Spoltud-1 Is a Chromatoid Body Component Required for Planarian Long-Term Stem Cell Self-Renewal

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Developmental Biology 328 (2009) 410–421

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Developmental Biology

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Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Jordi Solana a,⁎, Paul Lasko b, Rafael Romero a

a Departament de Genètica, Facultat de Biologia, Av. Diagonal 645, Ediﬁci Annex, Planta 1, 08028 Barcelona, Catalunya, Spain b Department of Biology and Developmental Biology Research Initiative, McGill University, 1205 Avenue Docteur Penﬁeld, Montreal, Quebec, Canada H3A 1B1

article info abstract

Article history: Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated Received for publication 20 October 2008 totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those Revised 29 January 2009 of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid Accepted 30 January 2009 bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Available online 9 February 2009 Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Keywords: Planarian Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of Schmidtea polychroa planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather Stem cells than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and Neoblasts differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal. Regeneration © 2009 Elsevier Inc. All rights reserved. Tudor Nuage Chromatoid bodies Germ line

Introduction A particular hallmark of neoblast cells is the presence of structures called chromatoid bodies, electron-dense perinuclear granules com- Planarian flatworms have an impressive power of regeneration. An posed of RNA and protein (Coward, 1974; Hori, 1982; Morita and Best, undifferentiated population of totipotent stem cells known as 1984; Morita et al., 1969). Chromatoid bodies are also present in neoblasts underlies this process (Baguñà et al., 1989; Handberg- planarian germ cells and in blastomeres during embryonic develop- Thorsager et al., 2008; Rossi et al., 2008; Sanchez Alvarado and Kang, ment (Cardona et al., 2005b, 2006) and resemble germ granules, often 2005). These cells are able to give rise to all differentiated cell types in called nuage, present in germ line cells from most animals (Eddy, the adult planarian and are maintained throughout the animal's life. 1975; Raz, 2000; Saffman and Lasko, 1999). The presence of these Neoblasts are defined on the basis of their proliferative capabilities structures not only in the germ line but in totipotent somatic stem and their morphological features, such as lack of differentiated cells suggest a role for chromatoid bodies in establishing or structures, relatively uncondensed chromatin and high nucleocyto- maintaining totipotency or an undifferentiated state (Auladell et al., plasmic ratio. Neoblasts are scattered throughout the mesenchyme of 1993). However, the precise role of chromatoid bodies in planarian the animal with the exception of the pharynx, located in the central stem cell biology is not clear yet. region of the body, and the region anterior to the photoreceptors. Interestingly, apparent orthologues of several genes that encode Analysis of proliferation markers such as PCNA immunohistochem- components of nuage, or that required for germ line establishment in istry (Orii et al., 2005), mcm2 in situ hybridization (Salvetti et al., more complex metazoans, are required for planarian neoblast 2000), anti-phospho-histone 3, and bromodeoxyuridine labeling function. Smed-bruno-like (bruli) is a RNA-binding protein that has (Newmark and Sanchez Alvarado, 2000) show a similar pattern to been recently shown to be required for stem cell maintenance in the that of neoblasts, since they are the only proliferative cell type in planarian Schmidtea mediterranea (Guo et al., 2006). Previously, two asexual planarians. Furthermore, neoblasts and all these markers are genes belonging to the PIWI/Argonaute family, smedwi-1 and specifically eliminated after high doses of irradiation, a behavior smedwi-2 were identified in the same species, and smedwi-2 was typical of proliferative cells. Thus, proliferation markers are a powerful directly implicated in neoblast differentiation control (Reddien et al., tool in order to identify neoblast cells. 2005). An additional piwi gene with a similar function, smedwi-3, has been recently found (Palakodeti et al., 2008). A pumilio homologue, ⁎ Corresponding author. DjPum, was found in the planarian Dugesia japonica, and was also E-mail addresses: [email protected], [email protected] (J. Solana). shown to be involved in planarian stem cell biology (Salvetti et al.,

