Dev Genes Evol (2006) 216: 335–346DOI 10.1007/s00427-006-0060-0

ORIGINAL ARTICLE

Leonardo Rossi . Alessandra Salvetti . Annalisa Lena .Renata Batistoni . Paolo Deri . Claudio Pugliesi .Elena Loreti . Vittorio Gremigni

DjPiwi-1, a member of the PAZ-Piwi gene family,defines a subpopulation of planarian stem cells

Received: 3 August 2005 / Accepted: 20 January 2006 / Published online: 11 March 2006# Springer-Verlag 2006

Abstract Planarian regeneration, based upon totipotentstem cells, the neoblasts, provides a unique opportunity tostudy in vivo the molecular program that defines a stemcell. In this study, we report the identification of DjPiwi-1,a planarian homologue of Drosophila Piwi. Expressionanalysis showed that DjPiwi-1 transcripts are preferentiallyaccumulated in small cells distributed along the midline ofthe dorsal parenchyma. DjPiwi-1 transcripts were notdetectable after X-ray irradiation by whole mount in situhybridization. Real time reverse transcriptase polymerasechain reaction analysis confirmed the significant reductionofDjPiwi-1 expression after X-ray treatment. However, thepresence of residual DjPiwi-1 transcription suggests that,although the majority of DjPiwi-1-positive cells can beneoblasts, this gene is also expressed in differentiating/differentiated cells. During regeneration DjPiwi-1-positivecells reorganize along the midline of the stump and noaccumulation of hybridization signal was observed eitherin the blastema area or in the parenchymal region beneath

the blastema. DjPiwi-1-positive cells, as well as theDjMCM2-expressing neoblasts located along the midlineand those spread all over the parenchyma, showed a lowertolerance to X-ray with respect to the DjMCM2-expressingneoblasts distributed along the lateral lines of the paren-chyma. Taken together, these findings suggest the presenceof different neoblast subpopulations in planarians.

Keywords Stem cells . Neoblasts . X-ray . Planarians .PAZ-Piwi proteins

Introduction

The ability of stem cells to self-renew and producedifferentiated progeny is critical for the development andmaintenance of a wide variety of tissues in metazoans (Linand Schagat 1997; Lin and Spradling 1997; Morrison et al.1997; Potten 1977). Stem cells divide asymmetrically togive rise to one cell identical to the mother and another cellfated to differentiate (Watt and Hogan 2000; Yamashita etal. 2005). Members of the Piwi/Argonaute/Zwille (PAZ)RNA-binding protein family are considered key regulatorsinvolved in stem cell self-renewal in various organisms(Deng and Lin 2001; Forbes and Lehmann 1998; King etal. 2001; Lin and Spradling 1997; Moussian et al. 1998;Sharma et al. 2001; Smulders-Srinivasan and Lin 2003;Tan et al. 2002). Drosophila Piwi modulates the rate ofstem cell division (Cox et al. 2000) and interacts with thepre-mRNAs or mRNAs of target genes in the nucleus toattenuate their processing, life span, and transport into thecytoplasm (Smulders-Srinivasan and Lin 2003). In partic-ular, it has been demonstrated that Piwi maintainsDrosophila germ stem cells by repressing bag-of-marbles(Bam) expression. In absence of Piwi, Bam downregulatesPumilio/Nanos function in repressing the translation ofgenes necessary for differentiation (Chen and McKearin2005; Szakmary et al. 2005). ZWILLE, the Arabidopsishomologue of Drosophila Piwi, is required for meristemplasticity, and it has been suggested that it buffers the shootapical meristem stability by ensuring the critical number

Communicated by M.Q. Martindale

L. Rossi . A. Salvetti . A. Lena . V. Gremigni (*)Dipartimento di Morfologia Umana e Biologia Applicata,Università di Pisa,Pisa, Italye-mail: gremigni@biomed.unipi.itTel.: +39-050-2219103Fax: +39-050-2219101

R. Batistoni . P. DeriDipartimento di Biologia,Università di Pisa,Pisa, Italy

C. PugliesiDipartimento di Biologia delle Piante Agrarie,Sezione di Genetica,Università di Pisa,Pisa, Italy

E. LoretiIstituto di Biologia e Biotecnologie Agricole,Consiglio Nazionale delle Ricerche,Pisa, Italy

of apical cells required for proper meristem function(Moussian et al. 2003).

