Article

Inheritance of a Nuclear PIWI from Pluripotent StemCells by Somatic Descendants Ensures
Differentiation by Silencing Transposons in
PlanarianGraphical Abstract

Authors
Norito Shibata, Makoto Kashima,
Taisuke Ishiko, ..., Haruhiko Siomi,
Mikiko C. Siomi, Kiyokazu Agata

Correspondence
shiba@mdb.biophys.kyoto-u.ac.jp (N.S.),agata@mdb.biophys.kyoto-u.ac.jp (K.A.)

In Brief
PIWI proteins are known to mediatetransposon silencing in the germline.
Shibata, Kashima et al. show that, in theplanarian Dugesia japonica, differentiatedsomatic cells, which themselves do not
express PIWI proteins, inherit a nuclear
PIWI from their pluripotent predecessors.
The PIWI protein is required fortransposon silencing during
differentiation and regeneration.

HighlightsdThree PIWI proteins temporally switch to repress TE during
PSC differentiationd

Nuclear DjPiwiB is required for differentiation of neoblasts(PSCs) in planariandDepletion of DjPiwiB causes activation of TEs in somatic
descendants of neoblastsdDjPiwiB produced in neoblasts is inherited and acts in theirsomatic descendants

Shibata et al., 2016, Developmental Cell 37, 226–237
May 9, 2016 ª 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.devcel.2016.04.009

Developmental Cell

ArticleInheritance of a Nuclear PIWI from Pluripotent StemCells by Somatic Descendants Ensures Differentiation
by Silencing Transposons in Planarian
Norito Shibata,1,4,* Makoto Kashima,1,4 Taisuke Ishiko,1 Osamu Nishimura,1,2 Labib Rouhana,1,5 Kazuyo Misaki,2Shigenobu Yonemura,2 Kuniaki Saito,3 Haruhiko Siomi,3 Mikiko C. Siomi,3,6 and Kiyokazu Agata1,*
1Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan
2Center for Life Science Technologies, RIKEN, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
3Department of Molecular Biology, Keio University School of Medicine, 3 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
4Co-first author
5Present address: Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA
6Present address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16, Yayoi, Bunkyo-ku,
Tokyo 113-0032, Japan*Correspondence: shiba@mdb.biophys.kyoto-u.ac.jp (N.S.), agata@mdb.biophys.kyoto-u.ac.jp (K.A.)
http://dx.doi.org/10.1016/j.devcel.2016.04.009

SUMMARY

Differentiation of pluripotent stem cells (PSCs) requires transposon silencing throughout the process.
PIWIs, best known as key factors in germline transposon silencing, are also known to act in somatic differentiation of planarian PSCs (neoblasts). However,
how PIWIs control the latter process remains elusive.
Here, using Dugesia japonica, we show that a nuclear
PIWI, DjPiwiB, was bound to PIWI-interacting RNAs
(generally key mediators of PIWI-dependent transposon silencing), and was detected in not only neoblasts but also their descendant somatic cells, which
do not express piwi. In contrast, cytoplasmic DjPiwiA
and DjPiwiC were detected only in neoblasts, inaccord with their transcription there. DjPiwiB was
indispensable for regeneration, but dispensable fortransposon silencing in neoblasts. However, transposons were derepressed at the onset of differentiationin DjPiwiB-knockdown planarians. Thus, DjPiwiB appears to be inherited by descendant somatic cells ofneoblasts to ensure transposon silencing in thosecells, which are unable to produce PIWI proteins.

INTRODUCTION
Pluripotency is the ability of stem cells to differentiate into alltypes of somatic (and germline) cells in an organism. Differentiation of pluripotent stem cells (PSCs), such as embryonic stem
cells (ESCs), to somatic cells requires dynamic alterations ofgene expression by drastic transitions of epigenetic modifications (Xie et al., 2013). To accomplish this, the chromatin at
specific loci in the genome may be ‘‘loosened.’’ This looseningof the genome, or euchromatinization, might, however, lead toconcomitant activation of transposable elements (TEs) at these
loci, threatening the integrity of the genome. Therefore, TEsshould be continuously silenced throughout the differentiation
process. Histone modifications may be involved in repressing
TEs during PSC differentiation (Leung and Lorincz, 2012). However, the detailed mechanism underlying the control of TEs during PSC differentiation remains unknown.
A number of invertebrates possess high regenerative ability,
enabling them to reproduce a whole animal body from a tiny
body fragment using adult PSCs (Agata et al., 2007; Sanchez Alvarado and Yamanaka, 2014). Planarian contains abundant
PSCs, called neoblasts, throughout most of its body, and so,theoretically, tiny fragments amputated from almost any part ofthe planarian body would regenerate into complete individuals
within a week or so, as long as these tiny fragments contained
a sufficient number of neoblasts and sufficient positional information from differentiated tissue to serve as a guide for regeneration (Agata et al., 2014; Agata and Watanabe, 1999; Reddien,
2011; Shibata et al., 2010). The establishment of state-of-theart experimental techniques has made planarian a highly useful
animal model in stem cell and regeneration research (Reddien,2013; Rink, 2013; Shibata et al., 2010).
Neoblasts in planarian express various genes that are normally
restricted to germline cells in non-regenerative animals (Rink,
2013; Shibata et al., 2010; Solana, 2013). One such example isthe piwi family of genes. These genes encode germline-specific
Argonaute members, PIWI proteins, that repress TEs by specifically associating with PIWI-interacting RNAs (piRNAs) (Iwasaki
et al., 2015; Juliano et al., 2011; Malone et al., 2009). piRNAs
are small non-coding RNAs enriched in the germline of various
animals, and most piRNAs possess sequences antisense to TE
transcripts (Iwasaki et al., 2015; Juliano et al., 2011; Malone
et al., 2009). Indeed, loss of piRNAs and PIWI proteins in the
germline causes TE derepression, leading to infertility of mutant
animals (Aravin et al., 2007; Khurana and Theurkauf, 2010). Thus,
PIWI-piRNA-mediated gene silencing machinery is indispensable for animal species that rely on a sexual reproductive
system.
Neoblasts in planarian Schmidtea mediterranea express threepiwi genes, smedwi-1, smedwi-2, and smedwi-3 (Palakodeti
et al., 2008; Reddien et al., 2005). Also, small RNAs that show

