Vasa Unveils a Common Origin of Germ Cells and of Somatic Stem Cells from the Posterior Growth Zone in the Polychaete Platynereis Dumerilii ⁎ N

Developmental Biology 306 (2007) 599–611 www.elsevier.com/locate/ydbio

Vasa unveils a common origin of germ cells and of somatic stem cells from the posterior growth zone in the polychaete Platynereis dumerilii ⁎ N. Rebscher a, ,1, F. Zelada-González b,1, T.U. Banisch a, F. Raible c, D. Arendt c

a Institute of Zoology, Philipps University Marburg, Karl von Frisch Strasse 8, 35032 Marburg, Germany b CIML, Marseille, France c EMBL, Heidelberg, Germany Received for publication 27 February 2006; revised 27 March 2007; accepted 27 March 2007 Available online 1 April 2007

Abstract

To elucidate the evolution of germ cell specification in Metazoa, recent comparative studies focus on ancestral animal groups. Here, we followed the germline throughout the life cycle of the polychaete annelid Platynereis dumerilii, by examining mRNA and protein expression of vasa and other germline-specific factors in combination with lineage tracing experiments. In the fertilised egg, maternal Vasa protein localises to the yolk-free cytoplasm at the animal pole. It then asymmetrically segregates first into the micromeres, then into the founder cells of the mesodermal posterior growth zone (MPGZ). Vasa transcripts initially show ubiquitous distribution, but then become progressively restricted to the MPGZ. The cells of the MPGZ are highly proliferative, as evidenced by BrdU pulse labelling experiments. Besides vasa, they express nanos along with the stem cell- specific genes piwi, and PL10. At 4 days of development, four primordial germ cells are singled out from within the MPGZ, and migrate into the anterior segments to colonise a newly discovered ‘primary gonad’. Our data suggest a common origin of germ cells and of somatic stem cells, similar to the situation found in planarians and cnidarians, which may constitute the ancestral mode of germ cell specification in Metazoa. © 2007 Elsevier Inc. All rights reserved.

Keywords: Vasa; Primordial germ cells; Platynereis; Germline; Evolution; Stem cells

Introduction Interestingly, and despite these obvious differences in strategy, key players involved in germ cell development are evolutiona- The separation of the immortal germ cells from the somatic rily conserved. For unraveling the ancestral role of these factors tissue has been the first diversification step in metazoan evolu- we should gain a broader view of different modes of germ cell tion. How did the segregation of the germline first occur in early specification in Bilateria. Metazoans, and how did germ cell specification evolve further? Here, we investigate germ cell development in Platynereis This is an unsolved issue because two different strategies are dumerilii, a polychaete annelid. Histological studies had so far encountered in conventional animal models (Extavour and suggested a mesodermal origin of germ cells for lophotrocho- Akam, 2003). Either, the germ cells are specified by the inhe- zoan molluscs, oligochaetes and polychaetes (Bürger, 1891; ritance of maternal cytoplasmic determinants localised in the Nusbaum, 1908; Malaquin, 1924; Meyer, 1929; Lieber, 1931; oocyte (“preformation”), as it is the case in Drosophila and in Woods, 1931; Pfannenstiel and Grünig, 1982; Hanske, 1989). Caenorhabditis elegans (reviewed in Saffman and Lasko, Putative primordial germ cells (PGCs) were identified by their 1999; Wylie, 1999). Or, inductive processes select the germ oval or spindle-like shape, by granular cytoplasm with perinuc- cells from undifferentiated cells at later embryonic stages (“epi- lear ribonucleotide particles, and a by large round nucleus with genesis”), as observed in the mouse (McLaren, 2003). peripheral chromatin aggregations. These studies however did not allow tracing back the PGCs to embryonic or larval stages. ⁎ Corresponding author. Fax: +49 6421 2823407. More recently the cloning of a nanos homologue in the leech E-mail address: [email protected] (N. Rebscher). Helobdella robusta has paved the way to address the origin of 1 Authors contributed equally. the PGCs in annelids by molecular tools. Although Nanos was

