Pfister Et Al. 2008

ARTICLE IN PRESS YDBIO-03711; No. of pages: 14; 4C: 3, 4, 5, 6, 7, 8, 9, 10 Developmental Biology xxx (2008) xxx–xxx

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Developmental Biology

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Flatworm stem cells and the germ line: Developmental and evolutionary implications of macvasa expression in Macrostomum lignano

Daniela Pﬁster a,⁎, Katrien De Mulder a,b, Volker Hartenstein c, Georg Kuales a, Gaetan Borgonie b, Florentine Marx d, Joshua Morris e, Peter Ladurner a,⁎ a University of Innsbruck, Institute of Zoology, Technikerstrasse 25, A-6020 Innsbruck, Austria b University of Ghent, Department of Biology, Ledeganckstraat 35, B-9000 Ghent, Belgium c University of California Los Angeles, Department of Molecular, Cell and Developmental Biology, Los Angeles, CA 90095, USA d Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3/II, A-6020 Innsbruck, Austria e Department of Biology and Chemistry, Azusa Paciﬁc University, Azusa, CA 91702, USA article info abstract

Article history: We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic Received for publication 23 August 2007 stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA Revised 19 February 2008 interference were developed for M. lignano to study gene expression and function. Macvasa expression was Accepted 20 February 2008 followed during postembryonic development, regeneration and in starvation experiments. We were able to Available online xxxx follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by Keywords: feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed Germline specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad Evolution fl Neoblasts formation and stem cell proliferation. Our results corroborate the exclusive nature of the atworm stem cell Planaria system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. Totipotent We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to Vasa unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells. © 2008 Elsevier Inc. All rights reserved.

Introduction while injection of differentiated cells could not undo irradiation effects (Baguñá et al., 1989). Third, incorporation of the thymidine Flatworms possess an exceptional stem cell system that gives rise analogue bromodeoxyuridine (BrdU) has shown neoblast prolifera- to all cell types during development and regeneration. These stem tion, migration and differentiation (Ladurner et al., 2000; Newmark cells – called neoblasts – are the only cells that divide in the adult and and Sánchez, 2000). In recent years, a number of genes involved in the are responsible for the enormous regeneration capacity and plasticity proliferation and regulation of flatworm stem cells such as e.g. MCM, of flatworms tissue. PCNA, pumilio or piwi have been identified (Orii et al., 2005; Salvetti et More than 100 years of research on planarian regeneration has al., 2005; Reddien et al., 2005a; Rossi et al., 2006). revealed a large body of evidence demonstrating the differentiation Germ cells in flatworms can be derived from stem cells when tiny potential of flatworm neoblasts. First, classical regeneration experi- pieces of tissue that lack any gonad structures regenerate into a ments have shown that a small fragment of a planarian body can complete animal (Agata, 2003; Reddien and Sanchez, 2004; Egger et regenerate into a complete animal (Newmark and Sánchez, 2002; Saló al., 2006). To study the origin and function of the germ cell lineage, the and Baguñá, 2002; Agata, 2003; Reddien and Sanchez, 2004). Second, DEAD-box family gene vasa is best characterized as a germ-cell irradiation experiments revealed neoblasts as a source for tissue specific marker throughout the animal kingdom (Linder et al., 1989; regeneration (Lender, 1962; Wolff, 1962; Reddien and Sanchez, 2004). Raz, 2000; Extavour and Akam, 2003). Vasa is an ATP-dependent RNA Notably, injection of enriched fractions of neoblasts into lethally helicase that exhibits eight characteristic sequence motifs including a irradiated hosts resulted in the rescue and long-term survival of the DEAD-box (Asp–Glu–Ala–Asp). DEAD-box proteins are found in other recipient together with the restoration of the regenerative ability invertebrates and vertebrates and play a role in the spliceosome, ribonucleoprotein rearrangement, and RNA processing (Luking et al., 1998; Linder, 2000). The vasa gene is highly conserved in all animals studied including Cnidaria (Mochizuki et al., 2001; Extavour et al., ⁎ Corresponding authors. Fax: +43 512 507 2930. E-mail addresses: Daniela.Pfi[email protected] (D. Pfister), [email protected] 2005), Planaria (Shibata et al., 1999; Mochizuki et al., 2001), Caenor- (P. Ladurner). habditis elegans (Gruidl et al., 1996; Kuznicki et al., 2000), the

Please cite this article as: Pﬁster, D. et al., Flatworm stem cells and the germ line: Developmental and evolutionary implications of macvasa expression in Macrostomum lignano, Dev. Biol. (2008), doi:10.1016/j.ydbio.2008.02.045 ARTICLE IN PRESS

