Smed-SmB, a member of the LSm protein superfamily, is...

Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid bodyorganization and planarian stem cell proliferationEnrique Fernandéz-Taboada, Sören Moritz, Dagmar Zeuschner, Martin Stehling, Hans R. Schöler, Emili Salóand Luca Gentile

There were errors published in Development 137, 1055-1065.

On p. 1056, Spoultd-1 should have been Spoltud-1.

On p. 1063, the Acknowledgements failed to mention support by the Max Planck Society.

The Isken and Maquat (2008) reference and corresponding citation were incorrect and should instead have referred to the following:

Isken, O. and Maquat, L. E. (2007). Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev. 21, 1833-1856.

We apologise to authors and readers for these mistakes.

Development 137, 1583 (2010) doi:10.1242/dev.051847

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1055DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE

INTRODUCTIONPlanarians (phylum Platyhelminthes, class Turbellaria) arebilaterian, triploblastic free-living flatworms best known for theirimpressive ability to regenerate lost body parts (Morgan, 1898; Salóet al., 2009; Forsthoefel, 2009). This extreme plasticity is due to thepresence of a large population (20-30% of the total number of cells)(Hay and Coward, 1975; Baguñà, 1976; Baguñà and Romero, 1981;Newmark and Sánchez Alvarado, 2000; Hayashi et al., 2006) ofsomatic pluripotent stem cells known as neoblasts (Dubois, 1949;Wolff, 1962). Apart from germ cells, neoblasts are the only cellscapable of proliferating in adult planaria (Morita and Best, 1984;Newmark and Sánchez-Alvarado, 2000). They reside in thethickness of the parenchyma (Baguñá, 1973; Newmark and SánchezAlvarado, 2000; Orii et al., 2005) and are distributed, with fewexceptions, throughout the entire body of the animal. Neoblastsmaintain tissue homeostasis (Baguñà et al., 1989), generate thegermline (Sato et al., 2006) and replace structures lost to damage oramputation through a complex process of pattern re-establishment(re-patterning) (Saló and Baguñà, 1984; Baguñà et al., 1989; Saló,2006), which begins with the formation of an undifferentiated,unpigmented tissue called blastema (Dubois, 1949; Morita and Best,1984; Saló and Baguñà, 1984; Newmark and Sánchez Alvarado,

2000). The blastema, however, does not contain proliferatingneoblasts. Instead, they are enriched in the region adjacent to theblastema, called post-blastema (PB) (Saló and Baguñà, 1984;Eisenhoffer et al., 2008).

Neoblasts are actively recruited to the PB through signals elicitedby the wound response and probably through other subsequentsignalling (apoptosis, Wnt-P1) (Pellettieri et al., 2010; Petersen andReddien, 2009). In the PB, neoblasts actively proliferate to regeneratethe lost body part, mainly in two temporally defined waves: the minorone after eight hours, the major one between 48 and 72 hours (Salóand Baguñà, 1984). A similar proliferative response was also foundfollowing feeding, especially after a period of starvation (Baguñá,1976; Newmark and Sánchez Alvarado, 2000). The molecularmechanisms underlying the regulation of neoblast proliferation anddifferentiation remain, however, largely unknown.

The LSm (like-Sm) RNA-binding protein superfamily is a set ofgenes involved in many aspects of the RNA metabolism (for areview, see Lührmann et al., 1990). LSm protein products are highlyconserved throughout metazoans and relatively well-conserved inbacteria and archaea (Schumacher et al., 2002). LSm proteins arecharacterized by the presence of the Sm fold, a chaperone-likedomain that allows a variety of RNA-RNA and RNA-proteininteractions (for a review, see Wilusz and Wilusz, 2005). Complexesof six or seven proteins (the LSm ring) (Khusial et al., 2005) interactwith different small nuclear RNAs (snRNAs) to generate smallnuclear ribonucleoproteins (snRNPs). LSm proteins take part inseveral processes such as Cajal body formation, telomere elongation(Fu and Collins, 2006) and ribosomal assembling (Kiss, 2004). LSmproteins are, however, best known for their role in mRNA processing(He and Parker, 2000). They are also involved in the organization ofthe major and minor spliceosome complexes, which are responsiblefor the splicing processes of the pre-mRNA (Mayes et al., 1999;

Development 137, 1055-1065 (2010) doi:10.1242/dev.042564© 2010. Published by The Company of Biologists Ltd

1University of Barcelona, Department of Genetics, IBUB, Av. Diagonal 645, 08028,Barcelona, Spain. 2Max-Planck-Institute for Molecular Biomedicine, Department ofCell and Developmental Biology, Röntgenstraße 20, 48149, Münster, Germany.3University of Münster, Faculty of Medicine, Domagkstraße 3, 48149, Münster,Germany.

*Authors for correspondence ([email protected]; [email protected])

Accepted 26 January 2010

SUMMARYPlanarians are an ideal model system to study in vivo the dynamics of adult pluripotent stem cells. However, our knowledge of thefactors necessary for regulating the ‘stemness’ of the neoblasts, the adult stem cells of planarians, is sparse. Here, we report on thecharacterization of the first planarian member of the LSm protein superfamily, Smed-SmB, which is expressed in stem cells andneurons in Schmidtea mediterranea. LSm proteins are highly conserved key players of the splicing machinery. Our study shows thatSmed-SmB protein, which is localized in the nucleus and the chromatoid body of stem cells, is required to safeguard theproliferative ability of the neoblasts. The chromatoid body, a cytoplasmatic ribonucleoprotein complex, is an essential regulator ofthe RNA metabolism required for the maintenance of metazoan germ cells. However, planarian neoblasts and neurons also rely onits functions. Remarkably, Smed-SmB dsRNA-mediated knockdown results in a rapid loss of organization of the chromatoid body, animpairment of the ability to post-transcriptionally process the transcripts of Smed-CycB, and a severe proliferative failure of theneoblasts. This chain of events leads to a quick depletion of the neoblast pool, resulting in a lethal phenotype for bothregenerating and intact animals. In summary, our results suggest that Smed-SmB is an essential component of the chromatoid body,crucial to ensure a proper RNA metabolism and essential for stem cell proliferation.