Planarians (Platyhelminthes, Tricladida), an invertebrategroup well known for their extraordinary regenerativeability, retain a population of totipotent stem cells, calledneoblasts, throughout their life. Because neoblasts possessthe ability to self-renew and to give rise to all differentiatedcell types, they represent an important system to study themechanisms defining a stem cell in vivo. Neoblasts aredistributed throughout the planarian parenchyma with theexception of the most anterior end of the cephalic regionand the pharynx. In addition, neoblasts are accumulated inmultiple clusters along the midline and the lateral lines inthe dorsal parenchyma, as demonstrated by the use ofproliferating cell markers, such as DjMCM2 and DjPCNA(Orii et al. 2005; Salvetti et al. 2000). During regeneration,neoblasts proliferate and accumulate beneath the woundepithelium, giving rise to the regenerative blastema(Baguñà 1998; Brφndsted 1969; Gremigni 1981; Salòand Baguñà 2002). Although neoblasts share a similarmorphology—small cells with a thin rim of undifferen-tiated cytoplasm surrounding a round nucleus—they areconsidered a heterogeneous cell population (Reddien andSánchez Alvarado 2004). However, up to now, no molec-ular marker has been found useful to distinguish neoblastsubpopulations. Recent studies have made significantadvances in the understanding of the totipotent stem cellsystem in planarians (Agata 2003; Reddien et al. 2005a,b).Recently we isolated DjPum, a planarian homologue ofDrosophila Pumilio. DjPum is preferentially expressed inplanarian stem cells and its knockdown by double-strandedRNA (dsRNA)-mediated genetic interference (RNAi)resulted in a dramatic reduction in the number of neoblasts,thus supporting the hypothesis of a key role for this gene inneoblast maintenance (Salvetti et al. 2005). In this study,we report the isolation and characterization of DjPiwi-1, aplanarian homologue of Drosophila Piwi, which isexpressed in a specific subpopulation of small cells,round in shape, distributed along the central parenchymaunder the dorsal epidermis. X-ray treatment dramaticallyaffects the viability of DjPiwi-1-positive cells suggestingthat most of them can be neoblasts. We also demonstratethat DjPiwi-1-positive cells, as well as the DjMCM2-expressing cells located along the midline, have a lowertolerance to X-ray irradiation than the clusters ofDjMCM2-expressing cells distributed along the lateral lines.

Materials and methods

Animals

Planarians utilized in this work belong to the speciesDugesia japonica, clonal strain GI (Orii et al. 1993).Animals were kept in autoclaved stream water at 18°C andstarved for 1 or 2 weeks before being used in theexperiments. Regenerating fragments were obtained asdescribed by Salvetti et al. (1998).

X-ray irradiation

Intact planarians were exposed to 30 gray (Gy) of hard X-rays (200 KeV, 1 Gy/min) using a Stabilipan 250/1instrument (Siemens, Gorla-Siama, Milan, Italy) equippedwith a Radiation Monitor 9010 dosimeter (RadcalCorporation, Monrovia, CA, USA). The animals werekilled 3, 24, 48 h, or 6 days after irradiation and processedfor whole mount in situ hybridization. Ultrastructuralanalysis was performed on X-ray irradiated planarianskilled 3, 6, 24, or 48 h after the treatment (dose: 30 Gy).Real-time reverse transcriptase polymerase chain reaction(RT-PCR) was performed on X-ray irradiated specimenskilled 1, 2, 4, or 7 days after irradiation (dose: 30 Gy).

Cloning of DjPiwi-1 and sequence analysis

The SMART RACE cDNA amplification kit (Clontech)was used to obtain the full-length DjPiwi-1 sequence.Amplification of the 5′ region was obtained with thesequence-specific antisense primer 5′-TTTGCGGTACTTGACACCCATTCTCTTT-3′. The 3′ region was amplifiedwith the sequence-specific sense primer 5′-TGGAACGAAACTATTTAACAGAGGTGA-3′. The PCR productswere TA-cloned using pGEM-T Easy Vector System(Promega). All clones were sequenced by automatedfluorescent cycle sequencing (ABI). Sequences related toDjPiwi-1 were identified with BLASTX (Altschul et al.1990). The BLAST2 sequence program was utilized toevaluate the nucleotidic and aminoacidic identity valuesbetween D. japonicaPiwi-like EST clones and DjPiwi-1.

The following primers have been utilized to evaluate theidentity between the Piwi-like EST clones:

– 917S : 5′-GAAGCTAGGGCATTCGATGG-3′– 917AS: 5′-AAGAACATTGTACACCATAGGA-3′– 378S: 5′-TTTAACACAATGCGTGACAACC-3′– 378AS: 5′-GAATACAGATGCTTGAATTGAC-3′– 725S: 5′-TCGGATTACTGATATTGATTGG-3′– 725AS: 5′-TTAAATACACTAATCCACCATCA-3′– 457S: 5′-GAAAATAAACCCCTCGAAATTG-3′– 457AS: 5′-TCAATTCTTGCTCTACGTCACT-3′– 967S: 5′-CAGAGTTCTAAATGATAATTCAGT-3′– 967AS: 5′-CAATACAAATGGTTGGTCACC-3′

In situ hybridization

Whole mount in situ hybridization was carried outaccording to the protocol described by Umesono et al.(1997). In situ hybridization on wax sections was carriedout according to Kobayashi et al. (1999). Transmissionelectron microscope (TEM) in situ hybridization wasperformed as described by Pineda et al. (2002). The clonesnamed: Piwi box (spanning from 1,911 bp to 2,379 bp ofthe DjPiwi-1 gene), DjMCM2 (Salvetti et al. 2000), DjPum550 (Salvetti et al. 2005), and Dj18S (Pineda et al. 2002)

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were used to obtain sense and antisense DIG-labeled RNAprobes using the DIG-RNA labelling kit (Roche).