2005). All of these genes are expressed in mesenchymal cells with a tertiary structure, and on the basis of their reciprocal blast hits to pattern similar to that of neoblasts and in the germ line, and abolish Drosophila tudor and mammalian TDRD1 and TDRD6 we finally regeneration capacity when inhibited through RNAi. selected spoltud-1 and spoltud-2 for further experiments (Accession In Drosophila melanogaster, the germ line is specified through numbers FJ655915 and FJ655916, respectively). We designed specific preformation (Extavour and Akam, 2003), meaning that it depends primers against S. mediterranea and used them to amplify the genes in upon the formation of a specialized cytoplasm that is derived from the a S. polychroa cDNA, made from total RNA using MMLV reverse products of genes expressed maternally during oogenesis. In Droso- transcriptase (Promega). In order to obtain the full cDNA sequence of phila, oskar RNA accumulates at the posterior pole of the developing spoltud-1 RACE reactions were carried out adding a poly-A tail to the oocyte, and nucleates the formation of pole plasm, which shares cDNA with TdT enzyme (New England Biolabs) and amplifying it by several components with nuage. Vasa and Tudor proteins are localized PCR by means of an anchor primer. The list and position of primers in this region in an Oskar-dependent manner and, like Oskar, both are used (5′–3′) are indicated in Fig. S1. Probes for Spoltud-1 in situ essential for germ line specification (Boswell and Mahowald, 1985; hybridization were generated from primers 3 and 4, dsRNA for RNAi Lasko and Ashburner, 1988). No homologue of oskar has been found in was generated from primers 1 and 2. a non-dipteran species so far. However, a PL10 gene (closely related to vasa) was identified in the planarian species D. japonica, and its In situ hybridization expression is described to be enriched in mesenchymal neoblasts (Shibata et al., 1999). Whole mount in situ hybridizations and in situ hybridization on Tudor contains a number of repeated elements called Tudor paraffin sections were carried out as described previously (Cardona et domains (Thomson and Lasko, 2005). Different Tudor domain- al., 2005a; Umesono et al., 1997; Zayas et al., 2005). Fluorescent in situ containing proteins have been described in mice and humans and, hybridizations were incubated overnight with either antidigoxigenin- among them, TDRD1, TDRD6 and TDRD7 have been shown to have a POD or antibiotin-POD (Roche) at a 1:100 dilution and developed with role in the germ line (Chuma et al., 2006; Hosokawa et al., 2007) TSA system (Perkin Elmer). For double fluorescent in situ hybridiza- although their orthology relationships are not clear. The Tudor domain tions slides were treated with 1% H2O2 in PBS for 1 h and washed 6 binds symmetrical dimethylated arginines (Cote and Richard, 2005), times for 15 min in PBS after first development prior to incubating and in D. melanogaster Tudor interacts with Valois, a component of the with the second POD-labeled antibody. When in situ hybridization methylosome (Anne et al., 2007). Some protein arginine methyl- was followed by immunohistochemistry permeabilization was carried transferases such as capsuleen in D. melanogaster and its homologue in out by heating the slides at 95 °C for 20 min in citrate buffer (10 mM mouse, PRMT5, have been demonstrated to have a role in germ line sodium citrate, 0.05% Tween 20, pH 6.0). formation (Ancelin et al., 2006; Anne et al., 2007), suggesting that Tudor domains and symmetrical dimethylated arginines could have a Immunohistochemistry crucial role in germ line biology. Due to the close relationship between the metazoan germ line and Slides were permeabilized with 0.4% pepsin in 0.1 N HCl at 37 °C or planarian stem cells we performed Tudor domain searches in as described above, blocked in 5% non-fat dry milk in PBS 0.05% Tween planarian databases in order to check their possible roles in neoblast 20, incubated overnight with primary antibodies in blocking solution, cells. Here we present the isolation of spoltud-1, a Tudor domain- washed in PBS 0.05 Tween 20, and incubated for 4 h at room containing gene in planarians, and show that it is expressed in temperature with Alexa Fluor secondary antibodies (Molecular neoblast cells. Animals periodically injected with spoltud-1 dsRNA are Probes) at a 1:400 dilution in blocking solution. The following able to regenerate during the first weeks but fail to produce primary antibodies were used: anti-PCNA (rabbit polyclonal kindly blastemata after 2 months of treatment. Consistently, they dramati- provided by Hidefumi Orii, 1:100) and anti-Spoltud-1 (rabbit poly- cally lose their neoblasts at the end of this treatment, suggesting a role clonal against a BSA-conjugated peptide, BSA-[C]FKGSDSFSRKSESNF, for spoltud-1 in the long-term maintenance of neoblasts. peptide underlined in Fig. S1, produced by Sygma Genosys, 1:200). Nuclei were stained with TO-PRO 3 (Molecular Probes). Materials and methods Immunohistochemistry for electron microscopy Animals Animals were fixed in 4% paraformaldehyde −1% glutaraldehyde We selected S. polychroa as species of work. S. polychroa is in phosphate buffer, cryoprotected in saccharose, cryosubstituted in convenient in order to study embryonic development and provides 0.5% uranyl acetate in methanol and cryoembedded in Lowicryl HM20 the possibility of comparing regeneration data to available embryonic for 48 h at −50 °C and 24 h at RT. Ultrathin sections were obtained development data (Martin-Duran et al., 2008; Solana and Romero, and collected on grids. Grids were blocked in 50 mM glycine in 0.1 M 2009). Furthermore, this species is closely related to S. mediterranea, PBS for 4 min, washed in 0.1 PBS, blocked in 3% BSA in 0.1 PBS for for which there is an ongoing genome project. Sexual S. polychroa 15 min and washed in 1% BSA for 3 min. Primary antibody was adults were collected in Sot de Ferrer (Castelló, Spain). Animals were incubated for 2 h at a 1:10 dilution in 1% BSA 0.1 M PBS. Following kept in glass containers with 1:1 tap water:distilled water, at 17 °C in incubation, grids were washed 5 times for 5 min in 0.1 M PBS 0.25% the dark, fed every week and starved at least 1 week prior to Tween 20, 3 min in 1% BSA 0.1 M PBS, incubated with gold-conjugated experiments. Sexually immature juvenile animals were selected for secondary antibody in 1% BSA 0.1 M PBS, washed 5 times for 5 min in some experiments. 0.1 M PBS 0.25% Tween 20, washed extensively in deionized water, dried, and stained with uranyl acetate. Grids were observed with a Isolation of spoltud-1 JEOL 1010 electron microscope and images were captured with a Bioscan (Gatan) digital camera. To identify S. polychroa genes containing Tudor domains we performed tBLASTn searches using several Tudor domains as query Imaging against the closely related species S. mediterranea genome. This ongoing genome project corresponds to a species sharing an Whole mount animals were observed with a Leica MZ16F extremely high similarity with S. polychroa. We found several stereomicroscope. Images were captured with a Leica DFC300FX candidate genes containing Tudor domains, based on their predicted camera. Fluorescent slides were mounted in Vectashield and imaged 412 J. Solana et al. / Developmental Biology 328 (2009) 410–421 with a Leica TCS SPE confocal microscope. Images were analyzed with Image J and Adobe Photoshop software. Deconvolved images were produced with Huygens Essential software by Scientific Volume Imaging and using a theoretical point spread function.