2 D. Pfister et al. / Developmental Biology xxx (2008) xxx–xxx amphipod Parhyale hawaiensis (Extavour et al., 2005), the polychaete Cloning of macvasa Platynereis dumerilii (Rebscher et al., 2007), Drosophila (Sano et al., fi A 403 bp fragment of M. lignano vasa gene was isolated by degenerate PCR with 2002), Xenopus (Ikenishi and Tanaka, 2000), zebra sh (Yoon et al., primers corresponding to aa sequences MACAQTG and DRMLDMGF, using cDNA made 1997; Krovel and Olsen, 2002; Wolke et al., 2002), mouse (Fujiwara et from total RNA. Primer sequences were 5′-ATGGCNTGYGCNCARACNG-3′ and 5′- al., 1994; Tanaka et al., 2000; Toyooka et al., 2000), and human AANCCCCATRTCNARCATNCKRTC-3′. Resulting PCR fragments were sub-cloned in (Castrillon et al., 2000). In the animal kingdom, vasa-like genes are pGEM®-T (Promega) and sequenced by MWG (Germany). The 3′-end was obtained by RACE-PCR with a M. lignano EST cDNA library (Morris et al., 2006). Primers were 5′- present in numbers from one to four (Sano et al., 2002; Rebscher et al., GAGCTGGACACCAACGATTC-3′,5′-ACAGGCTCTGGCAAGACTG-3′ and 5′-CCAAA- 2007; Kuznicki et al., 2000; Extavour et al., 2005; Shibata et al., 1999). CAGGCTCTGGCAAG-3′. The resulting fragment was 1444 bp long. 5′RACE experiments In all organisms cited above, vasa seems to be a specific germ cell were performed with cDNA from isolated mRNA (Smart RACE amplification kit, BD marker and allowed researchers to follow germ cell formation during Biosciences Clontech). Primers were 5′-GCCGAGAACATCACAATCTGC-3′,5′- ′ ′ ′ early development. Notably expression of DjvlgA in the planarian GAGGCGGGTCTTGTTCAGACT-3 and 5 -AATGTTCCTCTCGGCGAAGTC-3 and yielded a 1972 bp fragment and a second 5′ fragment (1159 bp) that is considered to be a splicing Dugesia japonica was also present in somatic stem cells of an asexual variant. Sequences were conceptually spliced together, resulting in full-length 2875 bp strain (Shibata et al., 1999). The authors of this work suggested that macvasa gene nucleotide sequence with an open reading frame of 2583 bp. Sequencing DjvlgA plays a role in neoblast maintenance and differentiation. GFP- was done by MWG or GATC (Germany) or with a 16-capillary sequenator from Applied vasa transgenic lines have been used to isolate germ line cells from Biosystems (ABI 3100) at the Division of Genomics and RNomics, Innsbruck Medical University. Sequences were analyzed by BLASTX searches of the GenBank database Drosophila (Shigenobu et al., 2006) and from rainbow trout (Takeuchi (http://www.ncbi.nlm.nih.gov/blast/). Amino acid and nucleotide alignments were et al., 2002). A nontransgenic GFP primordial germ cell labelling generated with Multiple Sequence Alignment Program CLUSTALW (http://align. method was applied to visualize germ cells in salmonid fish (Yoshizaki genome.jp/) using default alignment parameters. Alignments were hand edited for et al., 2005). In a broad variety of species, Vasa has been shown to be obvious alignment errors and used to calculate a phylogenetic tree (neighbor-joining, present in the perinuclear germ line specific organelle called nuage bootstrap 1000 fold) with TreePuzzle 5.2. (Ikenishi, 1998; Shibata et al., 1999; Knaut et al., 2000; Carre et al., Whole mount in situ hybridization 2002; Findley et al., 2003; Bilinski et al., 2004; Parvinen, 2005; Johnstone et al., 2005) — an evolutionarily conserved structure of Primers used for generation of PCR template for in situ riboprobe were 5′- unknown function. In flatworms a chromatoid body – a structure CCCGGTCGCTCTTATACTGACTCC-3′, corresponding to aa sequence PVALILT, and 5′- similar to nuage – was found in stem cells (Coward, 1974; Hori, 1982; GGTTGGCACGGAACATCCTCTC-3′, corresponding to aa sequence ERMFRAN. PCR Shibata et al., 1999; Salvenmoser, pers. comm.). Chromatoid bodies conditions were 3 min at 94 °C, 35 cycles (94 °C, 57 °C, 72 °C for 1 min each), 72 °C for 7 min. A 1067 bp digoxigenin-labelled riboprobe was generated using the DIG RNA disappear during neoblast differentiation but remain in the germ line Labeling KIT SP6/T7 (Roche) and used at a working concentration of 0.075 ng/μl. Whole (Coward, 1974; Hori, 1982, 1997). mount in situ hybridization was performed using a newly developed protocol for M. Two aspects of the germ line are currently extensively studied e.g. lignano (Pfister et al., 2007) based on a modified in situ protocol for Hydra (Grens et al., in Drosophila. First, the ontogenetic formation of germ cells, based on 1995, 1996). Color development was performed with a NBT/BCIP system (Roth) under visual control. Developing time was 10 min at 37 °C and 8 min at room temperature. cellularization of germ plasm and the corresponding molecular Stainings were analyzed and photographed using DIC optics on a Leica DM5000 pathways (Campos-Ortega and Hartenstein, 1997; Mahowald, 2001). microscope and a Pixera Penguin 600CL digital camera. Pictures were assembled and Second, the Drosophila germ line as a model for studying the biology edited with Adobe® Photoshop® 7.0 Software. of adult stem cells in vivo (reviewed in Fuller and Spradling, 2007). Both questions can be addressed in flatworms, taking advantage of the Immunocytochemistry epigenetic formation of the germ line during regeneration (reviewed Polyclonal Macvasa antibody was produced at Sanova Medical Systems (Vienna, in Saló and Baguñá, 2002; Reddien and Sanchez, 2004; Agata, 2003, Austria) using two selected peptides for rabbit immunization. Amino acid sequences 2006; Egger et al., 2006, 2007; Saló, 2006) and postembryonic were TTIHGDRLQREREISL (aa 696–712) and RRSVARDRFREQPDEG (aa 822–838). development from the totipotent stem cell system (Ghirardelli, 1965; Animals were incubated with primary antibody (rabbit-anti-Macvasa, 1:200 in BSA-T) Fedecka-Brunner, 1965; Baguñá et al., 1989; Zayas et al., 2005). overnight at 4 °C. Secondary antibody (FITC-swine-anti-rabbit, 1:150, DAKO) was fl used at RT in darkness for 60 min. Control experiments without primary antibody Here we describe the cloning and characterization of the atworm show unspecific background in pharyngeal glands and glands around the female Macrostomum lignano vasa-related gene macvasa and its use as a tool genital opening. Western blots with Macvasa were performed as previously described for answering questions concerning germ line formation. We show (Ladurner et al., 2005a). For BrdU labelling of S-phase cells after RNAi experiments, that macvasa mRNA and protein is expressed in male and female animals were soaked for 30 min in 5 mM BrdU (Bromodeoxyuridine, Sigma) before washing, fixation, dehydration by methanol series and storage at −20 °C. Animals gonads and in somatic stem cells in juvenile and adult worms. were thawed for in situ hybridization procedure as described above and after in situ Expression of macvasa was followed during postembryonic formation protocol, kept in PBS-T for 1 h and then treated as described in Ladurner et al. of gonads, during starvation and regeneration. We show the presence (2000), except for the use of Proteinase K. For double-labelling of juveniles with of small clusters of Macvasa-positive cells in freshly hatched worms. macvasa and BrdU, freshly hatched worms (b1 h) were pulsed for 2 h in 1 mM BrdU and fixed after 4 days chase. Animals underwent in situ treatment (see above) This suggests, in contrast to present knowledge, that the germ line μ fl with 0.25 ng/ l macvasa probe. A higher probe concentration was applied because of might be segregated earlier in atworms than generally assumed. using Fast Red (Sigma, F4648) for color development. Animals were further During regeneration, however, male and female gonads were rebuilt processed through standard BrdU staining protocol applying 0.2 mg/ml Protease from somatic stem cells. Functional knockdown by macvasa RNA XIV for 30 min (Sigma, P5147) (Ladurner et al., 2000). Mounted specimens were interference led to a depletion of macvasa mRNA and protein, but not photographed using either a Leica DM5000 or a Zeiss Axiovert LSM 510 confocal microscope. to phenotypic changes. Finally, we present a model for germ line development in flatworms and speculations on its evolutionary Regeneration, postembryonic development and starvation implications concerning preformation and epigenesis. For regeneration experiments, animals were cut at the post-pharyngeal level Materials and methods with a modified razor blade in order to remove gonads completely. Animals were left to regenerate under normal culture conditions with food. At given time points Animal culture and RNA isolation animals were relaxed, fixed and stored in 100% methanol at −20 °C as described in Pfister et al. (2007). For postembryonic development experiments, timed eggs were The animal used was the free-living flatworm M. lignano (Ladurner et al., 2005b). obtained from adult M. lignano. Eggs were monitored and young worms were Animals were kept in petri dishes with nutrient-enriched artificial seawater (Guillard's cultured until the required developmental time, fixed and stored in methanol. In f/2 medium, Anderson et al., 2005) on a layer of Nitzschia curvilineata. Climate chamber situ hybridizations and antibody stainings were performed as described above. For conditions were 20 °C, 60% humidity and a 14:10 day–night cycle (Rieger et al., 1988). starvation experiments, a sample of 260 adult worms were put in a clean glass Total RNA isolations were performed with batches of 200–500 animals with TRI® petri dish and washed once a week. At defined points in time, a batch of animals Reagent (T9424, Sigma), according to manufacturer's instructions. Animals were was removed, relaxed, fixed and stored in methanol as described in Pfister et al. starved for 2 days to prevent algae contamination. (2007).