KEY WORDS: LSm proteins, Proliferation, Stem cell, Chromatoid body, Regeneration, Planaria

Smed-SmB, a member of the LSm protein superfamily, isessential for chromatoid body organization and planarianstem cell proliferationEnrique Fernandéz-Taboada1, Sören Moritz2, Dagmar Zeuschner2, Martin Stehling2, Hans R. Schöler2,3,Emili Saló1,* and Luca Gentile2,*

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Patel et al., 2002). Although these complexes are mainly foundin the nucleus, virtually all metazoan germ cells displayribonucleoprotein (RNP) granules in the cytoplasm (Eddy, 1975),like the germ granules in Caenorhabditis elegans and Drosophilamelanogaster (reviewed in Strome and Lehmann, 2007) and thenuages or chromatoid bodies (CBs) in mammalian spermatids(reviewed in Parvinen, 2005).

A recent report showed that mammalian cell lines can also beinduced to form CB-like structures following the expression of acytoplasmic form of the prion protein PrP (Beaudoin et al., 2009).Two other studies proposed that LSm proteins regulate cellproliferation in the cytoplasm of mammalian cells through the minorspliceosome (König et al., 2007) and, if perturbed, can interfere withthe localization and subcellular distribution of the P-granules of C.elegans (Barbee et al., 2002). Planarian CBs are electron-dense RNPparticles characteristic for stem cells (Hay and Coward, 1975) andneurons (Yoshida-Kashikawa et al., 2007). Until now, only twoproteins have been described as components of the planarian CB:DjCBC-1 (Yoshida-Kashikawa et al., 2007) and Spoultd-1 (Solanaet al., 2009). Their function, not yet fully understood, is related toRNA processing and post-transcriptional modifications (Yoshida-Kashikawa et al., 2007).

In this study, we characterize the first member of the large LSmprotein superfamily, Smed-SmB, described in the planarian speciesSchmidtea mediterranea. Smed-SmB is the ortholog of mammalianSmB/B�/N, and is expressed by stem cells and neurons. Double-stranded RNA (dsRNA)-mediated silencing in intact andregenerating animals resulted in a quick reduction in the number ofdividing cells, which compromised both the ability to ensure normaltissue turnover and regeneration after amputation. A massivedegenerative process progressed from anterior to posterior in bothregenerating and intact animals and caused the animals to die withintwo to four weeks, respectively.

Our data show that inhibition of Smed-SmB impairs theorganization and function of the CB, to which the protein alsolocalizes. Furthermore, Smed-SmB RNAi affects the splicingefficiency of Smed-CycB, which indicates a failure to process pre-mRNAs necessary for cell cycle progression. The degeneration ofthe CBs and the impairment of the splicing efficiency seem to havea central role in the resulting proliferative failure we observed.

MATERIALS AND METHODSAnimalsAll planarians used in the present study were generated from a singleindividual of the asexual strain of Schmidtea mediterranea (clonal lineBCN-10), collected from a fountain in Montjuic (Barcelona, Spain).Animals were maintained in a 1:1 mixture of tap water treated withAquaSafe (Tetra Aqua) and MilliQ water (Millipore Iberica, Madrid, Spain)and fed twice a week with bovine liver, as previously described (Molina etal., 2007). Animals used for all the presented experiments were starved forone week. To prepare irradiated controls, planarians were g-irradiated at 75Gy (1.56 Gy/minute) with a Gammacell 1000 (Atomic Energy of CanadaLimited) (Saló and Baguñà, 1985). The animals were collected at differenttime points (at 12 hours, daily from days 1-11 and at days 14, 15 and 21)according to downstream applications.

Identification and cloning of Smed-SmBThe peptide sequence of Smed-SmB was identified among putativeneoblast-specific proteins (E.F.-T., G. Rodriguez-Esteban, E.S. and J.Abril, unpublished data), and confirmed by BLAST in a local databasegenerated from the S. mediterranea genome traces (sequenced byWashington University Genome Sequencing Center, USA). Primers weredesigned to amplify a 237 bp fragment by RT-PCR (forward primer,5�-CAACACTTCAAGATGGTCG; reverse primer, 5�-CCATTAACGCC-

TACTGAA) from total RNA preparation (TRIzol). 5�- and 3�-ends wereobtained by RNA ligase-mediated rapid amplification of cDNA ends(RLM-RACE)-PCR (5�RACE primer, 5�-GACUGGAGCAGGAGGA-CACUGACAUGGAGUGAAGGAGUAGAAA; 3�RACE primer, 5�-GCTGTCAACGATAGGCTACGTAACGGCATGACAGTG[T]18). Thecomplete sequence was confirmed by BLAST to the planarian genomedraft (accession number: GU562964).

Cell dissociation and FACSPlanarian cell dissociation protocol was modified from González-Estévez(González-Estévez et al., 2007). Briefly, animals were treated with 2% L-cysteine hydrochloride monohydrate (Merck, K23484539), pH 7.0, toremove the mucus, transferred in CMF buffer and mechanically dissociatedby gentle rocking. The suspension was then serially filtered through 70 and30 mm nylon meshes, stained for 2 hours with a mixture of 7.5 mg/mlHoechst 33342 (Sigma, B2261) and 0.05 mg/ml Calcein AM (Invitrogen,Karlsruhe, Germany), pelleted and resuspended for FACS (FACSARIA,BD, Heidelberg, Germany). Eventually, propidium iodide was added atconcentration of 1 mg/ml.