226 Developmental Cell 37, 226–237, May 9, 2016 ª 2016 Elsevier Inc.

A

3 dpa (10 d into RNAi)

piRNA features have been isolated by size fractionation, andmany of these putative piRNAs matched TEs in S. mediterranea(Friedlander et al., 2009; Palakodeti et al., 2008). Two otherplanarians, Dugesia japonica and Dugesia ryukyuensis, also
express three piwi family genes predominantly in neoblasts(DjpiwiA, DjpiwiB, and DjpiwiC in D. japonica and Drpiwi-1,
Drpiwi-2, and Drpiwi-3 in D. ryukyuensis) (Hayashi et al., 2010;
Nakagawa et al., 2012). Although functional analyses of smedwi
genes using RNAi suggested that smedwi-2 and smedwi-3 might
be involved in differentiation (Palakodeti et al., 2008; Reddien
et al., 2005), the specific roles of PIWI proteins in the differentiation of pluripotent neoblasts to specialized somatic descendants
in planarians remain unknown. Furthermore, although piRNAs in
S. mediterranea might target TEs (Friedlander et al., 2009; Palakodeti et al., 2008), the question of whether TEs are in fact
repressed by these PIWI proteins in the planarian PSC systemis still unanswered.
To address these questions, we examined how loss of individual PIWI proteins in D. japonica affects neoblast differentiation
to somatic cells. We found that two cytoplasmic PIWI proteins
and one nuclear PIWI protein act in neoblasts in D. japonica.
Knockdown (KD) of the nuclear PIWI, DjpiwiB, by RNAi caused
a severe defect in regeneration, whereas RNAi of DjpiwiA and/or DjpiwiC caused little or no regenerative defect, in accord
with the findings reported for S. mediterranea (Palakodeti
et al., 2008; Reddien et al., 2005). DjPiwiB-lacking neoblasts
proliferated and repressed TEs normally, but gypsy transposon
was derepressed at the onset of differentiation of neoblasts by
KD of DjpiwiB. All three PIWI proteins were observed in neoblasts, but only nuclear DjPiwiB was inherited by almost alldescendant differentiated cells, while transcription of DjpiwiBwas discontinued in these descendant cells during the differentiation process. This suggests that a DjPiwiB-solo function indifferentiating somatic cells ensures the repression of TE(s) during the differentiation process of planarian PSCs.

7 dpa (14 d into RNAi)

piwiC(RNAi)

piwiB(RNAi)

piwiA(RNAi)

GFP(RNAi)0 dpa (7 d into RNAi)B

6 dpa (7 days into RNAi)

GFP(RNAi)

DjpiwiB(RNAi)

6 dpa (7 days into RNAi)

C

7 days into RNAi
GFP(RNAi)

14 days into RNAi

piwiB(RNAi)
pH3

pH3

pH3

pH3 PiwiA

pH3 PiwiA

pH3 PiwiA

pH3 PiwiA

E

***

F

Relative expression level

N. S.

250
200
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100

Relative expression level

N. S.

300

1.0
0.8
0.6
0.4

50

0.2

0

0

piwiA(RNAi)
piwiB(RNAi)
piwiC(RNAi)RESULTS

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0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2

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PRMT

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piwiB

piwiC

iw

pcna

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14

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Number of pH3-positive cells / mm2

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***

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piwiB(RNAi)

pH3

D

7d

GFP(RNAi)

Figure 1. Effect of DjpiwiB RNAi(A) Regeneration of planarians after RNAi of various Djpiwi genes. dpa, day(s)post amputation.
(B) Regeneration of planarians amputated during the early period after the lastdsRNA feeding. Arrows, regenerated eyes. Scale bars, 100 mm.
(C) Immunofluorescence analyses of mitotic activity using anti-pH3 andDjPiwiA antibodies in control and DjpwiB KD planarians. Scale bars, 500 mm inwhole-body samples. Scale bars, 100 mm in magnified view of boxed regions inwhole-body samples.
(D) Number of mitotic cells estimated using anti-phosphorylated histone H3(pH3)-positive cell count after GFP control (white bars) or DjpiwiB (gray andblack [head regressed] bars) RNAi. ***p < 0.005.

DjPiwiB Is Required for Differentiation of Neoblasts into
Somatic Descendant Cells
We performed RNAi to knock down individual piwi genes in
D. japonica and examined how regeneration of the planarians
was affected by loss of the PIWI proteins. For this RNAi, planarians were fed food containing double-stranded RNA (dsRNA)
twice with a 2-day interval, and then amputated into three fragments on day 7 after the second dsRNA feeding. The regenerative ability of the body pieces was monitored at 0, 3, and 7 days
post amputation (dpa). We found that regeneration was particularly defective when DjpiwiB was depleted (Figure 1A). When planarians were amputated at 1 day after the last feeding, however,partial regeneration was observed at 6 dpa (Figure 1B), indicating that loss of regenerative ability occurred within 7 days
after RNAi. Counting the number of phosphorylated histone(E) Relative gene expression levels of neoblast-specific genes in DjpiwiB KD
planarians. The experiments were performed in triplicate and the SDs were
calculated using Microsoft Excel.
(F) Relative gene expression levels of piwi family genes in DjpiwiA, DjpiwiB, or
DjpiwiC KD planarians.

Developmental Cell 37, 226–237, May 9, 2016 227

A

BFigure 2. Analysis of DjPiwiB-Interacting
piRNAs

C

D

E

F

G

H3 (pH3)-positive cells, i.e., cells in the M phase of the cell
cycle (Newmark and Sanchez Alvarado, 2000), at 7 days into
DjpiwiB(RNAi) treatment revealed that DjPiwiB-depleted neoblasts proliferated normally in non-amputated animals (Figures
1C and 1D). At 14 days into DjpiwiB(RNAi), however, some planarians (n = 8/20) showed noticeable head regression (Figures
1C and 1D). This was accompanied by over a 50% decrease in
the number of neoblasts (Figures 1C and 1D). The expression
levels of neoblast markers (pcna, histone H2b [h2b]), and
PRMT (Sakurai et al., 2012; Shibata et al., 2012; Tasaki et al.,2011) were not affected at 7 days into DjpiwiB RNAi (Figure 1E).
RT-PCR confirmed that RNAi of DjpiwiB was specific to this geneand did not affect the expression levels of DjpiwiA or DjpiwiC(Figure 1F). Also, RNAi of DjpiwiA or DjpiwiC did not affect theexpression level of DjpiwiB (Figure 1F). In contrast to the effects
of DjpiwiB KD, KD of DjpiwiA or DjpiwiC had no effect on regeneration at 7 dpa (14 days into RNAi) (Figure 1A). However, at alater period (21 days into RNAi), the regenerated heads of
DjpiwiC(RNAi) planarians regressed, like those in DjpiwiB(RNAi)
planarians at 14 days into RNAi (Figure S1). These results agreewith the reported observations that smedwi-2 and smedwi-3 in
S. mediterranea (orthologs of DjpiwiB and DjpiwiC, respectively),
but not smedwi-1, are critical for the regulation of planarian stem
cells, suggesting distinct requirements for PIWI members during
228 Developmental Cell 37, 226–237, May 9, 2016(A) Analysis of IP proteins using anti-DjPiwiB, antiDjPiwiA, and control antibodies by silver staining.
Arrowhead indicates immunoprecipitated DjPiwiB
protein.
(B) Enrichment of small RNAs by IP using antiDjPiwiB antibody. ncRNA, RNA immunoprecipitated in the negative control. Arrowhead indicates
immunoprecipitated small RNA.
(C) Frequency of representation of various RNA sequences among the total immunoprecipitated RNAs.
(D) Pictogram of nucleotide frequency in piRNA.
Nucleotide frequency is represented by letterheight.
(E) The fractions of piRNAs matching categories ofD. japonica EST genes. Numbers indicate piRNA
reads matching each category. piRNAs matched toTEs (69,671) were further categorized by types of
TEs (pie chart on the right).
(F) Pie charts illustrating the percent of sense- or
antisense-orientation piRNAs mapped to all ESTrepresented genes and TEs. Pictograms showing
the nucleotide frequency of TE-matching piRNAs in
both antisense and sense orientations.
(G) Expression levels of DjpiwiB and gypsy-P1
detected by RT-qPCR analyses of DjpiwiB(RNAi)
relative to control GFP(RNAi) planarians. The
accession number of piRNAs bound to DjPiwiB
is DRA: DRA002837. The experiments were performed in triplicate and the SDs were calculatedusing Microsoft Excel.