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RNA interference designed; two of them included a T7 RNA polymerase promoter sequence. Primer sequences were: An RNA interference protocol combining injection and soaking was developed for adult M. lignano using dsRNA generated with an in vitro transcription system (T7 dsT7 1: 5′-GGATCCTAATACGACTCACTATAGGCCCGGTCGCTCTTATACTGAC-3′ RiboMax TM Express RNAi System, Promega). Four gene speciﬁc primers were dsGS 2: 5′-GGTTGGCACGGAACATCCTCTC-3′

Fig. 1. (A) Alignment of Macvasa and splice variant amino acid sequence of M. lignano with vasa genes of other species. Well-conserved helicase motifs are highlighted in orange (I– VIII), RGG repeats in green. Epitope recognition sites of Macvasa antibody are indicated with blue bars and in situ primer binding sites with black arrows. DjvlgA Dugesia japonica (AB017002) Hydra Hydra magnipapillata (AB047382), Danio Danio rerio (BAA22535), Mouse Mus musculus (Q61496), Drosophila Drosophila melanogaster (M23560). (B) Domain architecture of Macvasa. Zinc ﬁngers are indicated by yellow diamonds and helicase motifs I–VIII are shown with orange boxes.

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dsGS 3: 5′-CCCGGTCGCTCTTATACTGACTCC-3′ adults, hatchlings, juveniles and regenerates were performed in 24-well plates. Animals dsT7 4: 5′-GGATCCTAATACGACTCACTATAGGGGTTGGCACGGAACATCCTCTC-3′ were kept on algae and incubated with 250 μl dsRNA solution [3.0 μg/ml] in f/2 and antibiotics (Kanamycin and Ampicillin, 1:1000). Test group sizes were 50 animals per Macvasa dsRNA length was 1067 bp. dsRNA for negative control was produced from well. All supernatants were checked for degradation on agarose gels before and after fi fl a pGEM®-luc Vector (Promega) containing a re y luciferase sequence and was dsRNA changes twice a day. 1002 bp long. For injecting, dsRNA was diluted in PBS and for soaking in ASW or f/2 to a concentration of 3.0 μg/ml. There was no difference between the two methods for applying dsRNA (data not shown). Injected animals' test group size was 50–60 animals. Results Animals were injected on 1% agar plates in the morning for seven or 11 consecutive days with a pulled microcapillary filled with dsRNA solution. Injections were done by hand Molecular characterization of the vasa homologue macvasa under a dissecting microscope using an oil pressure injector system. The injected volume was 1.0–1.4 nl. After injection, animals were transferred to a petri dish and left We isolated a putative M. lignano vasa homologue by PCR and to recover for 4–5 h. For feeding, animals were put on algae for 1–3 h. Then animals were put in a 96-well plate for dsRNA soaking over night in darkness. Regenerates were RACE experiments. The composite sequence of the cDNA clones injected once before cutting and treated likewise. RNAi soaking experiments with reveals a 2875 bp fragment, designated macvasa (GenBank accession

Fig. 2. In situ hybridization of adult M. lignano with macvasa probe. (A) DIC picture of adult live M. lignano. Note gut ﬁlled with yellow–brown algae and eggs in dark brown. (B) In situ hybridization with macvasa antisense probe of adult animal. Signal was present in the testes (t), ovaries (ov), developing eggs (de), mature eggs (me) and in somatic stem cells (arrows). (C) In situ hybridization with macvasa sense probe. (D,E) Details of somatic stem cells (arrows) located below the epidermal layer (ep) of a subadult animal. (D) Region between the eyes and anterior tip of the left testis (E) Region between the posterior end of the left ovary and onset of the tail. (F) Enlargement of whole mount in situ staining of left testis, ovary and developing eggs (de). Note absence of signal in the center region of the testis because of sperm-ﬁlled cavity. (G) Horizontal histological section of in situ hybridization with macvasa riboprobe in the middle of the testis (t). e eyes. Anterior is to the left in panels D–G. Scale bars 100 μm in panels A–C and 20 μm in panels D–G.