Real time PCR

RNA was extracted using the Trizol reagent (Invitrogen). cDNA was obtainedfrom500ngoftotalRNAbyusingMMLVReverse Transcriptase (Promega). Elongation Factor 2 (EF2) was used as an internal control. Four animals were used per treatment and time point, and each sample was replicated three times in each real-time PCR experiment. PCR reactions were performed using the SYBR® Green RT-PCR kit (Applied Biosystems) and reactions were analyzed using an ABI Prism 7900HT (Applied Biosystems).

RNAi analysis

DsRNA was produced by in vitro transcription with T7 and SP6 enzymes (Roche) and injected into planarians as described before (Sanchez Alvarado and Newmark, 1999). Sexually immature juvenile animals were injected during 3 consecutive days and left for 11 days before starting a new round of injection, for a total of 2 months (4 rounds of injection). Control animals were injected with dsRNA of green ﬂuorescent protein (GFP). Animals were kept at 20 °C and observed daily with a Zeiss Stemi SV6 scope and images of living animals were captured with a Nikon Coolpix E995.

Results

Isolation of Spoltud-1

tBLASTn searches in the S. mediterranea genome identified several contigs containing open reading frames that would encode Tudor domains, which generally share a low conservation at the amino acid level. On the basis of their similarity to well established Tudor domain-containing proteins involved in germ line control we selected two of them and designed specific primers to amplify the orthologous genes in S. polychroa. RACE reactions allowed us to isolate a full-length cDNA of 2482 bp which we named Spoltud-1. The sequence of Spoltud-1 encodes a protein of 771 amino acids that contains an N-terminal RNA recognition motif (RRM1) and three Tudor domains (Fig. S1), and shares orthology with a previously described EST (05895_HH) from the planarian species D. japonica (Yoshida-Kashikawa et al., 2007). Spoltud-1 and EST 05895_HH have 65.0% identity and 88.0% similarity. The partial clone corresponding to Spoltud-2 comprises a Tudor domain. The related sequence in the S. mediterranea genome encodes a putative protein of 793 amino acids and contains three Tudor domains, with a very low amino acid identity with Spoltud-1 (less than 10% throughout the whole protein) Although other Tudor domain- containing genes exist in the genome of the closely related species S. mediterranea we have not found clear paralogue genes with high similarity to Spoltud-1. Furthermore, only the sequences corresponding to Spoltud-1 and Spoltud-2 gave reciprocal BLAST hits to D. tudor and related mammalian genes involved in germ line biology. Fig. 1. Spoltud-1 in situ hybridization on adult and juvenile planarians shows expression in the germ line, CNS and neoblasts. (A) Spoltud-1 whole mount in situ hybridization Spoltud-1 mRNA and protein expression on adult planarians. Anterior is to the left. Top: dorsal view. Bottom: ventral view. Expression in testes is seen as two dorsal rows indicated by asterisks. Expression in ovaries is indicated by arrows. Arrowheads indicate CNS expression in the brain and Spoltud-1 and Spoltud-2 mRNA expression was monitored by in ventral nerve cords. (B, C) Spoltud-1 in situ hybridization on paraffin sections of an situ hybridization. Spoltud-2 was found to be highly expressed in adult planarian showing testes, indicated by arrows (B), and parenchymal neoblasts ovaries and weakly expressed in testes but not in neoblast cells (Fig. (C). (D–G) Confocal maximum projection of a double in situ hybridization and immu- S2), and was therefore discarded for further experiments. In adult nohistochemistry of neoblast markers. (D) Spoltud-1 mRNA positive cells in green. (E) histone H2B mRNA positive cells in red. (F) Nuclear expression of PCNA protein sexualized animals Spoltud-1 was expressed in testes and ovaries, in neoblast cells in blue. (G) Overlay image showing colocalization of Spoltud1 mRNA central nervous system (CNS) and in the parenchyma with a pattern and both histone H2B mRNA and PCNA protein. te, testis. Scale bars: 1 mm in A; 50 μm similar to that of neoblast cells (Fig. 1A). Some animals also showed in B,C; 10 μminD–G. J. Solana et al. / Developmental Biology 328 (2009) 410–421 413 expression in the eyes (not shown), as has been described for some excellent marker to trace neoblasts, and its expression is well other genes expressed in neoblasts such as nanos (Handberg- characterized in planarians (Orii et al., 2005). Another stem cell- Thorsager and Salo, 2007). In situ hybridization on paraffin sections specific gene is histone H2B (Guo et al., 2006), which has been shown revealed that the expression in the testis was restricted to the to be irradiation sensitive and to localize to parenchymal cells in S. peripheral area (Fig. 1B) which contains the spermatocytes and the mediterranea asexual organisms. Our experiments have shown that spermatogonia, the germ line stem cells of the testis. Expression in the both PCNA protein and histone H2B colocalize in S. polychroa parenchyma was restricted to discrete cells (Fig. 1C) scattered neoblasts. throughout the body with the exception of the pharynx and the To further characterize parenchymal Spoltud-1 expressing cells we region anterior to the eyes. Juvenile animals lacking sexual structures developed a protocol to perform double in situ hybridization followed only showed expression in the CNS and parenchyma (data not shown). by immunohistochemistry. In S. polychroa juveniles Spoltud-1 mRNA Several neoblast markers have been described in recent years. One positive cells (Fig. 1D) expressed both histone H2B mRNA (Fig. 1E) and of them is Proliferative Cell Nuclear Antigen (PCNA), a protein PCNA protein (Fig. 1F). Therefore, we conclude that Spoltud-1 expressed in proliferative cells. Since neoblasts are the only pro- expressing cells in the parenchyma are neoblast cells. In adult liferative cell type in juvenile or asexual planarians, PCNA protein is an sexualized animals histone H2B and PCNA also colocalized with