Transmission electron microscopy

Transmission electron microscopy (TEM) was performedas previously described by Salvetti et al. (2005). Briefly,planarians were fixed with a 2.5% glutaraldehyde solutionin 0.1 M cacodylate buffer and postfixed with 2% osmiumtetroxide. Ultrathin sections were stained with uranylacetate and lead citrate and observed with a Jeol 100 SXtransmission electron microscope.

Mitosis analysis

Small pieces of tissue from central and lateral parts of thebody anterior to the pharynx were dissected as shown inFig. 8a. These pieces were directly placed on a microscopeslide and stained as described by Deri et al. (1999). Aftersquashing, slides were scored with a Zeiss Axioplanphotomicroscope. About 6,000 nuclei were examined foreach preparation and the number of mitotic figures wastabulated. Two different counting were carried on for eachslide. Six central and eight lateral regions from six differentanimals were analyzed. A statistical evaluation was madeusing the Student’s t test. The level of significance was setas p<0.05.

Real-time RT-PCR

Total RNA, from three animals for each experimentalpoint, was extracted with the Nucleospin RNA II Kit(Macherey-Nagel), according to the manufacturer’s in-structions. Antisense RNA (aRNA) was linearly amplifiedaccording to Wang et al. (2000) from 100 ng of total RNAin 9 μl of water containing 0.25 μl (1 μg/μl) oligo-dT (15)-T7 (5′-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGC-3′) primer. Total RNA was denaturedat 70°C for 3 min and primed while cooling to roomtemperature. T7 bacteria phage promoter was incorporatedinto cDNA synthesis in a reverse transcription (RT)reaction by adding 4 μl of first-strand reaction buffer,2 μl 0.1 M dithiothreitol (DTT; Gibco-BRL), 2 μl 10 mMdNTP, 1 μl SUPERase (Ambion), 1 μl (0.25 μg/μl)template switch primer (5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′) (Clontech), and 2 μl SuperscriptII (SSII) reverse transcriptase (Gibco-BRL). cDNA syn-thesis was completed at 42°C for 90 min. Full-lengthdscDNAwas synthesized by adding 106 μl of DNase-freewater, 15 μl Advantage PCR buffer (Clontech), 3 μl10 mM dNTP, 1 μl RNase-H (Promega), 3 μl AdvantagecDNA Polymerase (Clontech). The following temperaturecycle was used: 5 min at 37°C for RNA digestion, 2 min at94°C for denaturation, 1 min at 65°C for priming, and

30 min at 75°C for extension. Reactions were terminatedby incubation in 7.5 μl 1 M NaOH with 2 mM EDTA at65°C for 10 min. cDNA was extracted with phenol-chloroform-isoamyl, precipitated with ethanol in thepresence of 2 μl linear acrylamide (0.1 μg/μl, Ambion)and resuspended in 8 μl of water. Purified full-lengthdscDNAs were incubated with 2 μl of each 75 mM NTP(ATP, GTP, CTP, and UTP), 2 μl of 10× reaction buffer,and 2 μl of transcription enzyme mixture (T7 MegascriptKit, Ambion) in 20 μl at 37°C for 6 h. RNA recovery andremoval of template DNA was achieved by TRIZOLpurification. 0.5 μg of aRNAwere reverse transcribed intocDNA using 2 μg of random hexamer with 5 μl first-strandbuffer, 2 μl 0.1 M DTT, 1 μl SUPERase (Ambion), 2 μl of10 mM dNTP, and 2 μl of SSII. The reaction mixture washeated to 65°C for 10 min before adding SSII, thensynthesis was continued at 42°C for 90 min. Second strandcDNA synthesis was initiated by 1 μg oligo-dT-T7 primerin the conditions used in the first round. In vitrotranscription of aRNA was carried out as for the firstround in 40 μl as final volume. One microgram of eachaRNA was reverse transcribed into cDNA with theSuperscript First Strand Synthesis System for RT-PCR(Invitrogen) and random hexamer (Invitrogen). Real-timePCR amplification was carried out with the ABI Prism7000 Sequence Detection System (Applied Biosystems)using primer sense A1 5′-TCAAAGAAAGGTATAACATAAATTTAACAACAGAACAG-3′ and primer antisenseA2 5′-CATTATGTTCTTTTCTTTTATCTCTTCTCGCAA-3′ for DjPiwi-1; primer sense B1 5′-GCAATCGAAGACGTTCCATGTG-3′ and primer antisense B2 5′-CCAGGAAAAGTTGTTATAGTCCCAGTTT-3′ for DjEF2.The constitutively expressed elongation factor gene DjEF2was used as an endogenous control. Taqman probesspecific for each gene were used. Probe sequences are:5′-CAGCCATTTGTATTAACCC-3′ for DjPiwi-1 and 5′-CCAACAAGTCCACATATGTT-3′ for DjEF2. PCR reac-tions were carried out using 20 ng cDNA and TaqManUniversal PCR Master Mix (Applied Biosystems) follow-ing the manufacturer’s protocol. Relative quantification ofeach single-gene expression was performed using thecomparative CT method as described in the ABI Prism7700 Sequence Detection System User Bulletin No. 2(Applied Biosystems).