D. Pfister et al. / Developmental Biology xxx (2008) xxx–xxx 5 number AM411989). It contains the complete macvasa open reading the testis and are scattered along the outer margin. The pointed frame (ORF) of 2583 bp predicting a protein of 860 amino acids extension is composed of Spermatogonia and spermatocytes I followed by 295 bp of 3′ untranslated sequence. exclusively. Spermatocytes II are scarce and located with spermatids Macvasa exhibits all 8 sequence motifs (I–VIII) found in Vasa below the Spermatogonia-spermatocyte I layer. Towards the lumen of proteins (AXXXXGKT, PTREL, GG, TPGRL, DEAD, SAT, ARGXD, the testis gradually maturing spermatozoa are present. Finally, at the HRIGRTGR). In addition, 22 RGG repeats and 3 CCHC-type zinc fingers posterior end of the testis mature sperm enter into the vas deferens. are present in the N-terminal region. We conclude that macvasa likely The ovaries are paired organs located posterior to the testes. They constitutes a ATP-dependent RNA helicase and that we have identified consist of a compact mass of oogonia and oocytes I. Oogonia are a vasa orthologue from M. lignano. An alignment of the predicted located at the anterior region of the ovary, followed by oocytes I. amino acid sequence of macvasa with vasa genes of other species is Developing eggs of gradually increasing size leave the ovaries at the shown in Fig. 1A, together with a schematic drawing of domain posterior end. The ovary is bordered by a single layer of flat tunica cells architecture (Fig. 1B). Furthermore, phylogenetic analysis of Vasa and and extra cellular matrix. the closely related PL10 family with sequences of other animals clearly Macvasa expression in testes and ovaries was located in the placed macvasa in the vasa subgroup (Supp. Figs. 1 and 2). In addition cytoplasm of gonadal cells but not in mature sperm (Figs. 2B, F, G). to macvasa we have cloned a putative splicing variant (3082 bp) that Spermatozoa completely lacked signal (Figs. 2F, G). Spermatocytes and differs mainly in RGG-repeats sequence and lacks a zinc finger in the 5′ spermatogonia showed expression in the cytoplasm. Especially region (GenBank accession number AM411990). PCR surveys with Spermatogonia at the base of the anterior pointed extension of the degenerate primers were performed to detect an additional vasa gene testes exhibited a particularly strong signal (Figs. 2F, G). In contrast, or other DEAD-box family genes. Dozens of clones were picked and macvasa expression levels always appeared higher and more homo- sequenced. We could not isolate additional vasa-related genes from genous in cells of the ovaries (Figs. 2B, F, G). Specificity of the probe M. lignano. was confirmed by processing control animals hybridized with macvasa sense probes (Fig. 2C). Expression of macvasa mRNA and protein localization in adult animals A polyclonal Macvasa antibody was produced and showed immunoreactivity in male and female gonadal tissue (Figs. 3A, B), Expression of the macvasa transcript was examined in adult M. developing eggs (Figs. 3A, C) and in a subset of somatic neoblasts on lignano (Fig. 2A) by whole mount in situ hybridization (Figs. 2B–G) and the lateral sides of the animal (Figs. 3A, D and Supp. Fig. 3). Cross- a polyclonal Macvasa specific antibody (Fig. 3). In adult M. lignano, reactivity of the antibody with proteins in the nervous system was macvasa is expressed in the testes, ovaries, developing eggs, mature detected and unspecific background from the secondary antibody is eggs, and in somatic stem cells (Figs. 2B, D, E). When processing present around the female genital opening (Fig. 3A; see also Ladurner subadult and adult animals simultaneously for in situ hybridizations et al., 2005a; Pfister et al., 2007). Western blots (Supp. Fig. 7) revealed we consistently observed reduced staining intensity in somatic stem a band at about 90 kDa that may represent the Macvasa protein cells of adults (Fig. 2B) compared to the signal levels in subadults (Fig. (expected 89.5 kDa). The polyclonal antiserum reacts with other 4E). For a better understanding of the expression patterns of macvasa, proteins and therefore we hypothesize that the staining of the nervous the morphology of the gonads is described briefly. The testes are system is caused by cross-reactivity. paired oval shaped organs with a pointed extension at the anterior Corresponding to observations with in situ hybridization, in the end. Each testis is surrounded by flattened somatic tunica cells and sperm-filled cavity in the center of the testes antibody staining was extra cellular matrix. Spermatogonia and spermatocytes I are present lacking (Fig. 3A). In Macvasa-positive cells, the protein appeared to be in two proliferating centers at the anterior and the posterior region of located in small vesicles surrounding the nucleus, particularly clearly

Fig. 3. Macvasa antibody staining of adult M. lignano. (A) Confocal projection of whole mount. Testes (t), ovaries (ov), developing eggs (de) and somatic neoblasts (arrows) show immunoreactivity. Cross-reactivity was present in the nervous system (arrowhead) and unspeciﬁc background from the secondary antibody was visible around the female genital opening (fgo). (B) Confocal section of female gonad. (C) Confocal projection of stained developing eggs. (D) Confocal section of somatic neoblasts (arrows). Note perinuclear distribution of Macvasa protein in panels C and D. tp tail plate. Scale bars 100 μm in panel A and 20 μm in panels B–D.

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Fig. 4. Macvasa expression (A–E) and protein localization (F–K) during postembryonic development in M. lignano. (A,B) Whole mount in situ hybridizations of a 1-h-old hatchling (A) and a 24-h-old juvenile (B). Macvasa signal was present in gonad anlage (ga) and in somatic stem cells (arrows). (C) Three days after hatching the gonad anlage (ga) increased in size. See also inset. (D) Macvasa signal in a 5-day-old animal. Small ovary primordia (posterior clusters) were developed and testes (anterior clusters) increased in size. (E) Testes (t) and ovaries (ov) after 7 days of postembryonic development exhibited particular strong expression of macvasa. (F) Confocal projection of Macvasa antibody staining in 1-h-old hatchling. Immunoreactivity in gonad anlage (ga) and somatic neoblasts (arrows). Cross-reactivity was present in the nervous system (arrowhead) and unspecific background from the secondary antibody was visible in glands (asterisks). (G) Confocal section of the gonad anlage of hatchling in (F). Note subcellular location of Macvasa protein in small vesicles. (H) Enlargement of gonad anlage (ga) and somatic stem cells (arrows) of a 1-h-old hatchling. (I) Three-day-old juvenile. Somatic stem cells (arrows). Inset shows developing gonad anlage (ga) in the elongation phase, containing migrating cells. (J) Fluorescence microscopy image of 5-day-old juvenile. Number of cells in the gonad anlage has increased to about 30–40 cells. Unspecific red autofluorescence from ingested feeding algae (alg). Somatic stem cells (arrows). Inset shows detail of anterior cluster of gonad anlage. (K) Confocal projection of 7-day-old subadult with protein localization in developing testes and ovaries. Inset shows detail of anterior pointed extension of the left testis. Note unstained sperm-filled cavity of the right testis. Scale bars 100 μm in panels A–E and panels I–K, 50 μm in panel F, 30 μm in panel H, 10 μm in panel G and insets.

visible in developing eggs (Fig. 3C) and somatic neoblasts (Fig. 3D). gonads of 5-day-old juveniles (Figs. 4D, J). After 7 days, subadult The pattern of Macvasa antibody staining corresponds to the nuage/ animals showed macvasa expression and protein localization in chromatoid body-like structures observed by electron microscopy in almost mature testes and ovaries (Figs. 4E, K). M. lignano stem cells and germ cells (Salvenmoser, unpub.). We conclude that macvasa can be considered as germ line- and stem cell Expression of macvasa mRNA and protein localization during speciﬁc based on in situ hybridization and antibody staining. regeneration