Fig. 2. Spoltud-1 immunohistochemistry on adult and juvenile planarians shows localization to chromatoid bodies in neoblasts and testes and perinuclear granules in CNS. (A–D) Confocal maximum projection of an in situ hybridization and immunohistochemistry of Histone H2B and Spoltud-1. (A) Histone H2B mRNA. (B) Spoltud-1 protein. (C) nuclei. (D) Overlay image. (E) Deconvolution of a confocal series of a neoblast immunolabeled with anti-Spoltud-1. Labeled particles are perinuclear and are all around the nucleus. (F–H) Immunohistochemistry on electron microscope sections. (F) Low magniﬁcation of a neoblast cell displaying a chromatoid body. (G) Higher magniﬁcation of (F) showing an immunolabeled chromatoid body. (H) Detail of a chromatoid body immunolabeled with anti-Spoltud-1.(I–K, M–O) Confocal maximum projection of an anti-Spoltud-1 immunohistochemistry on brain sections (I–K) and testis section (M–O). (I, M) Spoltud-1 protein. (J, N) Nuclei. (K, O) Overlay images. (L, P) Deconvolution of a confocal section of an anti-Spoltud-1 immunohistochemistry on brain sections (L) and testis (P). Arrowheads in L indicate neural processes. nu, nucleus; cb, chromatoid body; mc, mitochondria; sg, spermatogonia; st, spermatids; sp, sperm cells. Scale bars: 5 μminA–E; 1 μm in F; 400 nm in G; 100 nm in H; 20 μminI–K; 7.5 μminL;25μminM–O; 7 μminP. 414 J. Solana et al. / Developmental Biology 328 (2009) 410–421

Spoltud-1 mRNA positive cells in the parenchyma and in testicular revealed labeling in these structures (Figs. 2F–H). Therefore, we spermatocytes and spermatogonia (data not shown). concluded that Spoltud-1 protein localizes to chromatoid bodies in To address the subcellular localization of Spoltud-1 protein we neoblasts. developed a polyclonal antibody against a peptide of the protein. Then we addressed the distribution of Spoltud-1 protein in CNS. Immunohistochemistry on paraffin sections of juvenile animals Interestingly, the pattern was shown to be similar to the one in stem revealed that histone H2B positive cells (Fig. 2A) displayed Spoltud-1 cells. Spoltud-1 protein localizes to perinuclear particles surrounding protein in perinuclear granules (Figs. 2B–D) in a pattern similar to that the nuclei of neurons in the brain (Figs. 2I–K), a pattern similar to that of chromatoid bodies and chromatoid body components such as of DjCBC-1. These particles are in general smaller than chromatoid DjCBC-1 (Yoshida-Kashikawa et al., 2007). Deconvolution of these bodies and more closely attached to the nucleus (Fig. 2L). Further- images showed that these perinuclear particles surround the whole more, some particles were observed in neuronal processes (Fig. 2L). nucleus and lie completely outside of it (Fig. 2E). We then examined To address the expression in testes, we performed immunohis- the localization of Spoltud-1 at the electron microscopic level. tochemistry on paraffin sections of sexualized planarians. The protein Neoblasts are easily identified in electron microscopy because of the was localized to perinuclear particles surrounding the nuclei of presence of chromatoid bodies, identified as electron-dense peri- spermatocytes and spermatogonia (Figs. 2M–O). No expression was nuclear structures. Immunohistochemistry on ultrathin sections found in spermatids or sperm cells (Fig. 2P).

Fig. 3. Spoltud-1 expression in S. polychroa developing embryos. (A–I) Schematic representation of S. polychroa stages of development, see text for explanation. Grey: syncytium. Light grey: ingested yolk mass. Dark blue nuclei: syncytial nuclei. Green cells: Zygote-derived blastomeres and embryonic cells (A–E). Red: embryonic (A–G) and deﬁnitive (G–I) pharynx. Orange: gastrodermal cells and gastrodermis. Magenta: nerve cells and nervous system. Green: Muscle cells and muscular system. (J–R) Spoltud-1 whole mount in situ hybridization in embryos. (J) Stage 2 embryo. (K) Stage 3 embryo. (L, M) Stage 4 embryos. (N) Stage 5 embryo. (O) Stage 6 embryo. (P) Stage 8 embryo, anterior is to the left, dorsal is to the top. (Q, R) Deconvolution of confocal sections of a Spoltud-1 immunohistochemistry on embryo parafﬁn sections. (Q) Stage 4 embryo. (R) Stage 7 embryo. Scale bars: 300 μmin J–O; 150 μminP;7μminQ,R. J. Solana et al. / Developmental Biology 328 (2009) 410–421 415