RNAi experiments

DjPiwi-1 Piwi box and DjPiwi-1 318 (1,592 bp to 1,910)were digested with ApaI and PstI to obtain sense andantisense RNA, respectively. dsRNA preparations wereobtained as described by Salvetti et al. (2005). Intactplanarians were injected with DjPiwi-1 Piwi box-, DjPiwi-1 318- dsRNA and processed, as described by Salvetti et al.(2005). Negative controls were done by injection of wateror β-Gal-dsRNA.

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Endogenous transcript analysis by comparativeRT-PCR

Total RNAwas extracted from planarian fragments injectedwith either DjPiwi-1 Piwi box or water, using theNucleoSpin RNAII kit (Macherey-Nagel). cDNA wasgenerated from 0.3 μg of total RNA using the SuperscriptFirst Strand Synthesis System for RT-PCR (Invitrogen).Injected fragments were killed for RNA extraction 4 daysafter the first or second transection. To assess the reductionof endogenous transcripts in the injected specimens, thefollowing primers were utilized:

– DjPiwi-1 forward 5′-TGAGAATTATGGTTTACCGAG-3′

– DjPiwi-1 reverse 5′-AGGAACTGGAACTCTAATGG-3′

Control reactions were performed in the absence ofreverse transcriptase. DjEF2 was amplified as an internalcontrol using forward 5′-TTAATGATGGGAAGATATGTTG-3′ and reverse 5′-GTACCATAGGATCTGATTTTGC-3′ primers. For each PCR reaction the concentration ofcDNA and the number of cycles used were optimized toobserve a quantifiable signal within the linear range ofamplification, according to the putative abundance of eachmRNA amplified and the size of the corresponding PCRproduct. The analysis was performed in duplicate with RNAextracted from at least two independent samples.

Results

Molecular cloning of DjPiwi-1

In the course of a whole genome PCR screening for DNAtarget sequences of an unrelated transcription factor, weserendipitously isolated, in Dugesia japonica, a cDNAfragment of 400 bp which showed similarity withDrosophila Piwi. We completed the sequence of theplanarian Piwi-related gene, called DjPiwi-1, by a 3′-5′RACE strategy. DjPiwi-1 cDNA is 2,544 bp long(accession no. AJ865376) and contains an open readingframe (ORF) encoding a putative protein of 818 aminoacids that shares significant similarity with members of thePAZ-Piwi family. Sequence conservation is remarkable in

the carboxy-terminal region including the Piwi box(residues 638 to 793) that is part of a larger Piwi domain(residues 520 to 816). The DjPiwi-1 central region containsa PAZ domain (residues 193 to 351), a sequence shared byall members of the PAZ-Piwi and Carpel Factory (CAF)families (Cerutti et al. 2000). Piwi and PAZ domains havebeen found to be implicated in RNAi processes and relatedphenomena, such as post-transcriptional gene silencing(PTGS) and transcriptional gene silencing (TGS) in severalorganisms (Doi et al. 2003; Ma et al. 2005; Pal-Bhadra etal. 2002; Parker et al. 2005; Tijsterman et al. 2002;Vaucheret et al. 2001). Although Drosophila Piwi is anucleoplasmic protein (Cox et al. 1998, 2000), the mousePiwi homologues, MILI, and MIWI, show a cytoplasmiclocalization (Kuramochi-Miyagawa et al. 2001). Nonuclear localization signal has been detected in DjPiwi-1by PSORT analysis (Nakai and Kanehisa, 1992), suggest-ing that the planarian protein has a cytoplasmic localiza-tion. The PAZ-Piwi protein most closely related (BLASTXE value: 1e−73) to DjPiwi-1 is Danio rerio Piwi-like 1protein (Tan et al. 2002). A cDNA fragment, containing theC-terminus of the Piwi domain of a planarian Piwi-likegene, has been isolated in the planarian Schmidteamediterranea (clone H.2.12c; Sánchez Alvarado et al.2002). The comparison between DjPiwi-1 and H.2.12cshows 65% identity at the amino acid level in theoverlapping region. In addition, some cDNA fragmentscoding for Piwi-related proteins (Table 1) have beenidentified in a head cDNA Expressed Sequence Tag (EST)collection of D. japonica (Mineta et al. 2003). Sequenceanalysis of these EST fragments revealed that at least twoother Piwi-related genes exist in the planarian genomebesides DjPiwi-1 (Fig. 1a). Indeed, clones gi 32903681, gi32904967, and gi 32904457, which differ from DjPiwi-1,are identical to each other in the overlapping regions,indicating that these fragments belong to the same Piwi-like gene. Clone gi 32903725 shows no significantsimilarity at the nucleotidic level with DjPiwi-1 andclone gi 32904967, suggesting that this sequence is afragment of another Piwi-like gene. Finally, gi 32899917and gi 32901378 clones differ from DjPiwi-1 and do notoverlap any other EST Piwi like fragment. Clone gi32902540 is identical to clone gi 32901378, except for twonucleotide deletions that change the reading frame. Todefine more precisely the number of Piwi-related genes in