neoblast differentiation (Palakodeti et al.,2008; Reddien et al., 2005).
We found that DjPiwiB-depleted neoblasts proliferated normally at 7 days
after DjpiwiB(RNAi), in accord with findings reported forS. mediterranea (Figures 1C and 1D) (Reddien et al., 2005). However, D. japonica’s regenerative ability disappeared within 7 days
after RNAi of DjpiwiB (Figures 1A and 1B). These results suggestthat neoblasts in D. japonica require DjPiwiB in order to differentiate into somatic cells but not to proliferate.
Comprehensive Analysis of piRNAs Associated with
DjPiwiBPIWI proteins select gene targets depending on the sequencesof piRNAs associated with them in vivo. To determine the sequences of DjPiwiB-associated piRNAs, we immunoisolated
the DjPiwiB-piRNA complexes from wild-type planarians using
an anti-DjPiwiB antibody we raised (Figure 2A). The specificityof the anti-DjPiwiB antibody was confirmed by western blotting
and RNAi (see below, and also Figure S2). We confirmedthat the immunoprecipitated protein was DjPiwiB by western
blotting (data not shown), and found by RNA extraction that
small RNAs were accumulated in the immunoprecipitation (IP)sample (Figure 2B). The most abundant piRNAs within the
immunoprecipitated DjPiwiB complexes appeared to be approximately 32 nucleotides long (Figure 2B). We determined thesequence of 4,177,817 of these small RNAs and mapped them
onto 2,629,514,899 base pairs of D. japonica shotgun genomesequence data (2.9-fold estimated genome coverage; Nishimura
et al., 2015), and also onto D. japonica expressed sequence tag(EST) sequences (Nishimura et al., 2012, 2015). Seventy-fourpercent of the reads (3,087,387 reads) were perfectly matchedto the D. japonica genome sequence and the D. japonica EST
sequence dataset. We then used these small RNAs for subsequent detailed analyses. Although these RNAs showed highsequence diversity (Figure 2C), as do piRNAs of mouse and fly(Aravin et al., 2006; Girard et al., 2006; Saito et al., 2006), about
80% of them possessed uracil as the first nucleotide (1U) (Figure 2D), a conserved feature of piRNAs (Iwasaki et al., 2015).
We could not find a ping-pong signature (matching of the firstten bases in the opposite orientation between piRNA basicallybound to distinct PIWIs, also a conserved feature of piRNAs;
Iwasaki et al., 2015).
Of the DjPiwiB-bound piRNAs, 206,992 matched the planarian
EST database (Nishimura et al., 2012), and 34% of them mapped
onto EST genes annotated as TEs, with 54%, 19%, and 15% ofthem classified as polinton, penelope, and gypsy, respectively(Figure 2E). Although the 1U tendency was observed for bothsense and antisense piRNAs that matched TEs, 60% of the
TE-sequence piRNAs were in the antisense direction, an enrichment compared with the 48% of total piRNAs that matched all
EST-represented genes (Figure 2F), suggesting that a fractionof DjPiwiB-piRNA complexes are capable of targeting TEs
in vivo. To see if such targeting indeed occurred, we examinedthe expression of TEs in DjPiwiB-depleted planarians. We particularly focused on TEs whose sequences were complementary to
the DjPiwiB-associated piRNAs that we identified in this study.
When DjPiwiB was depleted for 24 hr by RNAi, the expression
level of a gypsy transposon corresponding to cDNA clone
Dj_aH_208_K05 (DDBJ: FY934805; referred to as gypsy-P1)
was 4-fold higher than that in normal cells (Figure 2G). The antisense sequence of gypsy-P1 matched 71 piRNA reads (Table
S1). The level of gypsy-P1 expression became even higher
with prolonged RNAi treatment and reached an 8-fold increaseat 5 days into the RNAi treatment (Figure 2G).
Loss of DjPiwiB Derepresses TEs Only in Differentiating
Somatic Cells
To examine how the expression of gypsy-P1 was altered in various
parts of the animals by DjPiwiB depletion, we performed in situ hybridization experiments using a specific RNA probe hybridizing to
the transposon transcripts in the sense orientation. This examination revealed that, on day 3 into DjpiwiB RNAi, gypsy-P1-positive
cells were concentrated in the head region, particularly anterior to
the eyes, where no neoblasts are normally observed (Shibata
et al., 2010) (Figure 3A). The gypsy-P1-positive cells were DjPiwiA
negative (Figure 3B), suggesting that derepression of gypsy-P1
occurred only in differentiated somatic cells.
To determine whether those gypsy-P1-positive cells corresponded to cells that had already differentiated before the RNAitreatment, or cells that had differentiated de novo upon DjPiwiB
depletion, we tested the expression of gypsy-P1 in DjPiwiBdepleted animals after elimination of neoblasts by X-ray irradiation. X-ray-irradiated DjPiwiB-depleted planarians possessedno gypsy-P1-positive cells (Figure 3C). When planarians weretreated with DjpiwiB dsRNA together with U0126, an MEK inhibitor that blocks somatic differentiation from the neoblasts (Tasaki