In situ hybridizationWholemount in situ hybridization (ISH) protocol was modified from Agata(Agata et al., 1998). Briefly, after fixation and rehydration, planarians werepermeabilized in 0.1% Triton X-100 in PBS (PBST�0.1) added with 20mg/ml proteinase K and quenched with 2% glycine. Triethanolamine(Sigma, T9534) treatment was performed as previously described (Nogi andLevin, 2005) before hybridization, which was carried out at 55°C for 18 to30 hours, using digoxigenin-labelled RNA probes prepared using an in vitrolabelling kit (Roche, Mannheim, Germany) at a final concentration of 0.07ng/ml. ISH on dissociated cells was performed as previously described(González-Estévez et al., 2003).

ImmunohistochemistryWholemount immunohistochemistry (IHC) was performed as described(Cebriá and Newmark, 2005). Briefly, after fixation in Carnoy andrehydration, animals were blocked in 1% BSA in PBST�0.1 for 2 hours atroom temperature. The antibody against proliferating cell nuclear antigen(a-PCNA), kindly provided by Dr Hidefumi Orii (Himeji Institute ofTechnology, Hyogo, Japan), was used 1:1000 in blocking solution for 20hours under constant agitation at 4°C. Animals were then labelled for 14-16hours with FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch,1:200). PCNA-positive cells were counted using a Leica SP2 confocal laser-scanning microscope (Leica Microsystems, Barcelona, Spain).

RNA interference (RNAi)In vitro dsRNA preparation of Smed-SmB 5�-region (500 bp) and injectionprocedure were previously described (Oviedo and Levin, 2007). A total ofthree injections, distributed over 3 consecutive days (one injection per daywith three pulses of 32 nl of 400 ng/ml dsRNA solution) were performedusing a Drummond Scientific Nanoject injector (Broomall, PA, USA)targeting the gastrovascular system. The control group was injected withwater. Some animals were pre-pharyngeally amputated 1 or 3 days after thelast injection (t-cut).

RT-PCR and quantitative real-time RT-PCR (qRT-PCR)Total RNA was extracted using TRIzol (Invitrogen, Barcelona, Spain) andreverse-transcribed to cDNA with the High-Capacity cDNA ReverseTranscription Kit (AppliedBiosystems, Darmstadt, Germany). Transcriptlevels were determined on the ABI PRISM SDS 7900HT(AppliedBiosystems, Darmstadt, Germany) using custom-designedoligonucleotides for the 5�-nuclease assays (see Table S1 in thesupplementary material) and normalized to the endogenous control SmEf2that was chosen among other ubiquitously expressed genes for its stableexpression throughout the regenerative process, as well as for not beingaffected by the RNAi treatment performed (see Fig. S1, Fig. S2 in thesupplementary material). Relative quantification of gene expression wascalculated using the Ct method. Three technical replicates were used foreach real-time PCR reaction; a reverse transcriptase blank and a no-templateblank served as negative controls.

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For Smed-CycB RT-PCR of spliced and unspliced forms, the equivalentof 5 ng of reverse-transcribed RNA were amplified using oligonucleotidesspanning the second intron (forward primer, 5�-ATGCCGC CGA -AACTTTATACCTG; reverse primer, 5�-AACTTCTTCGA CTTTT -GCTGCAA), according to mk4.001494.00.01. Expected amplicon sizes forthe spliced and unspliced forms of the transcript were 136 bp and 219 bp,respectively.

Electron microscopy (EM)Post-blastema fragments of Smed-SmB dsRNA- and water-injectedplanarians were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH7.4, for 2 hours at room temperature. Specimens were post-fixed in 1% OsO4

and embedded after dehydration in epon. Ultrathin sections of 70 nm werecut (Leica-UC6 ultramicrotome, Vienna, Austria) and counterstained withuranyl acetate and lead citrate. Specimens were observed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands). Pictures weretaken using imaging plates (Ditabis, Pforzheim, Germany).

For statistical analysis, neoblasts in the PB area were inspected andnumber, size and morphology of their chromatoid bodies were accounted.

For immuno-EM, fragments of planarians were fixed with either 4%paraformaldehyde or a mixture of 2% paraformaldehyde and 0.2%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The specimen was thencryoprotected in 2.3 M sucrose and frozen in liquid nitrogen. Ultrathincryosectioning and immunogold-labelling were performed as described(Slot and Geuze, 2007), except a permeabilization treatment with 1 mg/mlsaponin in PBS, pH 7.4, for 10 minutes prior to blocking to improve theaccessibility of the antibodies to their epitopes.

Western blotAnimals were snap-frozen in liquid nitrogen and ground with a disposablepotter. After direct lyses in 50 ml 2� Laemmli buffer, samples wereloaded on a 10% acrylamide minigel and run under denaturing SDS-PAGE conditions for 3 hours under constant current. Proteins weretransferred on a PVDF Immobilon membrane (Millipore, Schwalbach,Germany) and processed for immunodetection with ECL plus chemistry(GE-Healthcare, Solingen, Germany). Antibodies used were: mouse anti-SmB monoclonal antibody (Sigma S0698) diluted 1:1000; rabbit anti-Gapdh polyclonal antibody (Abcam, ab36840) diluted 1:5000; and rabbitanti-Smed-SmB (residues 46-60) affinity-purified polyclonal antibodiesSmB310 and 311, both diluted 1:250,000. Secondary HRP-conjugatedantibodies were used at 1:50,000, according to the host species of theprimary antibodies. Band intensities were quantified using the Chemi-Doc XRS platform equipped with Quantity One software (BioRad,Munich, Germany).

StatisticsOne-way ANOVA with Dunnett’s post-test, Student’s t-test and Fisher’sexact test were performed using GraphPad Prism 4.03 (GraphPad software,La Jolla, CA, USA).