Table 1 Piwi-like clones selected in the Dugesia japonica EST collection

Gi number Homology BlastX E value Nucleotidic identity (%)vs DjIwi

Aminoacidic identity (%)vs DjIwi

32901378 Piwi-related protein(Tetrahymena thermophila)

8e−17 75 82

32903681 Cniwi (Podocoryne carnea) 8e−07 72 7132904967 Seawi (Strongylocentrotus purpuratus) 2e−09 74 7132904457 Cniwi (Podocoryne carnea) 8e−07 72 7132903725 Piwi-like 2 (Canis familiaris) 1e−31 Not found 2832902540 Seawi (Strongylocentrotus purpuratus) 8e−23 72 Not found32899917 Piwi-like 1 (Danio rerio) 2e−12 Not found Not found

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D. japonica, we designed several couples of primers toamplify cDNA regions included between the different genefragments. RT-PCR analysis demonstrated that both clonesgi 32899917 and clone gi 32903725 contain differentfragments of the same gene while clones gi 32901378, gi32903681, gi 32904967, and gi 32904457 contain frag-ments belonging to another gene (Fig. 1b).

DjPiwi-1 expression as shown by light and electronmicroscopy defines a subpopulation of neoblast-likecells

In situ hybridization experiments showed that DjPiwi-1transcripts were specifically accumulated in small paren-chymal cells located under the dorsal epidermis, distributedalong the midline and interrupted at the head and pharynxlevel (Fig. 2a–e). DjPiwi-1 hybridization signal appearedmore detectable in the prepharyngeal region than in thepostpharyngeal region (Fig. 2a). No DjPiwi-1 hybridiza-tion signal was observed in control specimens hybridizedwith DjPiwi-1 sense RNA probe (Fig. 2b). In situhybridization on wax sections demonstrated that DjPiwi-1 transcripts were specifically distributed in small paren-chymal cells round in shape, which were grouped under thedorsal epidermis along the central midline (Fig. 2c–e). NoDjPiwi-1-positive cell was detected in other parenchymalregions by using this technique.

In situ hybridization experiments performed on ultrathinsections obtained from the dorsal central region anterior tothe pharynx (Fig. 3a), showed that DjPiwi-1 transcriptswere localized in small cells with a high nucleo-cytoplas-mic ratio (Fig. 3b,c). DjMCM2- and DjPum-expressingneoblasts were also found in the central region (Fig. 3d,e).Positive controls with the ribosomal 18S riboprobe (Dj18S)showed that the nucleolus was labeled (Fig. 3f). Nosignificant cluster of gold particles was observed withDjPiwi-1, DjMCM2, and DjPum sense-strand probes, orafter RNase treatment followed by hybridization usingDjPiwi-1, DjPum, or DjMCM2 antisense-strand probes(Fig. 3g). Because the technical procedure for ultrastruc-tural in situ hybridization does not allow an optimalpreservation of the subcellular structures, we also madeTEM ultrastructural observations of the same region wherein situ hybridization was performed. Figure 4a shows thepresence of some neoblast clusters located under the dorsalepidermis along the midline of the anterior parenchyma.The neoblasts were distinguishable by their morphologicalhallmarks such as very small size and undifferentiatedcytoplasm with free ribosomes and chromatoid bodies(Gremigni 1981; Hori 1992; Hori and Kishida 2003;Pedersen 1972) (Fig. 4b).

Regenerant fragments, killed 1, 3, or 6 days aftertransection, showed a pattern of DjPiwi-1 expressionsimilar to that observed in intact organisms, independentlyof the orientation of the cut. DjPiwi-1 transcripts were still

Fig. 1 Analysis of D. japonica Piwi-like sequences. a Schematicrepresentation of the Piwi-like clones found in the D. japonica ESTcollection compared to DjPiwi-1. Arrows indicate the primersdesigned to define the number of Piwi-like genes in D. japonica.Primers are indicated in black. Numbers in black indicate theaminoacidic position of the clones with respect to DjPiwi-1. b RT-PCR analysis of the Piwi-like genes found in D. japonica.Amplifications between different clones were performed using the

following couples of primers: lane 1:457 S vs 378 AS, lane 2:917 Svs 378 AS, lane 3:917 S vs 725 AS, lane 4:725 S vs 378 AS, lane5:917 S vs 967 AS. Control amplifications within the same genefragment were performed using the following couples of primers:lane 6:378 S vs 378 AS, lane 7:967 S vs 967 AS, lane 8:457 S vs457 AS, lane 9:725 S vs 725 AS, lane 10:917 S vs 917 AS. Negativecontrols (lane 11 to 15) were performed with the same couples ofprimers (lanes 1 to 5) in the absence of cDNA

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Fig. 2 Analysis of DjPiwi-1expression in intact D. japonica.a,b Dorsal view of an intactplanarian, as visualized bywhole mount in situ hybridiza-tion. a DjPiwi-1 antisense RNA.Arrowheads indicate DjPiwi-1expression b DjPiwi-1 senseRNA. Scale bars 500 μm. c–e Insitu hybridization on transversewax sections of an intact pla-narian. c Schematic drawing ofthe level of the sections depictedin d and e. ep, epidermis; IpcDjPiwi-1-positive cells; g gut;ph pharynx. d DjPiwi-1 hybrid-ization signal is detected inparenchymal cells localizedunder the dorsal epidermis. e Acluster of neoblast-like cells thatexpress DjPiwi-1 transcripts.Scale bars 20 μm