Expression of macvasa mRNA and protein localization during We performed regeneration experiments in M. lignano by cutting post-embryonic development animals directly posterior to the pharynx, resulting in regenerates completely lacking any gonad tissue. We could demonstrate the Macvasa expression and protein localization was followed from reformation of gonads from stem cells by in situ hybridization and 1-h-old hatchlings to adult animals by whole mount in situ immunocytochemistry after different days of regeneration conﬁrm- hybridization (Figs. 4A–E) and Macvasa antibody stainings (Figs. ing epigenetic mechanisms of germ line formation (compare Fig. 8). 4F–K). In freshly hatched worms macvasa mRNA and protein was Shortly after cutting, no macvasa mRNA expression could be present in two small clusters posterior to the pharynx (gonad detected in regenerating heads (Fig. 5A) and few somatic neoblasts anlage) and in numerous somatic stem cells along the lateral sides remained stained after amputation (Fig. 5I). In such head stumps, no of the animal (Figs. 4A, F, G, H). An average of six Macvasa-positive more than a total of about 160 neoblasts were present (Egger et al., cells were present in the gonad anlage (Fig. 4G) and this number 2006), of which only a fraction was Macvasa positive (Fig. 5I). One- remained unchanged until 24 h (Figs. 4A, B, F, G, H). As deve- and two-day regenerates showed macvasa mRNA in the region lopment proceeded, expression and protein localization remained posterior to the pharynx, most prominently in the blastema (Figs. high in somatic stem cells and expanded within the prospective 5B, C). The number of antibody stained cells increased especially in male and female gonad region (Figs. 4C, D, I, J). In young juveniles, the blastema region (Figs. 5J, K, L). From day 3 or 4 on, two distinct Macvasa labelled cells formed elongated clusters of migrating cells macvasa expressing clusters of cells were present at the lateral sides (Fig. 4I; see also Fig. 8). The number of cells in the gonad anlage of the animals (Figs. 5D, E). In contrast, Macvasa protein seemed not increased during the migration phase to about 30–40 cells in the to be present in clusters at day 3 (Fig. 5L) but small aggregations of

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Fig. 5. Macvasa expression and protein localization in regenerating M. lignano after cutting at the post-pharyngeal level (indicated by dotted lines). Gonads were completely removed. Arrows indicate somatic stem cells in all panels. (A) Regenerating head shortly after cutting. No speciﬁc signal present. (B and C) 1- and 2-day regenerates with macvasa signal in the blastema. (D–F) Distinct macvasa expressing cell clusters at the lateral sides (arrowheads). (G) Testes were rebuilt before the ovaries in a 6-day regenerate. The left ovary showed macvasa signal, whereas the right ovary was not yet formed. (H) Seven days regenerate with macvasa expression in both regenerated gonads. (I) Macvasa antibody staining in freshly cut head. (J,K,L) Macvasa immunoreactivity in blastema and somatic stem cells. (M) and (N) show 4- and 5-day regenerates with Macvasa stained somatic stem cells and developing gonads (arrowheads). (O) Seven-day regenerate. Note lower signal intensity in somatic stem cells compared to earlier stages. (P) Fully regenerated animal. Note sperm- ﬁlled cavity in the testes was not stained. e eyes, ov ovaries, t testes, tp tail plate. Scale bars 50 μm except for 100 μm in panels G, H, O, P.

cells became apparent after 4 days of regeneration (Fig. 5M), The effect of macvasa RNA interference during development and suggesting a delay in transcription of protein. Five days after cutting, regeneration macvasa expressing clusters increased in size (Fig. 5F), and Macvasa protein became present in the developing gonad (Fig. 5N). The In order to elucidate the function of macvasa,RNAinterference number of Macvasa labelled somatic stem cells remained high at methods by injecting and/or soaking of macvasa dsRNA were established these stages of regeneration (Figs. 5L–N), compared to the number for M. lignano. We have treated adult animals, hatchlings and in completely regenerated animals (Fig. 5P). We consistently regenerating animals up to 21 days with dsRNA, which was designed observed the rebuilding of the testes before the ovaries (Fig. 5G). to knock down both — macvasa and the splice variant. Control Seven days after regeneration both, testes and ovaries expressed experiments with luciferase dsRNA were simultaneously performed. macvasa mRNA and showed immunoreactivity (Figs. 5H,O). Full For all stages and time points, the impact of macvasa RNAi on adult restoration of functional gonads was evident after 14 days of animals, during postembryonic development and regeneration was regeneration (Fig. 5P). We observed that the time of regeneration investigated using various approaches: (1) macvasa gene expression greatly depended on the cutting level. If amputations occurred very by in situ hybridization, (2) Macvasa protein localization with a close to the pharynx the time of regeneration increased significantly. specific Macvasa antibody, (3) cell proliferation by BrdU labelling, (4) A detailed analysis of M. lignano regeneration times and regenera- progression of gonad development with a spermatid-specific mono- tion potential is found in Egger et al. (2006). clonal antibody (MSp1) (Ladurner et al., 2005a,b) and propidium

8 D. Pﬁster et al. / Developmental Biology xxx (2008) xxx–xxx iodide stainings, (5) interference contrast images of selected stages for In summary, macvasa RNAi treatment led to the knock-down of analysis of variations in size, gonad development, tissue architecture macvasa mRNA (Fig. 6A) and protein in adult animals (Figs. 6C, D), and reproductive organs. regenerates (Figs. 6G, H) and during postembryonic development (Figs.