RESULTSDespite being represented throughout the planarian body andexperimentally well accessible, neoblasts are still largely defined bytheir morphology and by their sensitiveness to g-irradiation.Therefore, g-irradiated planaria are instrumental in the discovery ofnew neoblast-specific markers. Using a proteomic approach, wewere able to identify the first planarian member of the LSmsuperfamily as differentially expressed in wild-type versus g-irradiated regenerating animals (E.F.-T., G. Rodriguez-Esteban, E.S.and J. Abril, unpublished data). ClustalW alignment showed theconservation of the two Sm fold domains at the N-terminus of theprotein (see Fig. S3A in the supplementary material), whereas thebootstrap-based UPGMA method was used to calculate thephylogenetic tree that confirmed Smed-SmB similarity with theSmB proteins of different metazoans (see Fig. S3B in thesupplementary material).

Smed-SmB transcripts localize to neoblastsNeoblasts are evenly dispersed throughout the parenchyma, ascarcely differentiated tissue that lies under the epidermal andmuscle layers, and surrounds all the organs (Baguñá, 1973; Saló,2006). Wholemount in situ hybridization (ISH) against Smed-SmBtranscripts showed the typical neoblast distribution in adult intactplanarians (Fig. 1A, 1-3). Fluorescent ISH on dissociated cellsrevealed that Smed-SmB signal could be detected in small (5-10 mm)round cells with a high nucleus-to-cytoplasm (N/C) ratio (Fig. 1B-D), consistent with the morphological definition of neoblasts(Baguñà, 1981). Additionally, wholemount ISH against Smed-SmBon regenerating animals 48-72 hours after amputation produced astrong signal in the post-blastema (PB; Fig. 1E), where proliferatingneoblasts consequently accumulate to the major proliferation peakinduced by amputation (Baguñà, 1976).

Following g-irradiation, Smed-SmB expression was significantlydiminished, although irradiation-tolerant Smed-SmB-positive cellspersisted in the cephalic region (Fig. 2A), as reported for otherneoblast markers like Bruli (Guo et al., 2006) and Pumilio (Salvettiet al., 2005). This observation was further substantiated as therelative expression of the transcript varied depending on the portionof the body considered. Fourteen days following g-irradiation,animals were cut into head, trunk (pharynx and surrounding tissues)and tail fragments (Fig. 2B). The expression of Smed-SmB mRNAin the cephalic portion of irradiated animals was similar to the non-irradiated control (P0.5667), whereas in the trunk and tailfragments it was 29±3% (P<0.0001) and 33±3% (P0.0005),respectively (Fig. 2C). Western blot confirmed a similar trend for theexpression of Smed-SmB protein (Fig. 2D,E).

1057RESEARCH ARTICLESmed-SmB, chromatoid bodies and proliferation

Fig. 1. Smed-SmB expression in asexual S. mediterranea.(A)Wholemount ISH showing the expression pattern of Smed-SmB inintact wild-type animals. Smed-SmB mRNA was found in theparenchyma throughout the animals, with the exception of thepharynx (Ph) and the area anterior to the photoreceptors. Thispattern is usually referred to as a neoblast pattern. Sections of thespecimen in A showed abundance of Smed-SmB transcripts alsoaround the cephalic ganglia (cg in A1). Signal was otherwise absentfrom nerve cords (nc in A1), pharynx (Ph in A2) and gastro-vascularsystem (asterisks in A1-3). (B-D)Fluorescent ISH against Smed-SmBon isolated cells. Fluorescence (B) and Nomarski (C) images of aSmed-SmB-positive cell with neoblast morphology, where acytoplasmic signal was detected. Differentiated cells showed nosignal for Smed-SmB (D). (E)Wholemount ISH against Smed-SmB onregenerating wild-type animals 48 hours after amputation. Signal ofSmed-SmB transcripts in the post-blastema region (arrowhead) isstronger than in the rest of the body. Scale bars: 0.5 mm in A and E;0.1 mm in A1-3; 5mm in B-D. D

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Smed-SmB is essential for tissue homeostasis andregenerationIn order to understand the function of Smed-SmB, we performedRNAi experiments on regenerating and intact animals. RNAiefficiency was assessed at both the RNA and protein levels. qRT-PCR showed a sudden downregulation of the transcript (more than90%), which followed the same kinetics in both intact andregenerating animals (Fig. 3A). Smed-SmB protein expression wasalso clearly influenced by the RNAi, showing different kineticsbetween intact and regenerating animals. In regenerating animals,Smed-SmB protein was barely detectable as early as 2 days afteramputation. A comparable level of protein expression is attained inintact animals after 9 days from the last dsRNA injection (Fig. 3A).

All Smed-SmB dsRNA-treated animals (n95), which wereamputated 1 or 3 days after the third round of injections (t-cut),failed to regenerate and died within two weeks after amputation.Interestingly, in no cases could we observe blastema formation (Fig.3B). Within three to four weeks following RNAi, intact animals(n50) also died, with signs of degeneration becoming evident 10days after the RNAi treatment (Fig. 3C).

To understand whether Smed-SmB has a neoblast-specificfunction, we observed the changes induced by Smed-SmB RNAi tothe planarian cell populations. Largely based on the morphologicalfeatures of planarian cells, Hoechst 33342/Calcein AM double-staining is able to separate the irradiation-sensitive cells (X1 and X2)from the differentiated cells (Xin) on a FACS plot (Reddien et al.,

2005; Hayashi et al., 2006). In a similar way to g-irradiation, Smed-SmB RNAi induced a dramatic reduction of irradiation-sensitivefractions X1 and X2 in intact animals (Fig. 4A). Irradiation-sensitiveX1 and X2 cells were drastically reduced in number after 11 days(18±8.3% and 27±11.3% of the relative control, respectively;P<0.0001), with the decrease beginning after 7 days (P<0.05; Fig.4B,C). These data nicely correlate with the availability of Smed-SmB protein shown in Fig. 3A. FACS analysis of PB tissueseparated from the rest of the body (RoB) of regenerating RNAianimals showed that the reduction of X1 and X2 cells happenedmuch faster in the PB (day 3, P<0.05), where actively proliferatingneoblasts are enriched compared with the RoB (day 6, P<0.05; Fig.4D-G).