et al., 2011), the number of gypsy-P1-positive cells was
drastically reduced as early as 5 days into RNAi (Figure 3D).
Furthermore, we analyzed gypsy-P1 expression in regenerating
DjPiwiB-depleted planarians, because neoblast proliferationand differentiation increase after amputation to supply new somatic differentiated cells (Wenemoser and Reddien, 2010).
Expression of gypsy-P1 was readily detected in the posterior
stump region of DjPiwiB-depleted body fragments (Figure 3E)
but was detected only at a very low level in intact DjPiwiBdepleted animals at day 3 into RNAi treatment.
RNA-seq of DjpiwiB KD planarians was then performed andrevealed that multiple types of TEs whose antisense sequences
matched piRNA reads were upregulated. For these TEs, qPCR
and/or in situ hybridization showed that they had a similar
expression pattern to that of gypsy-P1 in animals with combined
DjpiwiB knockdown plus X-ray irradiation or U0126 treatment(Figures S3A and S3B). Based on these results, we infer thatthe expression of multiple TEs, including gypsy-P1, was activated in DjPiwiB-depleted planarians during somatic differentiation from the neoblasts but not in neoblasts themselves, which
possessed full pluripotency.
Neoblast DjPiwiB, but Not DjPiwiA or C, Is Inherited byDifferentiated Somatic Cells
Although transcription of piwi genes occurs preferentially inneoblasts in D. japonica (Hayashi et al., 2010), some weak
expression of DjpiwiB as well as DjpiwiC was also observed in
the head region, where neoblasts are absent. We therefore
examined which types of cells express DjpiwiB, and found thatDjpiwiB was expressed in neoblasts and in brain cells in thehead region but not in other types of somatic differentiated cells
throughout the entire body (Figures S4A and S4B). However,
DjPiwiB depletion induced derepression of gypsy-P1 in denovo differentiated somatic cells in the entire body after RNAi
(Figure 3). To understand how this derepression occurred, we
compared the pattern of immunohistochemical staining with
anti-DjPiwi antibodies before and after Djpiwi KD in the animals.
DjPiwiA and DjPiwiC were specifically detected in the cytoplasmof the neoblasts (Figures 4A, 4B, and S2). Notably, DjPiwiC wasobserved as punctate signals (Figure 4B), suggesting that
DjPiwiC might be localized in chromatoid bodies, a neoblastspecific RNA/protein organelle (Shibata et al., 2010). In contrast,
surprisingly, DjPiwiB was observed only in the nucleus in almost
all cells in the body, namely, in both neoblasts and differentiatedsomatic cells (Figures 4C–4E and S4C), whereas SMEDWI-2 waspreferentially observed in the nucleus of the neoblasts in
S. mediterranea (Zeng et al., 2013). Particularly strong DjPiwiB
signals were also detected in nucleoli (Figure 4D). Combinedin situ hybridization of DjpiwiB and immunostaining of DjPiwiA
and DjPiwiB confirmed that, in the trunk region, transcription of
DjpiwiB occurred only in neoblasts, although DjPiwiB protein
was detected in both neoblasts and differentiated somatic cells
there (Figures 4F, S2, and S4).
Immunostaining of DjPiwiB-depleted animals at day 7 into
DjpiwiB RNAi revealed that DjPiwiB had by then decreased in
80% of neoblasts, but 90% of differentiated somatic cells in
the trunk region were still positive for DjPiwiB (Figure 5A). The immunostaining signals of DjPiwiA and DjPiwiC disappeared fromplanarians after the respective RNAi (Figure S2), indicating theDevelopmental Cell 37, 226–237, May 9, 2016 229

A

Figure 3. Analyses of Cells Expressing
gypsy-P1

B

GFP(RNAi)

piwiB(RNAi)

gypsy-P1 PiwiA

gypsy-P1 PiwiA

gypsy-P1 PiwiA Hoechst

D
X-ray (-)
piwiB(RNAi)

5 d after RNAi
9/9

8/8

8/8

X-ray (+)

7/7

U0126
GFP(RNAi)

piwiB(RNAi)

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600

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400

200

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E

DMSOU0126DMSOU0126
GFP(RNAi) piwiB(RNAi)

piwiB(RNAi)

piwiB(RNAi)Int.

5 d after RNAi

GFP(RNAi)

5 d after RNAi

piwiB(RNAi)

5 d after RNAi

GFP(RNAi)**DMSO
GFP(RNAi)

Number of gypsy-P1 positive cells/mm2

C

Two successive dsRNA feedings were performed
at 3-day intervals after the first feeding for feeding
RNAi.
(A) Expression of gypsy-P1 in the head region of
DjpiwiB(RNAi) planarian at 3 days after the lastfeeding. Green, gypsy-P1-expressing cells; magenta,
DjPiwiA. Scale bar, 500 mm.
(B) Cells expressing gypsy-P1 (green) and neoblasts (magenta). Scale bar, 10 mm.
(C) Expression of gypsy-P1 in DjpiwiB(RNAi) planarians with or without X-ray irradiation treatment.
The first RNAi feeding of DjpiwiB was carried out
for 1 day after irradiation. Five days after the lastRNAi feeding, planarians were sacrificed for in situ
hybridization. Scale bars, 500 mm.
(D) Expression of gypsy-P1 in DjpiwiB(RNAi) planarians treated with U0126. Planarians were treatedwith 25 mM U0126 after the first dsRNA feeding, andcontinuously thereafter. Scale bars, 500 mm. Graphshows the number of gypsy-P1-expressing cells in
those planarians. **p < 0.01, ***p < 0.005. The experiments were performed in triplicate and the SDs
were calculated using Microsoft Excel.
(E) Expression of gypsy-P1 in intact and regenerating DjpiwiB(RNAi) planarians. Planarians
were amputated 1 day after the last dsRNA feeding
and fixed 2 days after amputation. Scale bars,
500 mm.

2 dpa

DjPiwiB was observed in somatic cells
that had newly differentiated from
DjPiwiB-lacking neoblasts (Figures 5D
and S5), i.e., in cells that probably
succeeded in differentiating without TE
derepression at an early time after RNAi.
8/8
6/6
7/7
5/6
5/7
4/5
To examine the possibility that gypsyP1 was actually repressed by inherited
neoblast-specific expression of those proteins and the speci- DjPiwiB, we performed immunostaining using anti-poly(ADPficity of the antibodies used as well as of the RNAi. Western ribose) (PAR), because it is known that poly(ADP-ribosyl)ation
blot analysis confirmed the persistence of DjPiwiB in planarians (PARylation) occurs immediately at DNA break sites after DNA
whose neoblasts had already been eliminated by X-ray irradia- damage (Zhang et al., 2015). X-ray irradiation in D. japonica
tion (Figure 5B). In contrast, the expression levels of DjPiwiA could induce PARylation (Figure 6A). In DjPiwiB-depleted cells
and DjPiwiC proteins were significantly lowered in those planar- in the head region after KD of DjpiwiB, the fraction of cells
ians after X-ray irradiation, as expected (Figure 5B). In fact, only with a strong PAR signal was significantly increased compareda few neoblasts that lacked DjPiwiB expressed gypsy-P1 with the fraction in control GFP KD animals or in DjPiwiB-posi(Figure 5C), whereas many differentiated somatic cells lacked tive cells of KD planarians (Figure 6B), suggesting that activation
DjPiwiB and expressed gypsy-P1 (Figure 5C). This suggests of TE might have resulted in DNA breakage.
that, when DjPiwiB was absent, derepression of gypsy-P1
We found that numerous gypsy-P1-expressing cells showed
occurred during de novo differentiation. In the trunk region, aberrant DNA staining compared with gypsy-P1-negative cells
DjpiwiB mRNA was predominantly expressed in neoblasts (Fig- (Figure 6C). It has been reported that cell death was induced
ure S4) (Hayashi et al., 2010). Therefore, we hypothesized that during early regeneration (Pellettieri et al., 2010). We detected
DjPiwiB protein produced in neoblasts was inherited by somatic degenerating cells, which showed abnormal nuclei and cytodescendant cells during their differentiation. The neoblasts are plasm, by electron microscopy observations in both control
the only cell population that continuously divides in planarian. and DjpiwiB KD planarians at 2 days after amputation
In contrast, differentiated cells never proliferate. This difference (3 days into RNAi) (Figure S6A-A0 , B-B0 , and C-C0 ). In some demight be one reason why DjPiwiB should be retained during generating cells of DjpiwiB KD planarians, however, virus-likethe process of cell differentiation. This hypothesis was further particles were observed (Figure S6C00 ). Also, the upregulation
supported by our observation that low-level accumulation of of two apoptosis-related genes in DjpiwiB KD planarians
230 Developmental Cell 37, 226–237, May 9, 2016