Fig. 6. RNA interference with macvasa in M. lignano. Effects on mRNA expression (A,B), protein localization (C,D), BrdU incorporation (E,F), during regeneration (G,H,K,M) and during postembryonic development (I,J,L,N). (A) Knock-down of macvasa expression after 7 days of dsRNA treatment. (B) luciferase dsRNA treated animal for mock control showing normal macvasa signal. (C) Confocal projection of antibody staining after 11 days of macvasa RNAi. No protein present in the gonads (left gonads outlined) except for a single Macvasa positive cell (arrow and inset). (D) Confocal projection of antibody staining of luciferase control animal. (E,F) BrdU staining of S-phase cells after 11 days of macvasa RNAi treatment (E) and untreated control (F). Note similar number and distribution of S-phase cells. In these experiments animals were processed for BrdU staining after in situ hybridization (see Supp. Fig. 5). (G) Confocal projection of antibody staining after 6 days of macvasa RNAi. Regenerating gonads (t,ov) were present (see also detail in panel K) but not Macvasa-positive, except for two single cells (arrows). Inset shows magniﬁcation of right cell. Somatic stem cells were Macvasa-negative. Cross-reactivity was present in the nervous system. (H) Confocal projection of antibody staining of luciferase control animal. Somatic stem cells (arrows). For detail of nuclei of regenerating gonad see (M). (I) Animal after 6 days of macvasa RNAi treatment. Gonads (t, ov) do not show immunoreactivity, except for four single cells (arrows). Right cell is shown in the inset. Somatic stem cells were Macvasa-negative. Cross-reactivity was present in the nervous system. (J) Luciferase treated control animal 6 days after hatching with immunostaining in the testes (t), ovaries (ov) and somatic neoblasts (arrows). (K,M) Gonad regeneration after 6 days: Nuclei staining with Propidium-Iodide of testes (t) and ovaries (ov) in macvasa-RNAi treated animal (K) and luciferase-treated control (M). Detail of panel M shows right gonads in panel H. (L,N) Gonads after 6 days of postembryonic development: Propidium-Iodide staining of nuclei of gonads of macvasa-RNAi treated animal (L) and luciferase-treated control (N). arrowheads nuclei of epidermal layer, de developing egg, e eyes, me mature egg, ov ovaries, t testes, tp tail plate. Scale bars 100 μm and 50 μm for K–N.

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6I, J). No obvious effect on S-phase cell number and distribution was group of hatchlings was compared to a non-presoaked group that was observed after RNAi (Figs. 6E, F; Supp. Fig. 5). Propidum iodide stainings, obtained from non-treated adult animals. The difference between immunocytochemistry with MSp1 antibody and interference contrast “pre-soak” and “non pre-soak” groups is apparent concerning residual microscopy did not reveal morphological changes in the phenotype, cells after RNAi-treatment (Supp. Fig. 6). The number of animals gonad development (Figs. 6K–N), cell differentiation or reproduction. displaying residual cells in the presoaked group was low, while most However, single residual macvasa-positive cells remained in adult of the juveniles in the non-presoaked group had residual cells after animals at the tip of the testes or within the developing ovaries (Fig. 6C). RNAi. Residual cells disappeared gradually with prolonged incubation After 21 days of RNAi, a group of animals was transferred to normal (Supp. Fig. 6). culture medium and analysis by antibody staining revealed the complete recovery of Macvasa protein (data not shown). Expression of macvasa during starvation All luciferase-treated control groups showed normal macvasa mRNA expression and protein localization pattern in the gonads, A total of 260 animals were starved for 8, 16, 22, 31, and 72 days, eggs and in somatic stem cells (Figs. 6B, D). We could not see any respectively, and processed for in situ hybridization. A control group of unspeciﬁc effect of mock control luciferase, PBS or FastGreen animals was not starved and showed normal macvasa expression (Fig. injections, when compared to non-treated animals (Supp. Fig. 4). 7A). After 8 days of starvation, macvasa signal was still present in In one set of RNAi experiments, hatchlings were obtained from testes and ovaries (Fig. 7B). After 16 days, animals showed consider- adult animals that were pre-soaked in dsRNA for 6 days. The able reduction of the in situ signal and only remnants of eggs were hatchlings of the pre-soaked parents were then continuously present (Fig. 7C). After 31 days, animals had shrunk and lacked incubated up to 15 days; this group was called “pre-soak”. This morphologically visible gonads (Fig. 7D). Likewise, macvasa signal was reduced to almost background levels. After 72 days of starvation, animals had completely degraded gonads (Fig. 7E) and had shrunk signiﬁcantly to 68% of the initial body size, resembling a hatchling with a high head-to-body size ratio and big eyes. When 72 days starved animals were fed for 41 days, they entirely restored all morphological characters of adult animals including the gonads and showed normal macvasa expression (Fig. 7F).

Somatic stem cells do not contribute to developing gonads

It is generally assumed that germ cells are derived from somatic stem cells postembryonically (Fig. 8A). We wanted to know if the gonad anlage of a freshly hatched worm proliferates and gives rise to the developing gonad with or without the contribution of somatic neoblasts. Initially, in freshly hatched worms the 4–6 macvasa labelled cells of the gonad anlage do not proliferate and the cluster remains small until day 3 posthatching (compare Figs. 4A, C, F, I) while on day 4, the gonad anlage elongates (Fig. 4I) and the cells migrate to the future prospective male and female gonad region. Therefore we labelled a freshly hatched worm for 2 h with BrdU and performed BrdU/macvasa double staining after 4 days chase period. If somatic stem cells participated in the formation of germ cells one would expect to ﬁnd BrdU/macvasa double labelled cells. Our observations, however, did not reveal double stained cells (Figs. 8D– I) suggesting the proliferation of the gonad anlage after the Brdu pulse, and that the formation of the developing gonad occurred without the contribution of somatic BrdU labelled neoblasts.

Discussion

Sequence analysis of the vasa homologue macvasa

Sequence analysis of macvasa indicates that it is a true vasa family member. Macvasa contains all eight characteristic amino acid motifs within the helicase domain, including the DEAD-box (Asp–Glu–Ala– Asp) motif (Fujiwara et al., 1994; Linder et al., 1989) and the well- conserved tryptophan (W) residues near the start methionine and the

stop codon. Moreover, the NH2-terminal glycine-rich region is present, containing 22 RGG repeats and three repeats of the zinc finger motif CCHC. The RGG repeats are known to be required for the subcellular localization of Vasa protein in nuage in Drosophila and zebrafish Fig. 7. Starvation experiments in M. lignano. In situ hybridizations were performed with (Liang et al., 1994; Wolke et al., 2002) and the zinc fingers are a DNA macvasa probe at day 0 (A), 8 days (B), 16 days (C), 31 days (D) and 72 days (E) of binding motif known to be present also in vasa homologs of other starvation. Animals have shrunk during starvation period and testes (t) and ovaries (ov) animals such as tunicates (Sunanaga et al., 2006)orPlatynereis were degraded. Macvasa signal decreased to background level after about 4 weeks. No dumerlii (Rebscher et al., 2007). Together with the repetitive RGG maturation of eggs occurred after 2 weeks of food deprivation. (F) Specimen refed for 41 days. Gonads were regrown and showed normal macvasa expression. e eyes, eg egg. motifs these are only found in vasa orthologs but not in the related Scale bar 100 μm. gene family PL10 (Rebscher et al., 2007).