Smed-SmB RNAi affects the expression ofplanarian cyclinBWe analyzed the expression of neoblast-specific markers in bothintact and regenerating animals and we found they were, in bothcases, significantly reduced following Smed-SmB RNAi. qRT-PCR of regenerating animals showed that Smed-Bruli (Guo et al.,2006) and Smedwi1 (Reddien et al., 2005; Rossi et al., 2006)expression rapidly disappeared, beginning 3 days after Smed-SmBRNAi (P0.0002 and P0.0013, respectively; Fig. 5A). Bycontrast, the expression of differentiated cell markers, likeSmed-Pax6 (Pineda et al., 2002) and Smed-Tmus (Cebriá et al.,1997), was essentially unaltered (P0.4551 and P0.0983,


Fig. 2. Effects of g-irradiation on Smed-SmB expression. (A)Intact wild-type planarians were irradiated and subsequently analyzed for Smed-SmB expression by wholemount ISH. Following g-irradiation (75 Gys), Smed-SmB expression decreased without disappearing, particularly in thecephalic portion of the body. (B,C)Real-time quantification of Smed-SmB transcripts in different body compartments (B) confirmed that most of theresidual expression found in irradiated animals comes from the cephalic area (C, orange line). In trunk and tail portions, the effects of the irradiationwere more prominent (C, blue and green lines, respectively). Mean ± standard deviation of three independent experiments is shown.(D,E)Commercially available antibodies against mammalian SmB and Gapdh efficiently cross-reacted with the planarian proteins (D) allowing areliable quantification. The chart in E shows the quantification of the blot shown in D. Scale bar: 0.5 mm in A. In D, + is irradiated, – is wt control.

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respectively; Fig. 5B). The expression of Smedwi2, involved inneoblast differentiation (Reddien et al., 2005), also suffered asignificant reduction (P<0.05), although milder and at a later time(day 5) than the other neoblast markers (Fig. 5A).

Given the incapacity shown by Smed-SmB knocked-downanimals to form a regenerating blastema, we decided toinvestigate neoblast proliferative potential. PCNA-positive cellswere reduced in number after 10 days from Smed-SmBknockdown in intact animals (P<0.0001; Fig. 5C-E). PCNA is anessential component of the DNA replication machinery thataccumulates in cells that are virtually able to proliferate (Bravo etal., 1987; Orii et al., 2005). Conversely, cyclinB is expressed onlyby actively cycling neoblasts of the X1 subpopulation (Reddienet al., 2005; Eisenhoffer et al., 2008). In wild-type regeneratingplanarians, its expression increases around day 2-3 ofregeneration (P<0.05; Fig. 5F), related to the major peak ofmitotic activity (Saló and Baguñà, 1984). As a consequence ofSmed-SmB RNAi, Smed-CycB expression was promptly reduced2 days after amputation (P<0.05), reaching a basal level (morethan 90% downregulation; P<0.0001) after 7 days (Fig. 5F).

Using as primer oligonucleotides able to amplify both the splicedand unspliced forms of the Smed-CycB transcript, we observed thatthe decrease in mature Smed-CycB mRNA expression wasaccompanied by an increase in the expression of its unspliced form

(Fig. 5G). The ratio between the expressions of unspliced andspliced transcripts in Smed-SmB RNAi animals increased up to 5-fold after 3 days of regeneration (Fig. 5H). This suggests that Smed-SmB RNAi does not prevent the expression of cyclinB, but rather, atleast in part, prevents the splicing of its transcript.

Loss of Smed-SmB impairs chromatoid bodyorganizationWe found that Smed-SmB is important to guarantee the proliferativeability of the neoblasts. We then had a closer look at theultrastructural level of stem cells in both intact and regeneratingSmed-SmB RNAi animals. We noticed that, after 5 days ofregeneration, the cytoplasm of the neoblasts was less-denselypacked (Fig. 6A) compared with water-injected animals (Fig. 6B).This change was accompanied by an increase in cellular membranesof the endolysosomal system and the Golgi (Fig. 6A, asterisks) –normally not present in neoblasts (Baguña, 1973). CBs are electron-dense granules not delimited by membranes (Fig. 6B, arrows),which resemble the appearance of heterochromatic nuclear spots(Auladell et al., 1993). A loss of integrity in the CB structure wasalso observed at higher magnification (Fig. 6A,C, white frame).Several small CB-like structures (Fig. 6C, arrowheads) appeared,probably owing to the fragmentation of pre-existing CBs.Occasionally, we also observed the presence of large autophagic


Fig. 3. Smed-SmB phenotype. (A)Animals were injected with double-stranded RNA to suppress Smed-SmB expression. Some were subsequentlyamputated and let regenerate (n90), whereas others were left intact (n50). A swift reduction of about 90% at the RNA level was followed by areduction of about 80% at the protein level, as shown by qRT-PCR and western blotting. Although the dynamics of downregulation of thetranscripts is identical in intact and regenerating animals, in intact animals, the time required for an effective protein downregulation was about 5�longer than in regenerating ones. (B)Water- and Smed-SmB dsRNA-injected animals were prepharingeally amputated into two pieces (head andtail) in the days following the third dsRNA injection and let to regenerate. We could not observe blastema formation in any of the amputated Smed-SmB RNAi animals, whereas in control animals, this took place normally (red dashed lines). Additionally, a degenerative process (*) began in dsRNA-injected animals after 10 days, which eventually led to death within 2 weeks. Differences in the phenotypes were not observed between anteriorand posterior regeneration, nor among animals cut at different times (1 or 3 days) after dsRNA injection. (C)Intact animals were also treated withdsRNA to inhibit Smed-SmB expression. A degenerative process (arrows) began to affect the head of the animals around day 10, and graduallyprogressed throughout the rest of the body, leading to death within 3-4 weeks. Scale bars: 0.5 mm.