Fig. 3 In situ hybridization onultrathin sections. a Schemedepicting the central region ofthe dorsal parenchyma used forthe experiments (black box).Embedded specimens weresquared on the basis of toluidineblue stained semithin transversalsections to specifically select thecentral dorsal area (dotted box).g gut; vn ventral nerve cord; phpharynx. b Low magnificationof the planarian dorsal centralregion showing cells that havethe morphometric requirementsto be considered neoblasts. neneoblasts. Scale bar 2 μm. cMagnification of b (black box)showing clusters of gold parti-cles (arrows) on the cytoplasmof a neoblast after hybridizationwith DjPiwi-1 antisense RNA.d A cluster of gold particles(arrow) is visible on a neoblastafter hybridization withDjMCM2 antisense RNA.e A cluster of gold particles(arrow) is visible on a neoblastafter hybridization with DjPumantisense RNA. f Clusters ofgold particles (arrows) on thenucleolus after hybridizationwith Dj18S antisense RNA.n nucleus. Scale bars 0.5 μm.g No cluster of gold particles isvisible on a neoblast hybridizedwith DjPiwi-1 antisense RNAafter RNAse treatment. n nucle-us. Scale bar 1 μm

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detected along the midline and no accumulation of DjPiwi-1 mRNAwas observed either in the blastema area or in theparenchymal region beneath the blastema (post-blastema)(Fig. 5). The analysis of DjPiwi-1 expression pattern insmall regenerating fragments obtained from regions of thehead and tail showed the presence of DjPiwi-1 hybridiza-tion signal mainly localized in the midline of the stump. Itis noteworthy that the hybridization signal was detectedalong the antero-posterior axis at levels where it could notbe observed both in intact planarians and larger head andtail regenerants (Fig. 5d,e).

DjPiwi-1-positive cells show a low radiotoleranceto X-ray

It is well known that X-ray treatment inhibits planarianregenerative capability by selectively destroying themitotically active stem cell population (Baguñà et al.1989; Lange 1968a,b; Orii et al. 2005; Shibata et al. 1999).Because X-ray induces a dramatic reduction in the numberof DjMCM2- and DjPum-expressing neoblasts (Salvetti etal. 2000, 2005), we explored the possibility that X-rayirradiation also causes changes in the pattern of DjPiwi-1expression. After X-ray irradiation at 30 Gy of intactplanarians, DjPiwi-1 hybridization signal was no longerdetectable by whole mount in situ hybridization (Fig. 6a).Real time RT-PCR analysis of DjPiwi-1 transcriptsrevealed that a significant reduction of the expression ofthis gene occurred 24 h after X-ray treatment. However, it

is interesting to note that a residual expression of DjPiwi-1was still detectable 2, 4, and 7 days after irradiation(Fig. 6b).

We compared the changes in the expression pattern ofDjPiwi-1 andDjMCM2 at different times after irradiation at30 Gy (Fig. 6a). By whole mount in situ hybridization,DjPiwi-1-positive cells were no longer detected 24 h afterexposure. At the same time, DjMCM2 expression patternalso changed. DjMCM2-positive cells located along themidline anterior to the pharynx, as well as those spread allover the parenchyma, were no longer detectable. On thecontrary, DjMCM2 hybridization signal could be stilldetected along both lateral regions and the midlineposterior to the pharynx. Some DjMCM2-positive cells,grouped in lateral regions of the parenchyma, were stillobserved 48 h after irradiation (Fig. 6a). TEM analysisshowed that, following X-ray treatment, clusters ofneoblasts, located under the dorsal epidermis along themidline of the anterior central parenchyma, exhibitedmorphological features typical of apoptosis, such asmarked cytoplasm shrinkage and chromatin condensation.Some isolated neoblasts undergoing apoptosis were alsoobserved 3 h after X-ray treatment in this area. Apoptoticcell clusters were visible starting from 6 h after irradiation(Fig. 7a). A small number of apoptotic cell clusters werestill observed 24 (Fig. 7b) and 48 h after irradiation. No

Fig. 4 Ultrastructural analysis of the dorsal parenchymal regionalong the midline of the intact D. japonica. a Neoblast-like cells(arrows) are visible under the epidermis. ep epidermis; bl basallamina. Scale bar 5 μm. b Magnification of a neoblast showingundifferentiated cytoplasm rich in ribosomes. Arrowheads indicatechromatoid bodies. Scale bar 0.5 μm

Fig. 5 DjPiwi-1 whole-mount in situ hybridization of regeneratingplanarians, 3 days after transection. Blue dashed lines indicate theblastema region. a–f Dorsal view of regenerating fragments. aAnterior regenerant. b Posterior regenerant. c Bidirectionalregenerant. d Small anterior regenerant. e Small posterior regener-ant. f Lateral regenerant. Scale bar 500 μm

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morphological change was observed in differentiated cellsafter X-ray treatment (Fig. 7b). Apoptotic cell clusters werenever observed in the same region in non-irradiatedspecimens. To assess whether the cells located along themidline are more responsive to irradiation, because of theirhigher mitotic activity, with respect to those located in thelateral body regions, we counted the number of mitosesfrom central and lateral tissue samples (Fig. 8a). Theaverage was calculated for all the tissue samples belongingto each single group. The results obtained indicate that themean of mitoses number is higher in the central than in thelateral regions (Fig. 8b).