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We found a putative splicing variant of macvasa that differs mainly postembryonic development, these data are not consistent with in the number of RGG-repeats sequence in the 5′ region. Multiple RGG findings in other flatworms (discussed below). boxes from duplications are reported for the vasa locus in zebrafish For regeneration experiments in M. lignano, we cut the animals at (Bartfai and Orban, 2003), as well as a sexually dimorphic expression the post-pharyngeal level, in order to remove gonads completely. The pattern of a splice variant of zebrafish vasa during gonadal develop- germ cells had to be rebuilt from the remaining stem cells. Animals ment (Krovel and Olsen, 2004). Also in Tilapia (Oreochromis niloticus), treated in this way have functional gonads and produce fertile a teleost fish (Kobayashi et al., 2002) vas-s and in Tribolium, Tc-vasa offspring (see also Egger et al., 2006). In regenerating D. japonica,an are thought to be one of many vasa splice variants. accumulation of one of the two vas-related genes, DjvlgA,was In the animal kingdom, vasa-like genes are found in numbers of observed in the growing blastema after 2 days (Shibata et al., 1999). four, two and one. C. elegans features four vasa transcripts (Kuznicki This is consistent with our findings in M. lignano where 1-day and 2- et al., 2000). In the planarian D. japonica (Shibata et al., 1999) and in day regenerates show macvasa signal in the posterior half of the the sea anemone Nematostella vectensis (Extavour et al., 2005)two animal. With progression of regeneration, germ cells single out and vasa transcripts are present. In P. dumerilii (Rebscher et al., 2007), give rise to testes and ovaries. This finding for M. lignano is comparable Drosophila (Sano et al., 2002), Xenopus (Ikenishi and Tanaka, 2000), to planarian regeneration (Agata, 2003; Reddien and Sanchez, 2004). zebrafish (Yoon et al., 1997), mouse (Fujiwara et al., 1994) or human (Castrillon et al., 2000), only one vasa transcript has been found. In macvasa RNA interference in M. lignano M. lignano we performed extensive PCR surveys with degenerate primers and searched 13.000 EST sequences. Nevertheless, we could RNA interference is a new method for M. lignano.RNAiasa not isolate additional vasa-related genes from M. lignano.We sequence-specific gene-silencing phenomenon was demonstrated by therefore suggest the possibility of the presence of only one vasa- Fire et al. (1998) in C. elegans and has been used in Platyhelminthes gene in M. lignano. since 1999 (Sánchez Alvarado and Newmark, 1999; Cebria et al., 2002; Bermange et al., 2003; Orii et al., 2003; Reddien et al., 2003; Mannini macvasa mRNA and protein localization et al., 2004; Reddien et al., 2005b; Salvetti et al., 2005; Guo et al., 2006; Cebria et al., 2007; Takano et al., 2007; Wang et al., 2007; Cebria The conserved vasa-family genes are expressed almost exclusively and Newmark, 2007; Orii and Watanabe, 2007). in germ line cells throughout all metazoans examined (Extavour and Our results with M. lignano demonstrate that dsRNA treatment – Akam, 2003). In M. lignano, the mRNA and protein of macvasa was either by injection and soaking or by soaking only – is an effective detected in both gonads, in developing and mature eggs and in a method for knocking down macvasa gene expression and disrupting subset of somatic stem cells. Cross-reactivity of the polyclonal protein production in M. lignano. The effects of RNAi with macvasa antibody seen in the nervous system could be explained by the were studied in adult animals, during postembryonic development antiserum recognizing other proteins in the Western Blot. and regeneration. Adult worms showed normal gonad morphology Shibata et al. (1999) showed that one of the two vasa-related genes and produced sperm, eggs and fertile offspring. Our experiments in in D. japonica, DjvlgA, was not expressed in germ cells only, but also in adults, during postembryonic development and regeneration indicate putative somatic stem cells in the mesenchyme. DjvlgA, however, was that macvasa is not required for stem cell maintenance, neoblast placed in the PL10 subgroup in later studies (e.g. Mochizuki et al., proliferation, gonad formation and gonad maintenance. We assume 2001; Extavour et al., 2005). that macvasa function might be partially redundant with other genes. The subcellular localization of Macvasa protein appears to be in Moreover, vasa gene function has not been shown to be sufficient for small vesicles, which is particularly clearly visible in developing eggs germ cell specification or development in other organisms studied and somatic neoblasts. A similar perinuclear distribution of vasa (e.g. Braat et al., 2001). Furthermore, vasa genes can operate during protein in small vesicles was also observed in zebrafish (Braat et al., certain developmental stages and therefore loss of function does not 2000; Knaut et al., 2000), in the scorpion-fly Panorpa communis show a phenotype at all times (Styhler et al., 1998). We want to state (Batalova and Parfenov, 2003), in the amphipod P. hawaiensis that we obtained clear phenotypes using the soaking method with (Extavour, 2005) and in nuage material in P. dumerlii (Rebscher et other genes (e.g. piwi, De Mulder, unpub.; actin,Pfister, unpub., sxl, al., 2007) — this latter observation parallels Macvasa protein in M. Sekii, unpub.). lignano. Nuage-like structures in M. lignano (Salvenmoser, unpub.) are The presence of single Macvasa-positive cells (residual cells) as morphologically different to chromatoid bodies as seen in planarians. seen in non-presoaked juveniles after RNAi treatment, might be due to In the teleost fish Carassius auratus gibelio the CagVasa protein was the extreme longevity of Macvasa protein. However, an alternative observed to be strongly expressed in spermatogonia, at reduced levels explanation of the “RNAi resistance” of these cells would be that they in spermatocytes and spermatids and absent in sperm (Xu et al., show an initial burst of mRNA expression – and as a consequence a 2005). In contrast, in M. lignano the intensity of Macvasa antibody high protein level – before they are silenced (C. Mello, pers. comm.). staining seems to be comparable in spermatogonia and spermatocytes This transient expression would mean that these cells show a lag and spermatids, but is also absent in mature sperm. response time to RNAi due to an unknown mechanism. We tested macvasa riboprobe as a molecular marker during post- RNA interference in M. lignano could elucidate processes of the embryonic development in M. lignano. Most strikingly, Macvasa signal neoblast system involved in stem cell function, regeneration or tissue was present in the gonad anlage right after hatching. As platyhelminth homeostasis. Moreover, RNAi experiments for inhibiting gene function gonad cells are thought to be derived from totipotent stem cells during in M. lignano will be possible on a large scale high throughput basis, as