Figure 4. IHC Using anti-DjPiwiB Antibody

PiwiA

PiwiC

Hoechst

merge

PiwiA

PiwiC

Hoechst

merge

PiwiA

PiwiB

Hoechstmerge

Whole body

A

NBs

B

Whole body

C

D

PiwiA

PiwiB

Hoechstferentiation of neoblasts might be disrupted due to cell death by KD of
DjpiwiB.
Thus, we propose that inheritance of
DjPiwiB from the neoblasts by theirdescendant differentiated somatic cells
is indispensable for repressing TEs
during stem cell differentiation (Figures
7A and 7B).
DISCUSSION

NBs
F

Differ. cells

E

merge

(A) Whole-mount co-immunofluorescence analyses using anti-DjPiwiA (magenta) and antiDjPiwiC (green) antibodies. Hoechst 33342 staining
is shown in blue. Scale bars, 1 mm in whole-bodysamples.
(B) Higher magnification of whole-mount sample of(A). Arrowheads indicate punctate signals of
DjPiwiC. Scale bar, 10 mm. NBs, neoblasts.
(C) Co-immunofluorescence analyses using antiDjPiwiA (magenta) and anti-DjPiwiB (green) antibodies. Hoechst 33342 staining is shown in blue.
Scale bars, 1 mm.
(D) Neoblasts (NBs) in whole-body samples.
Arrows indicate nucleoli. Scale bar, 10 mm.
(E) Differentiated cells (Differ. cells) in whole-bodysamples. Scale bar, 10 mm.
(F) Co-staining of DjpiwiB mRNA by in situ hybridization and DjPiwiB protein by immunostaining.
Blue shows DjPiwiA protein. Scale bar, 10 mm.

PiwiA

PiwiB

Hoechst

piwiB

PiwiB

PiwiA

was revealed by RNA-seq and qPCR of DjpiwB KD planarians(Figure S6D), suggesting that gypsy-P1-expressing cells
might be degenerated, which may cause the differentiation
defect. Actually, we found a significant reduction of earlyprogeny of neoblasts, as indicated by the expression of
Dj_aH_000_05955HH, a D. japonica homolog of an early
epidermal progenitor marker in S. mediterranea (Eisenhoffer
et al., 2008; Shibata et al., 2012; van Wolfswinkel et al.,2014), in the regenerating region at 6 days after amputation(7 days into RNAi) (Figure 6D), suggesting that proper early dif-Conserved Roles of PIWI Proteins inGermline Development and PSC
Differentiation
TE suppression by PIWI proteins andmergepiRNA is a fundamental mechanism toassure production of the next generation
via protection of genome integrity inthe germline. Our findings here using
planarian D. japonica revealed that a nuclear PIWI protein, DjPiwiB, plays a crucial
mergerole in TE repression during PSC differentiation to somatic cells, indicating a
conserved mechanism of PIWI-mediatedTE repression between germline development and PSC differentiation. Planarianregeneration is a type of asexual reproduction, in which neoblasts (like germline cells
in non-regenerative animals) are responsible for producing thenext generation. Indeed, germline cells in non-regenerative animals and neoblasts express many genes in common (Shibata
et al., 2010; Solana, 2013), suggesting commonality of the relevant
cellular and molecular features of those cells. This commonality is
not restricted to planarians: a considerable number of invertebrate
species that undergo asexual reproduction using a regenerative
path retain adult pluri- or multipotent adult stem cells in theirbodies, and these cells express genes restricted to germline cells
in non-regenerative animals, including piwi family genes (Juliano
Developmental Cell 37, 226–237, May 9, 2016 231

Figure 5. Inheritance of DjPiwiB by Differentiated Somatic Cells

A
HoechstGFP(RNAi)

Merge

HoechstpiwiB(RNAi)

PiwiA PiwiB

GFP(RNAi)
piwiB(RNAi)(%) ***
***
100

Merge

Fraction of PiwiB-positive cells

PiwiA PiwiB

80
60
40
20

C

B

(kDa)

M

anti-PiwiA

anti-PiwiB

anti-PiwiC

X-ray

X-ray

X-ray

-

+

M

-

+

M

-

ce
lls
er
.
iff

D

N
eo
bl

as
ts

0

gypsy-P1

PiwiA

PiwiB

Merge

+

120
100

Two successive RNAi feedings were performed at
3-day intervals after the first RNAi feeding for
feeding RNAi.
(A) Immunostaining of DjPiwiA and DjPiwiB in
DjpiwiB(RNAi) planarians at 7 days after the lastfeeding. Scale bar, 50 mm. Graph showing comparison of the number of DjPiwiB-positive neoblasts (left) and differentiated cells (right) between
control GFP(RNAi) (white columns) and DjpiwiB(RNAi) (gray columns) animals. ***p < 0.005. The
experiments were performed in triplicate and theSDs were calculated using Microsoft Excel.
(B) Western blot analysis using anti-DjPiwiA, -B,
and -C antibodies in intact (X-ray ) and X-rayirradiated (X-ray +) planarians. M, size markers.
(C) Expression of gypsy-P1 in a neoblast. The
arrowhead indicates a cell double positive forgypsy-P1 (green) and DjPiwiA (magenta). DjPiwiB isshown in blue. Scale bar, 100 mm.
(D) IHC using anti-DjPiwiB antibody in de novo
differentiated cells in partially regenerated planarians (see Figure S3). Arrows show DjPiwiB-negative and DjPiwiA-negative cells. Scale bar, 25 mm.