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vacuoles (Fig. 6D, asterisks), suggesting that the decrease in the X1and X2 populations might be regulated by this degenerative pathway(González-Estévez et al., 2007). Interestingly, we also found that thedegeneration of the CBs follows the Smed-SmB protein availability(Fig. 3A). In intact animals, we occasionally observed degeneratedCBs, but only after 11 days from RNAi treatment onwards. Inregenerating animals, however, the CB state reflected the neoblastposition in the planarian body. CBs observed in neoblasts of theposterior stripe began to degenerate after 5 days from amputation,whereas those found in neoblasts of the PB began to degenerate asearly as 1 day after cutting (P<0.05; Fig. 6E). As CBs degenerated,an increasing number of neoblast-like cells without CBs wereobserved in the PB. The percentage of these cells increased from11.8±1.8% at t-cut to 50.5±12.5% after 3 days (P<0.05), with a cleartrend (R20.923; Fig. 6E). Conversely, the percentage of healthyneoblasts with normal CBs dropped from 69.4±8.5% at t-cut to13.7±11.1% after 3 days (P<0.05; Fig. 6E).

The evidence we collected at the ultrastructural level supportthe idea that knockdown of Smed-SmB impairs the functionalorganization of the CB, as was also observed for the germ granulesof C. elegans after RNAi of either SmE or SmB (Barbee et al.,2002).

Smed-SmB protein localizes to neoblast nuclei andchromatoid bodiesThe dynamics of the event characteristics of the Smed-SmBphenotype showed that the degeneration of the CBs is one of thefirst outcomes of the phenotype, particularly when considering theneoblasts located in the PB (Fig. 6E; Fig. 7A). As several articlesproved the localization and functional role of the LSm proteins inCBs and CB-like structures (Parvinen, 2005; Strome andLehmann, 2007; Beaudoin et al., 2009), we tried to uncoverwhether Smed-SmB protein also localizes to the CB.Cryoimmuno-EM using two different affinity-purified rabbitpolyclonal antibodies (SmB310 and SmB311), specifically raisedagainst residues 46-60 of the Smed-SmB protein, showed goldparticle labelling of both the nucleus – except the nucleolus – andthe CB of wild-type neoblasts (Fig. 7B). These were mainlylabelled at the edge, probably as consequence of steric hindranceof the antigens in the condensed structure. Results were confirmedby using both the antibodies specifically raised. Nuclear Smed-SmB signal was not found in differentiated cells (data not shown).These results indicate with good confidence the presence of theSmed-SmB protein inside as well as outside the nuclearcompartment of planarian neoblasts.


Fig. 4. Dynamics of the planarian cell populations following Smed-SmB RNAi. (A)Planarians were gently dissociated and their cells weredouble-stained with Hoechst 33342 and Calcein AM. FACS analysis showed different populations according to size and nucleus-to-cytoplasm ratio.(B,C)Compared with water-injected control animals (B), in intact Smed-SmB RNAi animals, the irradiation-sensitive population X1 (red) and X2(green) gradually disappeared, beginning around day 7 (C). After 11 days, X1 and X2 populations were reduced by about 81% and 73%,respectively. (D-G)Compared with control (D) or intact Smed-SmB RNAi animals (C), in the post-blastema of Smed-SmB RNAi regenerating animals,neoblast reduction happened much faster, beginning as early as 3 days after treatment (E). By day 7, neoblast populations had decreased to levelssimilar to intact animals after 11 days (C,E). In the rest of the body, however, the effect of Smed-SmB RNAi progressed more gradually, X1 and X2populations were reduced by about 36% and 33% after 7 days, respectively (G). Means ± standard deviations in B and C refer to four independentexperiments. Means ± standard deviations in D-G refer to three independent experiments.

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Fig. 5. Smed-SmB RNAi affects the expression of neoblast markers. (A,B)The expression of two panels of genes, either specific to neoblasts(A) or to differentiated cells (B), was assessed by real-time qRT-PCR in regenerating animals. The neoblast markers Smed-Bruli, Smedwi1 andSmedwi2, suffered a consistent downregulation that began 3, 4 or 5 days after treatment, respectively. After 7 days, Smed-Bruli and Smedwi1expression were reduced by about 90% relative to a water-injected control, whereas Smedwi2 expression was only reduced by about 55% (A).Conversely, Smed-SmB RNAi had no apparent effect on the expression of Smed-Pax6 and Smed-Tmus (B). (C-E)As shown by wholemount IHC ofthe tail portion of intact animals, the number of PCNA-positive cells in Smed-SmB-inhibited animals was significantly reduced around day 10 (D)compared with control (C). Chart E presents means ± standard deviation of PCNA-positive cells from five independent experiments. A significantreduction took place between 6 and 10 days after RNAi treatment. (F)Smed-CycB expression was quantified by qRT-PCR. In regenerating controlanimals, cyclinB expression resembles the major mitotic event occurring between 48 and 72 hours after amputation (blue line). This peak ofexpression is virtually abolished in both g-irradiated and Smed-SmB RNAi regenerating animals (green and red lines, respectively). (G,H)The presenceof the unspliced form of Smed-CycB transcript was analyzed separately by conventional RT-PCR (G, upper panel) and compared with the expressionof the mature transcript (G, lower panel). As early as 1 day after Smed-SmB RNAi treatment, we could observe an increased ratio betweenunspliced and spliced forms of Smed-CycB transcript compared with control, which reached its peak (~5-fold) after 3 days (H). Error bars in A, B, E,F and H represent the standard deviation over a minimum of three independent experiments. PCNA-positive cell number was normalized against astandard volume (1mm3). In E: *, P<0.05; **, P<0.001. The samples checked by conventional RT-PCR for Smed-CycB (G) are the same as screenedby qRT-PCR (F). However, amplification products were checked at end-point on ethidium bromide gel, which means during the linear/plateauamplification phase. Although a decrease of expression of the spliced form of Smed-CycB can still be appreciated (G, lower panel), a precisequantification of the spliced transcript is only possible by real-time RT-PCR. Scale bar: 0.2 mm. D

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DISCUSSIONIn this study we characterized the function of the planarianSmB/B�/N ortholog, Smed-SmB, the first member of the conservedLSm superfamily identified in planarians.