Spoltud-1 expression in developing embryos and 7 the number of Spoltud-1 positive cells is reduced (Figs. 3O, P) to finally conform a pattern similar to that of neoblast cells in the stage 8 We next addressed the expression of Spoltud-1 in planarian pre-hatch juvenile worm (Fig. 3P). Spoltud-1 immunohistochemistry embryos. Some Tudor domain-containing proteins, such as TUD in on paraffin sections of embryos revealed that the distribution of the D. melanogaster, are expressed maternally and are necessary for protein in stage 2–4 is in particles distributed throughout the germ line determination (Boswell and Mahowald, 1985; Thomson and cytoplasm of the embryonic cells but concentrated upon the surface Lasko, 2004, 2005). We therefore investigated if Spoltud-1 was of the multilobed nucleus (Fig. 3Q). In contrast, stages 6–8 expressed during embryonic development. Planarian development is differentiating positive cells display a more neoblast-like distribution, still poorly studied and is highly derived compared to more basal with perinuclear particles attached to the nuclear membrane of an platyhelminthes and other model organisms (Cardona et al., 2005b, unlobed nucleus (Fig. 3R). 2006). Briefly, several fertilized zygotes are deposited in egg capsules surrounded by yolk cells. Around each zygote a yolk syncytium forms Spoltud-1(RNAi) animals fail to regenerate after 7 weeks of treatment by the fusion of yolk cells, and zygotes are cleaved into different blastomeres which freely spread through this syncytium without To address the function of Spoltud-1 in neoblast cells we silenced it staying in touch to each other (Fig. 3A). In stage 2 an embryonic using RNAi. Juvenile organisms were injected with Spoltud-1 dsRNA pharynx develops, as does the epidermis, covering the whole and control animals were injected with GFP dsRNA, a gene not present syncytium (Figs. 3B, C). In stages 3 to 5 the embryonic pharynx starts in the planarian. Animals were injected during three consecutive days to ingest external, but not fused yolk cells. Due to this ingestion, a and cut on the fourth, and then allowed to regenerate for 10 days and cavity is formed and the syncytium is restricted to the periphery of the injected again. Our results indicated that after the first three rounds of embryo, which is henceforth called the germ band (Figs. 3D, E). In injection animals regenerated properly and displayed no obvious stage 5, embryonic cells start to differentiate massively into all phenotypes, but after the fourth round of injection all animals definitive cell types (Fig. 3F). Due to the differentiation of muscle cells, (N=35) failed to regenerate (Fig. 4B), while this effect was not embryos, which were previously spherical, adopt an ovoid worm-like seen in control animals (N=35, Fig. 4A). This effect was considerably shape (Fig. 3G). During stages 6–8 the germ band collapses and delayed compared to other RNAi phenotypes described in planarians embryonic cells grow to form septa which would finally shape the gut for neoblast-related genes, in which usually animals fail to regenerate and muscle system (Fig. 3H). A definitive pharynx develops de novo after one single round of injection (Guo et al., 2006; Reddien et al., and CNS and muscle system complete their development (Fig. 3I). 2005; Salvetti et al., 2005). Whole mount in situ hybridization in S. polychroa embryos showed Interestingly, some Spoltud-1(RNAi) animals (N=14) were able to Spoltud-1 expression in blastomeres and embryonic cells. Spoltud-1 initiate a regenerative process after the fourth round of injection, but mRNA is detected in stage 2 embryos (Fig. 3J). In stage 3 positive failed to finish it properly. Two representative animals are shown in embryonic cells are restricted to the germ band and are visible in the Figs. 4D, E, along with a control animal (Fig. 4C). Four days after the periphery of the embryo (Fig. 3K). During stage 4 embryos grow in cut the animals have developed a blastema. After 5 days, control size and the number of positive embryonic cells increases (Figs. 3L, animals show visible eyes. The animal depicted in Fig. 4D never M). In stage 5 embryonic cells highly diversify in size and shape developed eyes, while the animal in Fig. 4E displayed a rudiment of a coinciding with the onset of differentiation (Fig. 3N). During stages 6 right eye 7 days post-amputation. In S. polychroa regenerants eye

Fig. 4. Spoltud-1(RNAi) animals fail to regenerate after 7 weeks of treatment. (A, B) Representative animals of the RNAi experiments after four rounds of injection and 7 days post- amputation (A) gfp(RNAi) control animal. (B) Spoltud-1(RNAi) animal. (C–E) Head regeneration of representative animals that could initiate a regenerative blastema but failed to regenerate completely. (C) gfp(RNAi) control animal. (D, E) Different Spoltud-1(RNAi) representative animals. Blastemata 4 days post-amputation are indicated by arrowheads. Eye rudiment in one individual is indicated by arrow. Anterior is to the top in A, B, and to the right in C–E. Scale bars: 120 μm. 416 J. Solana et al. / Developmental Biology 328 (2009) 410–421 development is not completely synchronic; some animals develop one could not be tested due to the lack of feasible experimental procedures eye hours before the other. The animal in Fig. 4D never developed the to perform RNAi analyses during planarian embryonic development, right eye completely and never displayed any rudiments of the left although signiﬁcant progress has been recently produced (Martin- eye. Interestingly, Spoltud-1(RNAi) animals pigmented the aberrant Duran et al., 2008). blastemata after 12–16 days, and these blastemata never regressed, suggesting that these cells were effectively differentiating. Smedwi-2 Spoltud-1 RNAi leads to a decrease in Spoltud-1 mRNA and protein levels RNAi and Smed-bruli RNAi animals are able to produce small regeneration blastemata that regress after some time (Guo et al., Since the phenotype observed in Spoltud-1(RNAi) animals was 2006; Reddien et al., 2005), indicating an effect in differentiating and considerably delayed, we examined if the RNAi was effectively differentiated cells that come from inhibited neoblasts. Our results inhibiting the function of the gene. In order to address this question show that during Spoltud-1 RNAi, differentiated cells coming from we performed immunohistochemistry with anti-Spoltud-1 in injected inhibited neoblasts are stable over time. These results strongly suggest animals. Animals after one round of injection still showed Spoltud-1 that the phenotype observed is caused by an effect in neoblast cells expression in chromatoid bodies (data not shown) but after a second rather than in differentiated cells. round of injection much of Spoltud-1 protein expression was lost both Due to their larger size and the difﬁculty to deliver dsRNA for such in neoblast cells (Figs. 5G–I, compare with control in Figs. 5A–C) and a long time, sexually mature animals were not tested in order to test a brain (Figs. 5J–L, compare with control in Figs. 5D–F). After a third possible effect in ovaries and testes. Similarly, the effect in embryos round of injection no Spoltud-1 protein was detected (Fig. S3). These