DjPiwi-1 RNA interference

To establish a possible role of DjPiwi-1 in planarian stemcell maintenance, we analyzed the effect of DjPiwi-1RNAi-mediated gene silencing during planarian regen-eration. Although a consistent reduction of DjPiwi-1endogenous transcripts was obtained as a consequence ofDjPiwi-1 dsRNA (Fig. 9a), we did not obtain a significantnumber of phenotypes showing morphological abnormal-ities either in intact or regenerating specimens. Indeed, weobserved that the majority of injected specimens were ableto regenerate and only 10% (15/150, from three indepen-dent experiments) ofDjPiwi-1 dsRNA-injected animals did

Fig. 6 Effects of X-ray irradia-tion on D. japonica stem cells.a Expression of DjPiwi-1 andDjMCM2 after 30 Gy expo-sures, visualized by wholemount in situ hybridization.Arrows indicate the presence ofresidual DjMCM2 hybridizationsignal 2 days after irradiation.Scale bar 500 μm. b Real-timeRT-PCR analysis of DjPiwi-1expression after 1, 2, 4, or7 days from irradiation. Expres-sion levels are indicated inrelative units assuming as uni-tary the value of untreatedspecimens (control). Each valueis mean±standard deviation ofthree independent X-ray treat-ments done in duplicate

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not show a visible blastema after the second transectioncompared with β-Gal dsRNA- or water-injected controls(Fig. 9b,c). The “no blastema” phenotype was seen only inanterior fragments that, however, rescued regenerativecapability in a short time after the last DjPiwi-1 dsRNAinjection. No significant difference in the type andpercentage of “no blastema” phenotype was found byinjecting dsRNA obtained from two independent clones,DjPiwi-1 Piwi box and DjPiwi-1 318, which targetdifferent regions of DjPiwi-1.

Discussion

In this study, we report the characterization of DjPiwi-1, aplanarian homologue of Drosophila Piwi, which encodes aprotein typified by the presence of conserved PAZ and Piwidomains. DjPiwi-1 is expressed in small parenchymal cellsdistributed as multiple clusters along the midline under thedorsal epidermis. X-ray treatment dramatically affects theviability of DjPiwi-1-expressing cells. A large body ofexperimental work has demonstrated that neoblasts, but notdifferentiated cells, are specifically eliminated by X-rayirradiation and for this reason this treatment is widelyutilized to determine planarian stem cell-specific geneexpression and/or stem cell involvement in biologicalprocesses (Cebrià et al. 2002; Ogawa et al. 2002; Orii et al.2005; Reddien et al. 2005a,b; Shibata et al. 1999). Timecourse expression analysis demonstrates a loss of DjPiwi-1-positive cells under the light microscope that parallels theloss of neoblasts observed under the electron microscope.Recently, Orii et al. (2005) demonstrated that the neoblast-specific degradation, observed as a consequence of X-raytreatment, well agrees with the loss of expression of theneoblast molecular marker DjPCNA. According to theresults obtained by in situ hybridization of irradiatedspecimens, a strong reduction of DjPiwi-1 expression wasobserved by real-time RT-PCR in specimens killed 24 hafter X-ray treatment. However, we noted the presence ofresidual DjPiwi-1 expression (50%) that remained un-

Fig. 7 Ultrastructural analysis of the dorsal parenchymal regionalong the midline of an intact D. japonica after X-ray irradiation(30 Gy). a A cluster of apoptotic cells 6 h after X-ray irradiation.Arrows indicate apoptotic cells. Scale bars 5 μm. b A normalneoblast (ne) along with an apoptotic neoblast (ane) and twodifferentiated cells (dc) recognized by the highly developedendoplasmic reticulum, are visible 24 h after irradiation. Scale bar3 μm

Fig. 8 Evaluation of the num-ber of mitoses in medially orlaterally located areas of theparenchyma. a Schematicdrawing of the body regionsselected for tissue squashpreparations. ph pharynx.b Distribution of mitotic figures(number of mitoses for 106

cells). Each bar shows themean±standard deviation fromsix different specimens. *Sig-nificant at p<0.05

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changed in the time (2, 4, and 7 days after irradiation).Because whole mount in situ hybridization experimentsperformed at the same time did not show any detectableDjPiwi-1 signal, it is possible to hypothesize thatDjPiwi-1-positive cells in the midline are neoblasts. The residualexpression could be due to the presence of differentiating/differentiated cells expressing this gene distributedthroughout the planarian body. Due to the low DjPiwi-1expression level we were unable to identify the distributionand identity of these cells. Drosophila Piwi and Arabi-dopsis ZWILLE play a key role in stem cell self-renewal/maintenance where they act as extrinsic factors, beingexpressed in structures surrounding the stem cells (Chenand McKearin 2005; Cox et al. 1998; Moussian et al.1998). On the contrary, the human Piwi homologue hiwi isexpressed in marrow CD34+ cells, but not in marrowstroma or marrow mesenchymal stem cells (Sharma et al.2001). We found that DjPiwi-1 is expressed both inneoblasts and in yet unidentified differentiating and/ordifferentiated cells. For this reason it is possible tohypothesize that DjPiwi-1 plays a role in the planarianstem cell self-renewal/maintenance both as an intrinsic andextrinsic regulator.