The data we collected suggest an essential role for Smed-SmB insustaining the proliferative potential of the planarian stem cellsduring regeneration and in tissue homeostasis.

Smed-SmB transcripts were mainly found in the parenchyma andaround the cephalic ganglia. Expression of Smed-SmB bypostmitotic cells of the cephalic ganglia explains the partialdownregulation we found after g-irradiation, which only affectsactively dividing cells.

The silencing of Smed-SmB, efficiently achieved through dsRNA-induced RNAi, produced a late degenerative phenotype in intactanimals (Fig. 3C) and an early non-regenerating phenotype inamputated ones (Fig. 3B). The phenotype was accompanied, in bothcases, by downregulation of the neoblast markers (Fig. 5B) andreduction in the number of neoblasts (Fig. 4A-G). These effects werealso described following the perturbation of other genes – likePumilio, Smedwi2 and Bruli – involved in neoblast maintenance ordifferentiation (Salvetti et al., 2005; Reddien et al., 2005; Guo et al.,2006). Compared with the latter, however, the Smed-SmB phenotypeshowed some peculiarities.

The degenerative effects produced by Smed-SmB knockdown inintact animals progressed at a slower rate compared with the othergenes essential for neoblast function. At the beginning of the fourthweek, more than 60% of the intact animals were still alive, albeitwith a degenerated anterior part (Fig. 3C). This implies thatmaintenance of the differentiated cells was virtually unaffected (Fig.4A-C), and that the delay in degeneration observed in intact animalswas due to the prevention of tissue turnover.

Also, in Smed-SmB RNAi regenerating animals, the inability toform a blastema was not affected by the length of time betweeninjection and amputation, as observed in the case of Smed-Bruli andSmedwi2 RNAi (Guo et al., 2006; Reddien et al., 2005). Animals cutat different time points after injection (1 or 3 days) invariably failedto form a blastema, suggesting that neoblasts could proliferate, andpossibly differentiate, for a short time after either Smed-Bruli orSmedwi2 RNAi, but they were unable to do so following Smed-SmBRNAi. On the contrary, proliferation was severely hampered, asdepicted by a decrease in the expression of both PCNA (Fig. 5C-D)and Smed-CycB (Fig. 5E). Although we cannot exclude an effect ofSmed-SmB RNAi on the early neoblast progeny, we concluded thatthe inability to regenerate a blastema as demonstrated by Smed-SmBRNAi animals resulted from the absence of actively proliferatingcells.

Another interesting observation was that the expression of agene, which was otherwise ubiquitously expressed and involvedin basal cell functions, was restricted in planaria to stem cells andneurons. Conversely, SmB is known to be a component of RNPgranules, also known as CBs, which are present in the germ cellsof virtually all metazoans (Eddy, 1975). Recently, their presencewas also observed in CB-like structures induced in mammaliancells by expressing the cytoplasmic form of the Prion protein(Beaudoin et al., 2009). The large RNP complexes found in thegerm cells share remarkable similarities with planarian CBs,which are a characteristic feature of neoblasts (Hay and Coward,1975), germ cells (Coward, 1974) and neurons (Yoshida-Kashikawa et al., 2007) and are expected to function in post-transcriptional gene regulation.

Like the germ cell granules, planarian CBs assemble componentsof the machinery to process mRNA in the cytoplasm, like DjCBC-1 (Yoshida-Kashikawa et al., 2007) and Spoltud-1 (Solana et al.,2009). They might also contain small non-coding RNAs (miRNAs,snRNAs) and members of the LSm protein superfamily. Followingneoblast differentiation, CBs gradually disappear, only to persist inthe germline (Coward, 1974) and in neurons (Yoshida-Kashikawaet al., 2007).

The Smed-SmB RNAi effects we observed at the CB levelshowed striking similarities with the knockdown of either SmE orSmB in C. elegans embryos, which alters the sub-cellulardistribution of the P-granules and leads to the inaccuratesegregation of the germline (Barbee et al., 2002). Our data suggestthat Smed-SmB RNAi results in a loss of CB structure and


Fig. 6. Smed-SmB RNAi produces loss of chromatoid bodyorganization. (A-C)Representative appearance of neoblasts of theposterior stripe belonging either to Smed-SmB dsRNA- or water-injected regenerating animals, 5 days after amputation. In neoblastsfrom water-injected controls (B), chromatoid bodies (CBs) could beobserved (arrows), depending on the sectioning level of the cell, in ahighly condensed cytoplasm. On the contrary, some portions of thecytoplasm of treated animals were less-densely packed and cellularmembranes began to develop (asterisks in A). In these areas, smallgranules with the aspect of CBs could be recognized (arrowheads in C)together with translucent vesicular membranes. (D)This micrographshows a neoblast with unusual autophagic vacuoles (asterisks), in aSmed-SmB RNAi regenerating animal 5 days after amputation.(E)Structural integrity and number of CBs were analyzed on ultrathinsections of post-blastema. We began to observe neoblasts either withdestructured CBs or without any CBs starting 1 day after amputation ofSmed-SmB RNAi animals. The number of neoblasts with normalmorphology, conversely, dropped dramatically. After 3 days ofregeneration, only 26% of the neoblasts screened (n20) showed thepresence of CBs, and in only 14% of these cells, CBs had a sizecomparable with control ones. Bars in E represent means ± s.e. of threeindependent experiments. Scale bars: 2mm in A,B,D; 0.5mm in C.DEVELO