Fig. 5. Spoltud-1 protein is lost after 3 weeks of treatment. (A–L) Anti-Spoltud-1 immunohistochemistry in control and Spoltud-1(RNAi) animals after second round of injection (3 weeks). (A–C) Parenchyma of a gfp(RNAi) control animal. (D–F) Brain of a gfp(RNAi) control animal. (G–I) Parenchyma of a Spoltud-1(RNAi) animal. (J–L) Brain of a Spoltud-1 (RNAi) animal. (A, D, G, J) Nuclei staining (blue). (B, E, H, K) anti-Spoltud-1 (green). (C, F, I, L) Overlay images. Anterior is to the bottom left. Scale bars: 30 μm. J. Solana et al. / Developmental Biology 328 (2009) 410–421 417 results suggest that the lack of phenotype during the first weeks can neoblast proliferation or differentiation. Animals after 7 weeks of be explained by a persistence of the protein but the latency observed injection are not able to produce blastemata or produce abnormal between the disappearance of the protein and the onset of the blastemata that are not able to complete regeneration because they phenotype has to be explained by other means. might contain insufficient neoblasts, but the abnormal blastemata Next, we analyzed Spoltud-1 mRNA levels during the RNAi produced are still able to generate differentiated cells, strongly treatment by means of real-time PCR. Spoltud-1 mRNA levels rapidly suggesting that proliferation and differentiation are not affected. drop from the first dsRNA injection (Fig. 6A) and remain low during Interestingly, some animals lost anterior neoblasts before losing the whole treatment. The total amount of Spoltud-1 mRNA in Spoltud- their posterior counterparts (Figs. 7P–R, compare with control in Figs. 1(RNAi) animals is approximately 5% of the amount in controls. 7M–O), a pattern similar to the one shown after inhibition of smedinx- 11, a neoblast gap junction gene the inhibition of which leads to Neoblasts are rapidly lost after 7 weeks of treatment neoblast loss and lack of regeneration (Oviedo and Levin, 2007). Nevertheless, animals after a fifth round of injection (N=10) lost In order to analyze neoblasts in RNAi animals we performed His- nearly all neoblasts (data not shown). These results suggest that tone H2B in situ hybridization and anti-PCNA immunohistochemistry. anterior neoblasts are lost consistently before posterior ones during After three rounds of injections animals showed a normal pattern of RNAi of different stem cell genes, perhaps reflecting a difference in neoblasts (Figs. 7E–H, compare with control in Figs. 7A–D). No between these neoblast subpopulations. During smedinx-11 RNAi differences were detected either in animals after the first and second treatment (Oviedo and Levin, 2007) neoblasts are lost in an anterior– rounds of injection (data not shown). Nevertheless, after the fourth posterior progression. A mitotic gradient exists in planarians, with round of injection the number of neoblasts was dramatically reduced anterior neoblasts having more mitotic activity than posterior ones (Figs. 7I–L), coinciding with the moment in which animals lost their (Baguñà, 1976; Oviedo and Levin, 2007). We also observed a specific regeneration capability. These results indicate that, although protein loss of anterior neoblasts prior to posterior, suggesting that the more function might be absent from the third week on, neoblast cells are mitotically active cells are first lost due to a failure in stem-cell not affected at that time and continue to proliferate and differentiate maintenance capability. until the fourth round of injection, when a rapid decay in neoblast We analyzed pcna mRNA levels in RNAi animals to confirm number is seen. Taking together all these results, we suggest that the presence and function of neoblasts by means of real time-PCR during Spoltud-1 RNAi neoblast self-renewal is affected rather than (Fig. 6B). mRNA in Spoltud-1(RNAi) animals did not significantly

Fig. 6. Spoltud-1 and pcna mRNA expression levels in Spoltud-1(RNAi) animals. (A) Spoltud-1 mRNA relative levels by means of real time PCR during RNAi treatment, showing the efficiency of the dsRNA injections. (B) pcna mRNA levels during RNAi treatment. Pcna levels in Spoltud-1(RNAi) animals are not significantly different from control animals during the first 5 weeks. In the seventh week there is a decrease in pcna mRNA levels. w1, week 1; w3, week 3; w5, week 5; w7, week 7. 418 J. Solana et al. / Developmental Biology 328 (2009) 410–421

