aub and Ago3 in ovaries and testes differently. (A) Ovaries: the control
and krimp mutant ovaries were immunostained with anti-Aub, anti-Ago3,
and anti-Tud antibodies [(a): control/anti-Aub; (b): krimp mutant/anti-Aub;(c): enlarged image of the part boxed with a white line in (b); (d):control/anti-Ago3; (e): krimp mutant/anti-Ago3; (f): enlarged image ofindicated region in (e); (g): control/anti-Tud; (h): krimp mutant/anti-Tud; (i):enlarged image of indicated region in (h)]. Scale bars in all ovary images
indicate 10 μm. (B) Testes: the control and krimp mutant testes were
immunostained with anti-Aub, anti-Ago3, anti-Tud, and anti-Krimp antibodies[(a): control/anti-Aub; (b): krimp mutant/anti-Aub; (c): control/anti-Ago3; (d):krimp mutant/anti-Ago3; (e): control/anti-Tud; (f): krimp mutant/anti-Tud;(g): control/anti-Krimp; (h): krimp mutant/anti-Krimp]. Scale bars in all testis
images indicate 20 μm. All immunohistochemical analyses were performedat least three times and representative images are shown.

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FIGURE 5 | Krimp nuage localization in the piRNA mutant ovaries
and testes. (A) Ovaries: the control (a) and aub (b), ago3 (c), and tud (d)mutant ovaries were immunostained with an anti-Krimp antibody. Scalebars in all ovary images indicate 10 μm. (B) Testes: the control (a) andaub (b), ago3 (c), and tud (d) mutant testes were immunostained with

tud mutant testes (Figure 5C). Thus, the requirement of Tud in
Krimp nuage localization is different between males and females.
Why loss of aub and tud expression causes a severe reduction in
the Krimp expression requires further investigation. Krimp was
hardly detected in the tud mutant testes. However, Ago3 was able
to accumulate in the nuage, possibly because Aub was, at least
partially, localized to the nuage in tud mutant testes. It could be
postulated that Ago3 nuage localization necessitates either Aub or
Krimp in the nuage.
The nuage localization of piRNA factors, Aub, Tud, Krimp, andAgo3, in ovaries and testes from wt and the piRNA factor mutant
ﬂies was summarized (Figure 6A) and their molecular hierarchy
was compared (Figure 6B). Their expression patterns in wt ovary
and testis were also summarized (Figure 6C). An obvious difference in the hierarchy is the location of Krimp between Aub andAgo3 in ovaries but at the same level as Aub and upstream of Ago3
in testes. Krimp expression in testis is limited to developmentally
early stage cells, such as germline stem cells, gonialblasts, and spermatogonia (Figure 6C) and this may be the cause of the genderdifference observed in the location of Krimp in the molecularhierarchy.
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piRNA factors in male nuage

an anti-Krimp antibody. (C) Western blotting shows that the expressionof Krimp is hardly detected in aub and tud mutant testes. Scale bars in all
testis images indicate 20 μm. All immunohistochemical analyses were
performed at least three times and representative images areshown.