80

DjpiwiB(RNAi)

GFP(RNAi)

DjPiwi proteins in neoblasts, but only nuclear DjPiwiB, and not the two cytoplasmic PIWIs, was also observed inalmost of all of the somatic descendantDcells of neoblasts (Figures 6A and 6B).
newly differentiatedpre-existing
newly differentiatedpre-existingThis cell-type-dependent PIWI expression
PiwiA PiwiB
PiwiA PiwiB
PiwiA PiwiB
PiwiA PiwiB
is reminiscent of PIWI expression in the fly
germline. In the fly ovary, germ cells express three PIWI proteins: one nuclear(Piwi) and two cytoplasmic (Aubergine[Aub] and Ago3) (Brennecke et al., 2007;
Gunawardane et al., 2007). In contrast,
HoechstHoechstHoechstHoechstsomatic follicle cells, as well as a cultured
ovarian somatic cell line, express only nuclear Piwi (Brennecke et al., 2007; Gunawardane et al., 2007; Saito et al., 2009).
Aub/Piwi and Ago3 in germ cells interact
with TE transcripts that match the strand
opposite to piRNAs, and cleave the tranand Wessel, 2010; Juliano et al., 2010; Ross et al., 2014). Although script using an endonuclease (slicer) activity to repress themno evidence has been reported yet to support it, it seems likely that at the post-transcriptional level (Brennecke et al., 2007; GunaPIWI proteins have a conserved function of repressing TEs in any wardane et al., 2007; Saito and Siomi, 2010). In contrast, nuclear
cells that are responsible for producing the next generation. Actu- Piwi represses TEs at the transcriptional level via epigenetic
ally, germ cells of the related planarian species Dugesia ryukyuen- modification (Sienski et al., 2012). Germ cells and somaticsis, which are also derived from neoblasts during sexual conver- cells are derived from different origins, namely, germline stem
sion from the asexual to sexual state, express three piwi family cells and somatic stem cells, respectively, and so the twogenes, Drpiwi-1, -2, and -3 (orthologs of DjpiwiA, B, and C, cells are not kindred. In contrast, somatic cells in planarians
respectively) (Nakagawa et al., 2012). This may support our spec- are descendants of neoblasts. Thus, these two cases are
ulation about the link between the expression of piwi genes and not absolutely parallel, but it is reasonable to imagine thatthe responsibility of cells for producing the next generation.
DjPiwiB might be the counterpart of Piwi, and so it transcriptionally silences TEs in the nucleus, while cytoplasmic DjPiwiA
How Do Planarian PIWIs Repress TEs?
and DjPiwiC might be the counterparts of Aub and Ago3, or
Immunofluorescence analysis of the whole bodies of D. japonica vice versa, and implement TE silencing at post-transcriptional
using anti-DjPiwi antibodies that we raised detected all three levels.
232 Developmental Cell 37, 226–237, May 9, 2016

A
PAR

X-ray (+)

piwiA

PAR

piwiA

Fraction ofPAR-strongly-positive cells

X-ray (-)

Figure 6. Influence
gypsy-P1

***

100%
80%
60%
40%
20%
0%

X-ray (-) X-ray (+)

piwiB

***

80%

NS

60%
40%
20%

piwiB(RNAi)

PAR

piwiB

piwiB(RNAi)

PAR

100%

GFP(RNAi)

piwiB(RNAi)

GFP(RNAi)

Fraction ofPAR-strongly-positive cells

B

C

(-)
iB

(+
iB

w
Pi

w
Pi

Pi

w

iB

(+

)

)

0%

D

0

GFP(RNAi)

piwiB(RNAi)

Hoechst (+) Hoechst (-)

Why Is DjPiwiB, but Not Other PIWIs, Inherited bySomatic Cells during PSC Differentiation?
Neoblasts proliferated normally, and a TE was silenced in themeven when DjPiwiB was depleted by RNAi. These results suggest that DjPiwiA and DjPiwiC compensate for the functionalloss of DjPiwiB in neoblasts in an epistatic manner, although
the method of DjPiwiB-mediated silencing is supposedly
different from that of silencing by DjPiwiA and DjPiwiC (Figure 6A). Depletion of DjPiwiB caused a severe defect in regeneration, indicating that the role of DjPiwiB at the onset of neoblast differentiation is crucial (Figure 6B). The differentiating
cells expressing gypsy-P1 appeared to die, and perhaps
consequently differentiated cells that were needed to regulate
the differentiation or maintenance of neoblasts could not be
supplied. Another possibility is that other target(s) of DjPiwiB
might affect survivability of the neoblasts. These effects might
have caused the severe defect or decrease of neoblasts at alater period after RNAi. Interestingly, DjPiwiB appears to beneither transcribed nor translated in the differentiating neo-

Number of early-23
progeny cells/10 mm

Depressionof(A) IHC using anti-PAR antibody in intact or X-rayirradiated planarians. Graph shows the fraction of
PAR strongly positive cells in DjPiwiA-positive cells.
Scale bars, 10 mm. Graph shows the fraction of
PAR strongly positive cells (arrowheads) in
DjPiwiA-positive cells. ***p < 0.005.
(B) IHC using anti-PAR antibody in control or
DjpiwiB(RNAi) planarians. Graph shows the fractionof PAR strongly positive cells in DjPiwiB-positive or-negative cells. Arrowheads indicate cells withstrong signals. Scale bars, 10 mm. Graph shows thefraction of PAR strongly positive cells in DjPiwiBpositive or -negative cells. ***p < 0.005.
(C) In situ hybridization of gypsy-P1 with Hoechst
staining. Arrow indicates double-positive cells. Arrowheads indicate gypsy-P1 single-positive cells.
Scale bar, 10 mm. Lower graph shows the numberof cells double positive for gypsy-P1 and Hoechst,
and the number of gypsy-P1 single-positive cells.
(D) In situ hybridization of early progeny marker(Dj_aH_000_05955HH) and immunostaining ofsynaptotagmin in control and DjpiwiB(RNAi) planarians. Dashed lines show regenerated head.
Graph shows the number of early progeny markerpositive cells in the regenerated head region. Scalebar, 500 mm. *p < 0.05.
The experiments were performed in triplicate andthe SDs were calculated using Microsoft Excel.

*
400

blasts, or in the resulting somatic cells,
despite its indispensability for repressing300
TEs in those cells. This requirement torepress TEs during the somatic differenti200
ation process might be a reason toemploy the inheritance of DjPiwiB from
neoblasts by their somatic descendant
100
cells that have exited from the cell cycle
in D. japonica. In sexually reproducing
0
animals, PIWIs are germline specific;
namely, piwi genes are normallyrepressed in somatic cells of the animals. Thus, our observationthat the differentiating neoblasts, or resulting somatic cells, aretranscriptionally silent for piwi is reasonable and not surprising.
However, this might cause a fatal problem for planarians, andthus the animals might have acquired a system for the inheritance of DjPiwiB from neoblasts to somatic descendant cells
during differentiation. However, no DjPiwiA or DjPiwiC is inherited by the fully differentiated somatic cells, although remaining SMEDWI-1 or DjPiwiA was observed in early progenyof neoblasts in both S. mediterranea and D. japonica (Guoet al., 2006; Yoshida-Kashikawa et al., 2007). Why is DjPiwiB
the only PIWI protein selected to remain in descendant cellsof neoblasts during differentiation in D. japonica? One possibility is that DjPiwiB is a nuclear PIWI, which, as suggested
above, might repress gene expression at the transcriptional
level via an epigenetic mechanism such as has been observedin fly. Repression of gene transcription by a single protein in
nuclei would seem likely to be simpler and safer than translational repression of genes by two proteins via their interaction
piwiB(RNAi)100

neuronal cells

GFP(RNAi)