Fig. 7. Neoblasts are rapidly lost after 7 weeks of treatment. (A–L) Confocal maximum projection of a Histone H2B in situ hybridization and anti-PCNA immunohistochemistry in control and Spoltud-1(RNAi) representative animals. (A–D) gfp(RNAi) control after third round of injection (5 weeks). (E–H) Spoltud-1(RNAi) animal after third round of injection (5 weeks). (I–L) Spoltud-1(RNAi) animal after fourth round of injection (7 weeks). (M–R) Confocal maximum projection of an anti-PCNA immunohistochemistry in sagital sections of control and Spoltud-1(RNAi) representative animals. (M–O) gfp(RNAi) animal. (P–R) Spoltud-1(RNAi) animal. (A, E, I, M, P) Nuclei staining (blue). (B, F, J, N, Q) anti-PCNA (green). (C, G, K) Histone H2B fluorescent in situ hybridization (red). (D, H, L, O, R) Overlay images. Anterior is to the left. Scale bars: 50 μminA–H; 30 μminI–L; 200 μminM–R. differ from control animals during the first 5 weeks. Nevertheless, in Discussion the seventh week, and coinciding with the onset of the neoblast loss, a significant decrease in pcna mRNA levels is detected. Consistent Spoltud-1 is a component of chromatoid bodies in germ cells with the phenotype observed the pcna mRNA levels of Spoltud-1 and neoblasts (RNAi) animals in the seventh week do not reflect a complete disappearance of neoblast but an ongoing process of loss. These Several proteins with key functions in the germ line in different results confirm a late disappearance of neoblast cells in Spoltud-1 animals have been recently shown to be related to neoblast biology in (RNAi) animals. planarians (Guo et al., 2006; Reddien et al., 2005; Salvetti et al., 2005; J. Solana et al. / Developmental Biology 328 (2009) 410–421 419

expressed in neoblast cells and CNS resulted in a nervous system RNAi phenotype (Guo et al., 2006; Salvetti et al., 2005). DjCBC-1, described in the study by Yoshida-Kashikawa and cow- orkers, is a chromatoid body component with an expression pattern extremely similar to that of Spoltud-1. It corresponds to a Me31B homologue which belongs to the DEAD-box RNA helicases (Ladomery et al., 1997; Nakamura et al., 2001; Weston and Sommerville, 2006). Interestingly, it has been recently shown that Me31B in D. melanogaster forms a complex together with TUD (Thomson et al., 2008). DjCBC-1 is also expressed in the brain, in perinuclear particles surrounding the nuclei of neurons. At the ultrastructural level, DjCBC-1 can be seen to be localized to electron dense chromatoid body-like structures attached to the neuron nucleus (Oosaki and Ishii, 1965; Yoshida-Kashikawa et al., 2007). These structures might reﬂect the close relationship between neoblasts and neurons. Neuronal granules have been described in many animals and are thought to be responsible for the transport of silenced mRNAs from the cell bodies to the synaptic surface where they can be speciﬁcally translated. They share, together with germ granules and other types of RNA granules (Anderson and Kedersha, 2006), a common machinery including several RNA-binding protein such as Staufen (Ramasamy et al., 2006;

Fig. 8. Schematic drawing of Spoltud-1 effect on juvenile planarians. Relative levels of Thomas et al., 2005), translation factors (Amiri et al., 2001; Kedersha Spoltud-1 mRNA (blue) and protein (green) compared to neoblast number (red). et al., 2002, 2005), and many more (Thomson et al., 2008). Time points indicated on bottom, regeneration, proliferation and differentiation on top. Two different periods of protein persistence and latency are deﬁned. The embryonic origin of neoblasts

Shibata et al., 1999). The similarity between neoblasts and the germ Our results show that during the embryonic development of S. line also extends to the presence of chromatoid bodies in planarian polychroa there are two different types of Spoltud-1 expression. The stem cells. These structures resemble the structures present in the first type exists during the first stages of development (stages 1–4) germ line of most animals (P granules in C. elegans, polar granules in and is present in blastomeres and embryonic cells derived from them. D. melanogaster and chromatoid body in mammals, often called nu- They are relatively big in size and possess a multilobed nucleus. age) and its presence in planarian somatic stem cells implies a Spoltud-1 protein is present within them in granules distributed relationship with maintenance of the undifferentiated state and throughout the cytoplasm but concentrated on the periphery of the genome integrity. In asexual species of planaria, reproduction is multilobed nucleus. It has been recently proposed that these early carried out exclusively by regeneration. Thus the neoblasts must stages could represent a cryptic larval stage that feeds on maternal possess properties similar to those of germ line stem cells in other resources (Cardona et al., 2006). This putative larval stage extends animals. Different Tudor domain-containing proteins have been over the period in which the first type of Spoltud-1 expression is related to the germ line in many different organisms (Chuma et al., detected. 2003, 2006; Hosokawa et al., 2007; Ikema et al., 2002). Our results During stage 5 a wave of differentiation occurs within the demonstrate that Spoltud-1 protein is a component of chromatoid developing embryo and Spoltud-1 expression is seen as a second bodies in planarian stem cells. type of expression. Most of the cells generated during this stage are smaller and differently shaped, perhaps corresponding to the onset of Spoltud-1 expression in CNS their differentiation. During stages 6 and 7 Spoltud-1 expressing cells display a more neoblast-like pattern of distribution of Spoltud-1. Contrary to what was proposed for the ortholog EST 05895_HH Nevertheless, most of these cells might correspond to differentiating from D. japonica in a recent study (Yoshida-Kashikawa et al., 2007), cells rather than neoblasts, although an undifferentiated population we have found that Spoltud-1 is consistently expressed in CNS. This must exist during these stages. It is important to emphasize that difference can be explained either by species variability or different during stage 8 only a restricted pool of cells displays Spoltud-1 protein sensitivities of techniques. Interestingly, several genes expressed in and mRNA. This pool might correspond to the neoblast cells present in neoblasts have been also shown to be in CNS (Guo et al., 2006; a hatchling. Salvetti et al., 2005). Furthermore, 39 out of 60 putative RNA- binding proteins were described as expressed in neoblasts and CNS Spoltud-1 is required for neoblast long-term self maintenance in the above mentioned study (Yoshida-Kashikawa et al., 2007). Only 6 out of 60 were described as expressed specifically in Consistent with the expression of Spoltud-1 in neoblast cells we neoblasts and not in CNS, including the ortholog EST of Spoltud-1. found that Spoltud-1(RNAi) animals failed to regenerate after a long These data strongly suggest a relationship between neoblast and treatment, but could effectively regenerate during the first weeks. neuron gene expression patterns. Some proteins classically related to Furthermore, these cells are lost at the end of the treatment (Fig. 8). germ line development have been recently found to be involved in These results suggest that Spoltud-1 function in neoblasts is neuronal function and development. Translational regulators such us important for their long-term maintenance. The fact that in the Nanos and Pumilio, for example, are involved in neuronal excit- absence of detectable Spoltud-1 protein neoblasts were able to ability, dendrite morphogenesis, and long-term memory in D. proliferate and differentiate strongly suggests a function in stem cell melanogaster (Mee et al., 2004; Menon et al., 2004; Muraro et al., self maintenance. Furthermore, some Spoltud-1(RNAi) animals after 2008; Schweers et al., 2002; Ye et al., 2004). Although our the fourth round of injection were able to produce blastemata that experiments with Spoltud-1 did not reveal any obvious nervous did not conclude a proper regeneration, but the cells present in these defects we cannot discard the existence of a neuronal function for blastemata were able to differentiate organs such as the eye and the protein. Similarly, none of studies of genes described to be ultimately acquire pigmentation, a sign of terminal differentiation. 420 J. Solana et al. / Developmental Biology 328 (2009) 410–421