Fig. 8. Concepts of germ line specification during development and regeneration in flatworms. Neoblasts (blue), primordial germ cells (black), male committed germ cells (red with black rim), female committed germ cells (green with black rim), male germ cells (red); female germ cells (green). Inductive signal for germ line specification (black filled arrow), inductive signal for male gonad specification (red arrow), inductive signal for female gonad specification (green arrow). (A) For planarian species it is generally assumed that neoblasts develop into germ line precursors that give rise to male and female gonads directed by inductive signals during both, postembryonic development and regeneration. Germ cells are specified in a similar way during regeneration in Macrostomum (B). (C) In contrast, in Macrostomum the germ line is specified during embryogenesis followed by postembryonic proliferation, migration and commitment of male and female gonads. (D–F) Confocal projections of 4-day-old juvenile labelled with BrdU (pulse-chase). Three different confocal planes are shown: Dorsal side of the animal (D) 3.68 μm projection, middle region (E), where the gonad anlage is located (9.2 μm projection) and ventral side (F) (9.2 μm projection). Arrowheads point out the level of the eyes. Remnants of the most ventrally located gonad anlage (red) visible. (G–I) Detail of the same juvenile stained for macvasa by in situ hybridization (G) and BrdU (H). Note the absence of double stained cells in the developing gonads (I). Arrow indicates one BrdU labelled cell that is not macvasa stained, most likely representing a somatic envelope cell of the gonad anlage. Anterior to the left; ga gonad anlage.

12 D. Pfister et al. / Developmental Biology xxx (2008) xxx–xxx the soaking approach allows processing of large numbers of animals induced at later stages by inductive signals from neighboring tissues and many genes in RNAi screens. (epigenesis) (reviewed in Extavour and Akam, 2003). The first mechanism (preformation) is found in the model organisms Droso- Effects of starvation on macvasa expression phila melanogaster (Williamson and Lehmann, 1996), C. elegans (Kimble and White, 1981) and Danio rerio (Yoon et al., 1997). The Starvation of M. lignano leads to a successive decrease in body size, second mechanism (epigenesis) is reported from mice (Lawson and the reduction of gonads and a decline in the number of mitoses Hage, 1994; Tam and Zhou, 1996), axolotls (Nieuwkoop, 1947) and also (Nimeth et al., 2004). This is a similar response to starvation as found from the planarian S. mediterranea (Baguñá et al., 1989; Zayas et al., in the planarians Girardia (Dugesia) tigrina (Baguñá and Romero, 1981) 2005). Basal metazoans all derive their germ line from epigenetic and Schmidtea (Dugesia) mediterranea (Baguñá, 1976). In these animals mechanisms, from endodermal or mesenchymal cells (reviewed in the cell size remains constant during starvation (Baguñá and Romero, Extavour et al., 2005; Extavour, 2007a,b). Our data from macvasa 1981; Oviedo et al., 2003), suggesting that starving animals reduce mRNA and protein localization in hatchlings led to the speculation their number of neoblasts. With M. lignano, we followed the that the germ line in M. lignano might be separated during embryonic expression of macvasa over 72 days of starvation and after refeeding development by preformation. Recent data on Smednos in S. for 41 days. The results showed that macvasa expression subsequently mediterranea embryos (Handberg-Thorsager and Saló, 2007) indicate decreases. After 72 days of starvation, animals show no signal and a similar mechanism. Also studies of M. lignano embryonic develop- have extremely shrunken in size. The expression of macvasa in the ment (Morris et al., 2004) have not described a separation of germ line germ cells is highly affected by feeding conditions and correlates with during embryogenesis. However, to answer the question when exactly the decrease and regrowth of the gonads. These data are consistent the segregation happens, embryonic data is needed to support these with the high flexibility of the neoblast system concerning feeding assumptions. conditions of M. lignano (Nimeth et al., 2004). Acknowledgments Germ cell development We gratefully acknowledge the following people: R. Weiler-Görz More than hundred years of research on the development and for help with cloning, D. Jacobs (UCLA) for lab space, R. Rieger for regeneration of various flatworm species has led to the suggestion comments, W. Salvenmoser for histology. This study was supported by that the germ line is formed from neoblasts during postembryonic FWF grant P18099 (Austria) and TWF grant UNI-0404/87 to P.L. An development (Gremigni, 1974, 1983; Dubois, 1949, Fedecka-Brunner, FWO grant (Belgium) supported K.D.M and an APART-fellowship 1965, 1967; Kobayashi and Hoshi, 2002; Sánchez Alvarado, 2003; 10841 (Austria) P.L. Sánchez Alvarado and Kang, 2005; Fig. 8A). First, to our knowledge there is hardly any description of an early Appendix A. Supplementary data segregation of the germ line during embryonic development of flatworm species studies so far (for Melander, 1963, see discussion in Supplementary data associated with this article can be found, in Gremigni, 1974). Second, a large body of regeneration experiments the online version, at doi:10.1016/j.ydbio.2008.02.045. (reviewed in Agata, 2003, 2006; Egger et al., 2006, 2007; Saló and Baguñá, 2002; Saló, 2006; Reddien and Sanchez, 2004) in a broad References variety of flatworm species allows the conclusion that germ cells are formed from neoblasts during regeneration. Third, recent advance- Agata, K., 2003. Regeneration and gene regulation in planarians. Curr. Opin. Genet. Dev. 13, 492–496. ments using molecular markers support the concept of a postem- Agata, K., Nakajima, E., Funayama, N., Shibata, N., Saito, Y., Umesono, Y., 2006. Two bryonic specification of germ cells in planaria (Ogawa et al., 1998; different evolutionary origins of stem cell systems and their molecular basis. Semin. Zayas et al., 2005). Notably the dedicated germ cell marker Smed- Cell Dev. Biol. 17 (4), 503–509. — nanos has not been observed in very young hatchlings of S. Anderson, R.A., Berges, R.A., Harrison, P.J., Watanabe, M.M., 2005. Appendix A Recipes for Freshwater and Seawater Media; Enriched Natural Seawater Media. In: mediterranea (Wang et al., 2007). Similarly, Djnos appears for the Anderson, R.A. (Ed.), Algal Culturing Techniques. Elsevier Academic Press, first time in juveniles of D. japonica (Sato et al., 2006). On the contrary, Burlington, USA, p. 507. Smednos was detected in stage 8 embryos and newly hatched Baguñá, J., 1976. Mitosis in the intact and regenerating planarian Dugesia mediterranea n. sp. I. Mitotic studies during growth, feeding and starvation. J. Exp. Biol. 195, juveniles (Handberg-Thorsager and Saló, 2007). 53–64. During regeneration we find – in accordance with the germ cell Baguñá, J., Romero, R., 1981. Quantitative analysis of cell types during growth, degrowth specification in planarians – that neoblasts give rise to the germ line in and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia 84, 181–194. Macrostomum (Fig. 8B). Baguñá, J., Saló, E., Auladell, C., 1989. Regeneration and pattern formation in planarians. 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