No DjPiwi-1 expression has been detected at the headand pharynx level by whole mount in situ hybridizationexperiments. The hybridization signal appeared localizedonly in the dorsal parenchyma along the midline beingmore intense in the area anterior to the pharynx. It ispossible that the stronger hybridization signal observedanterior to the pharynx is due to the presence of cellsexpressing higher level ofDjPiwi-1 transcripts with respectto the DjPiwi-1 positive cells located posterior to thepharynx. As a further possibility, it could be due to agreater number of DjPiwi-1-positive cells, preferentiallyaccumulated along the prepharyngeal region. It has beenrecently proposed that clusterized neoblasts could begermline stem cells that give rise to spermatocytes andoocytes after sexualization (Orii et al. 2005). We do notthink that the neoblasts clusterized along the midlinerepresent reminescent germ stem cells because they arelocalized outside the developmental and topological posi-tion of reproductive organs (Kobayashi and Hoshi 2002).The Piwi-like genes recently isolated in S. mediterraneashow a widespread expression in the parenchyma (Sánchez

Alvarado et al. 2002; Reddien et al. 2005a,b). Although thedifferent expression pattern of Piwi-like genes in the twoplanarian species (i.e., D. japonica and S. mediterranea)may be due to species-specific differences, it is likely thatthese genes represent different members of the planarianPAZ-Piwi gene family. The discovery of two additionalPiwi-like genes expressed in D. japonica strongly supportsthis hypothesis.

During regeneration DjPiwi-1 hybridization signalreorganizes in a short time along the midline of thestump. DjPiwi-1-positive cells appeared in the midline ofregenerating fragments obtained from body regions that didnot show DjPiwi-1 hybridization signal in intact animals.This reorganization suggests that dynamic morphogenesisoccurs throughout the stump. However, we cannotestablish whether DjPiwi-1 plays a role in the maintenanceof the body plan.

The peculiar expression pattern, as well as the differentresponse to the regenerative stimuli of DjPiwi-1-positivecells, when compared with that observed in DjMCM2- orDjPum-positive cells (these cells accumulate in thepostblastema: Salvetti et al. 2000, 2005), further supportsthe hypothesis that DjPiwi-1 represents a molecular markerspecific for a distinct subpopulation of neoblasts. Althoughsome DjPum-positive neoblasts also express DjMCM2transcripts (Salvetti et al. 2005), the possibility that DjPum,DjMCM2, and DjPiwi-1 may be coexpressed in the samecells located along the midline remains an open question.In fact, we were unable to perform successful double in situhybridization experiments, due to the low expression levelof DjPiwi-1.

Although some DjPiwi-1 dsRNA-injected planarianheads, unable to form a visible blastema, were observedafter the second transection, the reduced percentage of no-blastema phenotypes cannot be considered significantaccording to the criteria introduced by Reddien et al.2005a,b. The presence of multiple Piwi-like genes inplanarians, supported by the discovery of two additionalPiwi-like genes in the D. japonica EST collection, mightexplain the difficulties in obtaining DjPiwi-1 RNAi-induced phenotypes. In fact, other Piwi-like transcriptsmight not be cross-silenced by DjPiwi-1 RNAi and theirpresence compensate for the loss of DjPiwi-1 function.

Fig. 9 Effects of DjPiwi-1 RNAi. a Visualization of a comparativeRT-PCR experiment in planarians injected with either DjPiwi-1dsRNA or water. DjEF2 is used as an internal amplification control.b–c Brightfield images of the injected organisms. Dorsal view.

Dashed lines indicate the blastema region. b Anterior fragmentinjected with DjPiwi-1 dsRNA, 4 days after the second transection. cWater-injected anterior fragment 4 days after the second transection.Scale bars 500 μm

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DjPiwi-1-positive neoblasts, as well as those expressingDjMCM2 distributed along the midline anterior to thepharynx, show a lower radiotolerance than the DjMCM2-positive neoblasts located along the lateral lines and in themidline posterior to the pharynx. We believe that thisdifference can be ascribed to a higher cycling rate of theneoblasts localized along the midline. Indeed, we demon-strated a significant difference in the number of mitoses inthe cells located in the parenchymal region including themidline with respect to those located in the lateralparenchyma. Stem cells have mechanisms that protect theintegrity of their genomes (Aladjem et al. 1998; de Waardet al. 2003; Hong and Stambrook 2004; van Sloun et al.1999; Xu 2005). It is suggestive to hypothesize thatactivation of specific gene expression profiles regardingthe DNA repair systems may also occur in distinct neoblastsubpopulations.

Acknowledgements We are especially grateful to Kiyokazu Agatafor providing us with the planarian GI clonal strain and for the in situhybridization protocol. We also thank Claudio Ghezzani for technicalassistance with TEM and Mrs. Tamar Shanks for English revision.Grant Sponsor: Programmi di Ricerca di Interesse Nazionale, MIUR,Italy. Leonardo Rossi and Alessandra Salvetti have equallycontributed to this work.

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