Taken together, these results suggest that in the absence of Spoltud-1 income or stability in the chromatoid bodies is impaired, although the protein cells are able to proliferate and differentiate but not able to RNA therein might be highly stable. This RNA may consist of self-renew, ultimately reaching a state in which nearly all neoblasts determinants for neoblast differentiation and the presence of this have differentiated and there are no stem cells left for a proper RNA in chromatoid bodies may constitute a determinant of undiffer- conclusion of regeneration. Nevertheless, we cannot rule out a entiated state. While Spoltud-1 function is affected, these RNAs may possible redundancy in function by other Tudor domain-containing be slowly mobilized and translated cytoplasmically. After RNA in proteins present in planarian species, though this possibility is chromatoid bodies is exhausted, neoblast cells are no longer able to unlikely due to the low similarity exhibited by them. Spoltud-2, self-maintain an undifferentiated state and after some time differ- another Tudor-domain containing gene similar to D. tudor and entiate without renewing the neoblast pool. Thus, animals are related mammalian gene, was found to be not expressed in neoblast depleted of stem cells. cells, and, furthermore, its similarity to Spoltud-1 is very low (less than 10%). Acknowledgments The late phenotype seen in Spoltud-1(RNAi) opens the door for a new understanding of neoblast biology. RNAi-mediated knockdown of We are grateful to: Francesc Cebrià and Aziz Aboobaker for critical other genes expressed in planarian stem cells result in no obvious review of the manuscript, Jon Permanyer for valuable help with initial phenotypes on the first weeks of treatment, but these analyses did not BLAST searches, Mette Handberg-Thorsager for help in developing the extend for as long a time as ours (Reddien et al., 2005; Yoshida- double in situ hybridization technique, Hidefumi Orii for kindly Kashikawa et al., 2007). We speculate that some of these other providing anti-DjPCNA antibody, and members of the Romero and knockdowns might in fact result in a phenotype upon a lengthier Lasko lab for helpful discussion and for kindly sharing reagents and exposure to RNAi, in a manner similar to Spoltud-1. samples. Electron and Confocal microscopy and Real-Time PCR were performed at the Serveis Cientificotècnics of the University of Function of Spoltud-1 in planarian chromatoid bodies Barcelona and under the supervision of their professional experts, in particular, Manel Bosch, Carmen López-Iglesias, Gema Martínez, We have demonstrated that Spoltud-1 activity is essential for Elisenda Coll, Ramón Seminago and Amaya Amador. J.S is supported neoblast maintenance. Nevertheless, neoblasts retain their function in by a BRD grant from the University of Barcelona and a travel fellowship the absence of Spoltud-1 protein for an extended period of time. The from Development journal. This work was supported by a BFU07- timing of this effect occurs in a very long fashion. Our experiments 63209 grant to R.R. indicate that during the first 3 weeks of treatment Spoltud-1 protein is still present but after the third round of injection the protein is no Appendix A. Supplementary data longer detectable (Fig. 7). Thus, the persistence of the protein in the first weeks can account for the lack of phenotype during this period. Supplementary data associated with this article can be found, in the Nevertheless, the appearance of the phenotype after a fourth round of online version, at doi:10.1016/j.ydbio.2009.01.043. injection has to be explained by other means. Chromatoid bodies are believed to be very stable structures which References contain RNA and are sites of translational regulation. Our results Amikura, R., Hanyu, K., Kashikawa, M., Kobayashi, S., 2001a. Tudor protein is essential indicate that Spoltud-1 protein, a component of chromatoid bodies, is for the localization of mitochondrial RNAs in polar granules of Drosophila embryos. highly stable over time and this fact might be related to chromatoid Mech. Dev. 107, 97–104. body stability. mRNAs contained in these structures are likely to be Amikura, R., Kashikawa, M., Nakamura, A., Kobayashi, S., 2001b. 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