Number of gypsy-P1-positive cells
with/without Hoechst staining
200

early progeny

6 dpa (7 days into RNAi)

gypsy-P1 HoechstofDevelopmental Cell 37, 226–237, May 9, 2016 233

A

Neoblast
Cyt
piRNA

piRNA

Descendent
Nuc

PiwiA

piRNA

PiwiC

Cyt

Nuc

Inheritanceof PiwiB

PiwiB

piRNA

TEs

PiwiB

piwiA mRNA (+)

piwiA mRNA (-)

piwiB mRNA (+)

piwiB mRNA (-)

piwiC mRNA (+)

piwiC mRNA (-)(A) Normal differentiation from a neoblast.
(B) Differentiation from a neoblast in DjPiwiBdepleted planarian.
T bars indicate repression of TEs.

antibodies were obtained from the rabbit sera. Allprocedures were conducted by MBL. The aminoacid sequences of the peptides were DjPiwiA(EPALQPETIIDKVGKDG, corresponding to aminoacids 27–43), DjPiwiB (KKPMRRERRKPGEEDKE,
corresponding to amino acids 303–319), and
DjPiwiC (FENSEKPTTSKFRRREH, corresponding
to amino acids 172–188).

TEs

B

piwiB(RNAi) planarian
Neoblast

Cyt
piRNA

piRNA

Figure 7. Schematic Drawing of Our Working Hypothesis about the Mechanism ofRepression of TEs by Inherited DjPiwiB during the Process of Somatic Differentiationfrom the Neoblasts

Normal planarian

Descendent
Nuc

PiwiA

piRNA

PiwiC

PiwiB

Cyt

Nuc

No inheritanceof PiwiB
piRNA

TEs

TEs

piwiA mRNA (+)

piwiA mRNA (-)

piwiB mRNA (±, -)piwiB mRNA (-)

piwiC mRNA (+)

piwiC mRNA (-)

in the cytoplasm. Recently, distinctive functions of SMEDWI-1
and SMEDWI-3 apart from TE repression, namely, the involvement of these cytoplasmic PIWIs in the localization of histonemRNAs in neoblast-specific nucleoprotein granules, havebeen reported (Rouhana et al., 2014), suggesting that cytoplasmic PIWIs might have particular neoblast-specific functionsin addition to TE repression in planarians. Thus, DjPiwiB might
be produced in neoblasts, and act in their somatic descendants
as epigenetic memory.
EXPERIMENTAL PROCEDURES
Biological Samples
A clonal strain of planarian D. japonica, sexualizing special planarian (SSP)(2n = 16) (Shibata et al., 2012), was cultured at 23 C in highly diluted artificial
seawater (0.05 g/l Instant Ocean Sea Salt powder; Instant Ocean). Chicken
liver was fed to cultured planarians once every 1 or 2 weeks. Animals werestarved for at least 1 week before all experiments.
X-Ray Irradiation
Animals were placed on wet filter paper on ice and irradiated with 160 R of
X-rays using an X-ray generator (SOFTEX B-5; SOFTEX).
Preparation of Antibodies
For production of anti-DjPiwiA, DjPiwiB, and DjPiwiC rabbit polyclonal antibodies, peptides corresponding to a part of each DjPiwi family protein weresynthesized and injected into rabbits. Affinity-purified polyclonal anti-peptide

234 Developmental Cell 37, 226–237, May 9, 2016

Western Blotting
Western blotting was performed as described by
Tasaki et al. (2011). Anti-DjPiwiA, -B, or -C antibody
was used at a dilution of 1/500, 1/1,000, or 1/1,000,
respectively.

PiwiB

Whole-Mount Immunohistochemistry
Whole-mount IHC was performed as described
previously (Yoshida-Kashikawa et al., 2007). The
antibodies against DjPiwi proteins and PAR (Trevigen) were diluted 1/1,000. The samples wereobserved with a confocal microscope (FLUOVIEW
FV10i; Olympus) or a fluorescence stereoscopic
microscope (M205FA T-RC 1; Leica).

Whole-Mount In Situ Hybridization
Whole-mount in situ hybridization was performed
as described previously (Takano et al., 2007;
Umesono et al., 1997). Signals were detected using a TSA kit #2 (Molecular
Probes).

qPCR Analysis of cDNA
qPCR was performed as described by Sakurai et al (Sakurai et al., 2012).
The forward and reverse primer sets for the investigated genes were (50to 30 ):
DjG3PDHFW: ACCACCAACTGTTTAGCTCCCTTAG
RV: GATGGTCCATCAACAGTCTTTTGC
pcna
FW: ACCTATCGTGTCACTGTCTTTGACCGAAAA
RV: TTCATCATCTTCGATTTTCGGAGCCAGATA
h2b
FW: ATTCAAACATCCGTCCGTCTRV: TTTTGTAACAGCCTTCGTTCC
PMRT
FW: AGTCAATAACGGTGAAGAGATAACTGG
RV: CCTTCCGACCTACCTCATTCG
DjpiwiA
FW: CGAATCCGGGAACTGTCGTAG
RV: GGAGCCATAGGTGAAATCTCATTTG
DjpiwiB
FW: ATGGATCCCATGGCTCCTAATG
RV: TGCACAGGGACAGGTACACG
DjpiwiC
FW: GGCCTGGAACTGTTAGAGTACCTG
RV: CAAACGGTCGCACAATAAATGAC

Gypsy-P1
FW: GTCTTTTCTGAAACGTTCAACGAAC
RV: CAGCCCATTTACATTTTGTAGGCTTFeeding RNAi
dsRNA was synthesized basically as described by Rouhana et al. (2013). The
primers for the PCR reaction were as follows (50 to 30 ): Zap Linker + T7, GATCAC TAA TAC GAC TCA CTA TAG GGG AAT TCG GCA CGA GG; M13 Rv,
GTT TTC CCA GTC ACG ACG TTG TAA.
Feeding RNAi was carried out as described previously (Rouhana et al., 2013;
Sakurai et al., 2012). Control animals were fed dsRNA containing the EGFP
cDNA sequence.
Preparation of Planarian Extract for Immunoprecipitation
Two hundred and fifty microliters of Medium Salt Buffer (MSB; 50 mM Tris-HCl,
150 mM NaCl, 0.05% NP-40) containing Mini Complete protease inhibitors(Roche Diagnostics) was added to 20 animals and the samples were quickly
frozen in liquid nitrogen. The frozen samples were thawed on ice, homogenized with a plastic pestle, and further dissociated by pipetting with a 200-ml
pipette. Then freezing and thawing were repeated once more, and the samples
were centrifuged at 18,000 3 g for 10 min at 4 C. The supernatant wascollected in a new tube (store 1). After that, 200 ml of MSB solution containing
protease inhibitor was added to the pellet, and pipetting, freezing, thawing,
and centrifugation were repeated under the same conditions as describedabove. Then the supernatant was collected in a fresh tube (store 2). This seriesof operations was repeated once more, and the supernatant was collected in a
new tube (store 3). Stores 1, 2, and 3 were mixed and used for IP.
Prior to IP, 300 ml of Protein G Sepharose 4 Fast Flow slurry (GE Healthcare)
was centrifuged and resuspended in 1 ml of MSB solution, mixed well by rotation for 5 min, and centrifuged. Then, the supernatant was removed and 650 ml
of MSB solution was added. The solution was separated into 100-ml aliquots,
and 50 ml of anti-DjPiwiB antibody (or pre-immune serum control) was added
to each aliquot and brought up to a final volume of 1 ml with MSB. The mixture
was rotated for 3 hr at 4 C and washed with MSB solution four times for 1 mineach with centrifugations at 5,000 3 g at 4 C.
Two hundred microliters of planarian extract was added to the anti-DjPiwiB
antibody bound to Protein G Sepharose, and MSB solution was added to a
final volume of 1 ml. The mixture was rotated for 1 hr at 4 C and washedfive times with 1 ml of cold MSB solution for 3 min each, with centrifugation
at 4 C at 5,000 3 g. Finally, the supernatant was removed completely andthe immunoprecipitate was used for protein electrophoresis or collection ofRNAs.