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organization, which subsequently translates into a loss of function(Fig. 6C). As generally believed, CBs store the transcripts toprovide the cell with a quick response to physiological needs(Kotaja and Sassone-Corsi, 2007). Hence, it seems plausible thatthe observed degeneration of the CBs triggers the proliferativefailure of the neoblasts and the inability of the animals to form ablastema. This is in contrast to the Spoltud-1 phenotype, where theCB is probably unaffected, but the neoblast population is depletedregardless, even though after a much longer time (Solana et al.,2009). The observed increase of unspliced Smed-CycB transcriptssuggests that Smed-SmB RNAi additionally leads to a splicingdefect of genes essential for neoblast proliferation. The moderateincrease in the expression of cyclinB pre-mRNA is probablyunderestimated owing to its active degradation by nonsense-mediated decay following the introduction of stop codons into thetranscript sequence by the unspliced introns (for a review, see Iskenand Maquat, 2008). Strikingly, the degeneration of the CBs inneoblasts of the PB occurs in parallel to Smed-SmB proteindownregulation. Therefore, we propose that CB degeneration is notthe effect of a general splicing defect caused by the loss of Smed-SmB. Such an indirect effect would require the degradation of boththe mRNAs and proteins of Smed-SmB splicing targets, a processwe would expect to take longer to produce the degeneration anddisappearance of the CBs that we observed.

Without the possibility to trace planarian cells and look at theirfate, we could only speculate about the fate of Smed-SmB RNAineoblasts. Nonetheless, we frequently observed lysosomes andautophagic vacuoles (Fig. 6A,D) in Smed-SmB RNAi neoblasts,suggesting that these cells might undergo resorption by autophagy(González-Estévez et al., 2007). It is interesting, however, thatneoblasts are able to survive longer if not actively cycling, as in thecase of intact RNAi animals. Non-regenerating animals rely on theirpool of stem cells to guarantee cellular turnover, and this is thereason why the neoblast population of intact animals is virtuallyunaffected by Smed-SmB knockdown for up to 7 days after the

RNAi treatment (Fig. 4A-D). This capacity to survive the Smed-SmBRNAi for an extended period of time is probably owing to the longhalf-life of the Smed-SmB protein (Fig. 3A; Fig. 7A).

In this work, we have shown that a member of the LSmsuperfamily, Smed-SmB, is required for the proliferation andmaintenance of the adult stem cells of Schmidtea mediterranea.Smed-SmB is expressed by stem cells and neurons. Knockdown ofSmed-SmB expression, however, apparently affects only neoblasts,causing the downregulation of the expression of specific neoblastmarkers like Smedwi1 and Smed-Bruli. Expression of theproliferation markers PCNA and Smed-CycB was also quicklyimpaired, resulting in a massive proliferative failure and drasticreduction of viable neoblasts. As a direct consequence, regeneratinganimals do not form blastema and die within 2 weeks. Smed-SmBknockdown also leads to the death of intact animals, presumably asconsequence of a lack of cellular turnover. At the subcellular level,we observed a severe degeneration of the CBs. Within 3 days, thenumber of neoblasts with ‘healthy’ CBs in the PB was reduced to 1out of 8. By immunogold-labelling, we could localize the Smed-SmB protein to the nucleus of planarian stem cells and to the CB. Inaddition, we also found an increased expression of unspliced Smed-CycB transcripts. The latter is indicative of a failure of the splicingmachinery, which further impairs the proliferative capacity of thestem cells.

AcknowledgementsWe thank H. Orii for providing DjPCNA antibody, C. Ortmeier and G. Verberkfor real-time qRT-PCR, K. Mildner for technical assistance with the electronmicroscopy sample preparation, J. Müller-Keuker for assistance with the figuresand S. Kölsch for proofreading. This work was supported by grant BFU-2005-00422 and BFU2008-01544 from the Ministerio de Educación y Ciencia (Spain)and grants 2005SGR00769 and 2009SGR1018 from AGAUR (Generalitat deCatalunya, Spain) to E.S., grant DAAD D/06/12871 to H.R.S., and E.F.T.received an FPI fellowship from the Ministerio de Ciencia y Cultura, Spain.

Competing interests statementThe authors declare no competing financial interests.


Fig. 7. Dynamics of the cellular and molecular events leading to the Smed-SmB phenotype in intact and regenerating animals, andsubcellular localization of Smed-SmB protein. (A)In both intact (I) and regenerating (R) animals, Smed-SmB transcripts were drastically reducedshortly after Smed-SmB knockdown. Smed-SmB protein, however, showed a small decay in intact animals, which only became prominent after 7days of treatment. In regenerating animals, by contrast, Smed-SmB protein downregulation shortly followed RNA downregulation, resulting in asevere reduction of protein availability after 2 days. Remarkably, the downregulation of Smed-CycB expression, the reduction of X1 neoblasts andthe number of neoblasts with CBs were correlated with – and consequent to – Smed-SmB protein downregulation, not only regarding the timing,but also the dynamics: gradual for intact animals, abrupt for regenerating ones. (B)Electron micrograph of an ultrathin cryosection (55 nm)immunogold-labelled against Smed-SmB protein, showing detail of a neoblast. Owing to EM protocol, membranes appear white. ProteinA-coupledgold particles are 15 nm in size. The nucleus (n) revealed gold labelling mainly localized to uncondensed chromatin. Cytoplasm labelling occurredrarely, whereas CBs (dimension emphasized by arrowheads) were mainly labelled at the rim. Mitochondria (mi) are devoid of labelling. Scale bar:200 nm.

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Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.042564/-/DC1

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