We reported previously that, in testes, Ago3 functions in theampliﬁcation loop with Aub for producing transposon-derived
piRNAs (Nagao et al., 2010). However, most Ago3-associated testis
piRNAs corresponding to Suppressor of Stellate [Su(Ste)] genesthat function in silencing the Stellate gene, are antisense-oriented
and are also detected in piRNAs associated with Aub (Nagao et al.,2010). In addition, the vast majority of Ago3-associated piRNAs
derived from the AT-chX locus on chromosome X, some of which
may function in silencing vasa, are also antisense-oriented and arefound among Aub-associated piRNAs (Nagao et al., 2010). The
piRNAs that show characteristics similar to those of AT-chX andSu(Ste) piRNAs have so far not been found in ovaries (Nagao
et al., 2010). Our current ﬁnding, that Krimp is located in a different position in the nuage localization hierarchy in ovaries andtestes, may further support the idea that some mechanisms ofpiRNA biogenesis differ in ovaries and testes. Our interpretationis that Krimp may be one of the important factors for producing AT-chX and Su(Ste) primary piRNAs and may enabling theirloading into not only Aub, but also Ago3, in testes. In ovaries,
Krimp is placed between Aub and Ago3 in the hierarchy and thismay be why primary piRNAs are speciﬁcally loaded into Aub, but
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FIGURE 6 | Molecular hierarchy of the piRNA factors in their localization
to nuage in ovaries and testes. (A) The nuage localization of piRNA factors,
Aub, Tud, Krimp, and Ago3 in wild-type (wt ) and their mutant ovaries andtestes: n.d.: none detected. Since none of the piRNA factors in this ﬁgure
were detected in the nucleus of wt ovaries and testes, therefore they wereconsidered to be cytoplasmic proteins. In tud mutant ovaries and testes, Aub
is only weakly accumulated in the nuage; thus, the nuage is indicated with athinner line. The Krimp signals are hardly detected in aub and tud mutant
testes. (B) The nuage localization hierarchy: Aub is placed upstream of Ago3 inboth ovaries and testes. However, Krimp shows gender-speciﬁcity; in ovaries
Krimp lies between Aub and Ago3, in testes Krimp is placed at the same levelas Aub in testes. Tud is required for Aub localization in both male and female
nuage. However, Ago3 can be localized to the nuage without tud function inboth males and females. It is likely that Ago3 nuage localization occurs in both
Aub-dependent and Tud-independent manners. (C) The expression patterns ofAub, Tud, Krimp, and Ago3 in ovaries and testes. GSCs, germline stem cells;
CBs, cystoblasts; NCs, nurse cells; Oo, oocyte; GBs, goniablasts; SG,spematogonia.

not into Ago3. Krimp has a Tud domain and, thus, it is reasonable
to postulate that Krimp associates with Aub and/or Ago3 throughtheir sDMA modiﬁcations in both ovaries and testes. However,
the mode of their associations could be different in the two tissues: the extent of sDMA modiﬁcation of Aub and Ago3 may
be different and/or the sDMA requirements in the association

between Krimp and Aub/Ago3 may be different. These possible differences could be the cause of the gender difference for
Krimp in the piRNA factor hierarchy. We are currently investigating the biochemical function of Krimp in piRNA biogenesis
and its relationship with Aub and Ago3, as well as the modiﬁcation status of Aub and Ago3 in both ovaries and testes, whichwww.frontiersin.org

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will help further our understanding of piRNA biogenesis in general and of the gender differences in the molecular mechanisms inparticular.

ACKNOWLEDGMENTS
We thank T. Okada, A. Shibuya, M. Komai, and H. Kotani fortechnical assistance and other members of the Siomi laboratory

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Conﬂict of Interest Statement: The
authors declare that the research waswww.frontiersin.org

piRNA factors in male nuage

conducted in the absence of any commercial or ﬁnancial relationships that
could be construed as a potential conﬂict of interest.

Received: 08 July 2011; accepted: 12
August 2011; published online: 30 August
2011.

Citation: Nagao A, Sato K, Nishida
KM, Siomi H and Siomi MC (2011)
Gender-speciﬁc hierarchy in nuage localization of PIWI-interacting RNA factors
in Drosophila. Front. Gene. 2:55. doi:
10.3389/fgene.2011.00055
This article was submitted to Frontiers in
Non-Coding RNA, a specialty of Frontiersin Genetics.

Copyright © 2011 Nagao, Sato, Nishida,
Siomi and Siomi. This is an open-accessarticle subject to a non-exclusive license
between the authors and Frontiers MediaSA, which permits use, distribution andreproduction in other forums, provided
the original authors and source are credited and other Frontiers conditions arecomplied with.

August 2011 | Volume 2 | Article 55 | 9