onto the D. japonica shotgun genome and EST sequence dataset was performed with BWA (0.6.1-r104; http://bio-bwa.sourceforge.net/) with default
settings (Li and Durbin, 2009). According to the mapping results (allowing
for at most two mismatches), piRNAs corresponding to the D. japonica
genome or transcripts were used for the subsequent analyses. Nucleotide
frequency at each nucleotide position of the piRNAs was calculated and illustrated using the seqLogo package of Bioconductor (http://www.bioconductor.
org/packages/2.12/bioc/html/seqLogo.html).
Transposon Annotation of Dugesia japonica Transcriptome
A survey of transposon taxonomic distribution was conducted by matching the
EST unigenes to the protein sequences in Repbase (ver. 16.08; http://www.
girinst.org/downloads/) using BLASTX software (ver. 2.2.25) with default settings. Only the top hits with E-value less than 1 3 106 were used forannotation.
RNA-Seq Analysis of DjpiwiB-Knockdown Planarians
mRNAs were prepared from planarians at 7 days after RNAi of GFP and
DjpiwiB. RNA-seq was conducted with GS FLX + (Roche Diagnostics), Miseq(Illumina), or Hiseq2500 (Illumina). All sequence data were deposited in
PRJDB4258. De novo transcriptome assembly for making reference sequences using reads obtained with Roche 454 and Miseq (Table S2) was conducted with Trinity with the CuffFly option (version r20140413p1) (Henschelet al., 2012). The assembled reference sequence was deposited as DRA:
DRZ007413 and DDBJ: IAAB01000001-IAAB01137201. The reads obtained
by Hiseq (Table S2) were mapped onto the reference sequence by usingalign_and_estimate_abundance.pl distributed with Trinity, by using Bowtie(Hatem et al., 2011) and eXpress (Roberts and Pachter, 2012). The rounded
estimated read counts were used for differentially expressed gene analysis
as described by Sun et al. (2013). The analysis result was deposited as
DRA: DRZ007434. For details of specific procedures, see Supplemental
Information.
Treatment with MEK Inhibitor Followed by Feeding RNAi
MEK inhibitor treatment was carried out as described by Tasaki et al. (2011).
ACCESSION NUMBERS
The accession number for the piRNA-seq result reported in this paper is DRA:
DRA002837. The accession number for RNA-seq and the results of the in silico
analysis reported in this paper is DRA: PRJDB4258.
SUPPLEMENTAL INFORMATION

Protein Electrophoresis and Silver Staining
Two hundred microliters of sample buffer was added to beads containing
immunoprecipitate and the mixture was boiled for 10 min at 95 C and centrifuged at maximum speed in a tabletop centrifuge at 4 C. The lysate wassubjected to SDS-PAGE. Silver staining was conducted using a SilverQuest
Staining Kit (Invitrogen) according to the manufacturer’s instructions.
Co-immunoprecipitation and Sequencing of
DjPiwiB-Interacting RNA
The immunoprecipitate obtained using anti-DjPiwiB antibody was washedwith MSB solution, and 150 ml each of H2O and phenol/chloroform was added.
After vortexing, the sample was centrifuged for 10 min, and the aqueous layer
was transferred to a new tube. The addition of phenol/chloroform and centrifugation were repeated. After transferring the aqueous layer to a new tube,
500 ml of chloroform was added. After mixing, centrifuging, and transferring
the aqueous layer to a new tube, RNAs were precipitated by ethanol precipitation. The RNAs obtained were labeled with 32P and detected by electrophoresis. Immunoprecipitated RNAs were sequenced by Hokkaido System Science using an Illumina sequencer. The piRNA-seq data is deposited in DRA:
DRA002837.
Analysis of the DjPiwiB-Interacting piRNAs
After adapter trimming with a handmade Ruby script, reads longer than 33 bp
were removed. To select reads derived from D. japonica, mapping of piRNA

Supplemental Information includes Supplemental Experimental Procedures,
six figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.devcel.2016.04.009.
AUTHOR CONTRIBUTIONS
N.S. and K.A. designed the study. N.S., M.K., and T.I. performed the study.
M.K. and O.N. performed in silico analysis of the piRNAs. N.S., L.R., K.S.,
H.S., and M.S. performed IP and isolation of piRNA. S.Y. and K.M. performed
the transmission electron microscopy observations. N.S., M.S., and K.A. wrote
the manuscript.
ACKNOWLEDGMENTS
We are grateful to Elizabeth Nakajima, Hayoung Lee, Nobuyoshi Kumagai,
Yuki Sato, and Takeshi Inoue for critical reading of the manuscript and to
Prof. Yoshio Koyanagi of the Institute for Virus Research, Kyoto University,and Prof. Koichi Sano of Osaka Medical College for commenting on the transmission electron microscopy analysis. We also thank all of our laboratory
members for their help and encouragement. This work was supported by aGrant-in-Aid for Scientific Research on Innovative Areas to K.A. (22124001),
a Grant-in-Aid for Creative Scientific Research to K.A. (17GS0318), Global
COE Program A06 of Kyoto University, the Naito Foundation, a Sasakawa

Developmental Cell 37, 226–237, May 9, 2016 235

Scientific Research Grant, the Future Development Funding Program of Kyoto
University Research Coordination Alliance, a Grant-in-Aid for Scientific
Research on Innovative Areas ‘‘RNA regulation’’ (20112006) to N.S., a JSPS
Research Fellowship to M.K., and a Minority Postdoctoral Research Fellowship from the NSF (No. 0804021) to L.R.
Received: September 25, 2015
Revised: January 18, 2016
Accepted: April 11, 2016
Published: May 9, 2016

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