0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.03.521 600 N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 proposed to play primarily a role in axis formation in this Materials and methods species (Pilon and Weisblat, 1997), in situ hybridisation revealed 11 nanos expressing paired spots in the germinal Animals plate of stage 9 and 10 embryos (Kang et al., 2002). These spots, P. dumerilii were cultured in aerated artificial seawater (ASW, Tropic Marin derived from the mesodermal lineage as judged by lineage 3.2% w/v, pH 8.2) at 18 °C as described previously (Hauenschild and Fischer, tracer experiments, were proposed to represent the PGCs. 1969). However, the future fate of the PGCs in this species could not be followed based on nanos expression. Cloning of Platynereis genes Besides Nanos, the Vasa transcript or protein, respectively is now widely used as a molecular marker for germ cells in Total RNA was extracted from 48 h post fertilisation (hpf) larvae using Trizol various organisms (Extavour and Akam, 2003; Johnson et al., Reagent (Invitrogen). First strand cDNA was synthesized with Super Script II Reverse transcriptase (Gibco). A 443 bp Pdu-vasa fragment was obtained by PCR 2003b; Wylie, 1999; Saffman and Lasko, 1999). Vasa has a using degenerated primers (5′-gayytnatggcntgygcncarac-3′), (5′-gcnacnttyccngar- general and conserved role in establishing and maintaining the garathca-3′)and(5′-cknccdatnckrtgnacrtantcrtcdat-3′)(seeFig. 1A). Amplifica- dichotomy of germline and soma in animal development tion parameters were: 94 °C for 2 min, 5 cycles of 94 °C for 1 min, 43 °C for 2 min, (Lasko and Ashburner, 1988; Roussell and Bennett, 1993; Ko- 72 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 48 °C for 2 min, 72 °C miya et al., 1994; Fujimura and Takamura, 2000; Raz, 2000; for 4 min, and finally 72 °C for 10 min. RACE PCR was performed using the specific primers (5′-gccactggggagatcgta-3′ and 5′-gcagccactgaggtagcaa-3′)ona Castrillon et al., 2000; Mochizuki et al., 2001, Shirae-Kura- 48 hpf larvae λZAP cDNA library (Stratagene). The 3′ end of the gene was bayashi et al., 2006). Vasa is an ATP-dependent RNA helicase obtained from random sequencing of a 48 hpf cDNA library (F. R., D. A., of the DEAD-box family, capable of unwinding double- unpublished data). From the same EST-sequencing project, a piwi orthologue was stranded RNA loops to promote translation of germline-specific obtained. Information on the genomic organization of Pdu-vasa was retrieved target mRNAs (Hay et al., 1988; Lasko and Ashburner, 1988; from sequencing a clone (CH305_59A6) identified from a genomic Platynereis library (CH305; BACPAC Resources Center, CHORI, http://bacpac.chori.org/). Johnstone and Lasko, 2001; Carrera et al., 2000; Raz, 2000). It Degenerated PCR for Pdu-vasa also yielded two Pdu-PL10 fragments. forms part of RNA-rich ribonucleoprotein (RNP) particles that RACE was performed using specific primers (5′-ggtggaggagcccgacaaga-3′,5′- are involved in processing, localisation, and regulation of aatgcttctggtccggattc-3′)and(5′-ccagcacgggggtctttcc-3′,5′-gggcctcctctc- germline mRNAs. By electron microscopy, these particles have gctccttc-3′). A nanos homologue was obtained using the degenerated primers “ ” (5′-tgygtnttytgymgnaayaay-3′) and (5′-ggrcartayttdatngtrtgngc-3′). The primers been identified as electron-dense germinal granules that ′ ′ ′ ′ “ for RACE were (5 -tctacacatcccacgtcctga-3 ) and (5 -tccgtgggctccgcagttt-3 ). together with mitochondria and ribosomes constitute the germ Alignments were performed with CLUSTAL-X and used to calculate a 1000-fold plasm” characteristic for germline cells (Eddy, 1975). One of bootstrapped phylogenetic tree (neighbour-joining method) in Tree-Puzzle 5.2. the few known targets of Vasa translational control is the nanos All sequences have been submitted to EMBL nucleotide database (Pdu-vasa transcript that itself encodes another translational regulator AM048812, Pdu-PL10a AM048813, Pdu-PL10b AM048814, Pdu-nanos (reviewed by Johnstone and Lasko, 2001). In Drosophila, AM076486, Pdu-piwi AM076487). Nanos suppresses somatic differentiation (Deshpande et al., Developmental RT-PCR 1999; Hayashi et al., 2004) and, by translational repression prevents premature differentiation of germline stem cells and Total RNAwas extracted with TRIZOL reagent (Invitrogen) according to the primordial germ cells into gametes. Therefore as a translational manufacturer's instructions. First strand cDNA synthesis was carried out using regulator Vasa acts (at least in part) through Nanos to establish the Revert Aid First strand cDNA synthesis kit (Fermentas). Primers used were ′ ′ ′ ′ ′ the future germ cells and to maintain them in an undifferen- 5 -tgtgtttgtgactgtcggacgag-3 and 5 -gcagccactgaggtagcaa-3 (Pdu-vasa), 5 - aatgcttctggtccggattc-3′ and 5′-gggcctcctctcgctccttc-3′ (Pdu-PL10), and 5′- tiated state. In some species, germ cells also express piwi and ggagacgatgctcccagagct-3′ and 5′-aactctcaattcattgtagaaggtgt-3′ (Pdu-actin). Am- PL10, demarcating undifferentiated cells. Piwi is involved in plification parameters were: 96 °C for 2 min, 30 cycles of 96 °C for 1 min, 60 °C asymmetric cell divisions and stem cell renewal in different for 45 s, 72 °C for 45 s, and 72 °C for 10 min. For negative controls, either species (Cox et al., 1998; Bosch, 2004; Seipel et al., 2004). reverse transcriptase or template was omitted. PL10 is, like Vasa, a DEAD box RNA helicase active in RNA Northern analysis splicing and translation by interaction with translation initiation factors (Mochizuki et al., 2001; Lüking et al., 1998). Total RNA from embryos, larvae, or worms (8 μg per lane) was subjected to We find that in the fertilised Platynereis egg maternal Vasa northern analysis according to standard procedures (Sambrook et al., 1989). protein localises to the ‘yolk-free cytoplasm’ (YFC), clear cy- Digoxygenin-labeled antisense probes (10 ng/ml) were hybridised over night at toplasm that fractionates into the cleaving micromeres (Dorres- 63 °C. Chromogenic detection was performed with alkaline phosphatase teijn, 1990). Vasa protein is then specifically enriched in the coupled anti-digoxygenin antibodies and NBT/BCIP as a substrate. founder cells of the mesodermal growth zone (MPGZ), a highly Whole mount In situ hybridisation proliferative region of undifferentiated cells. Along with the germ cell markers vasa and nanos these cells also express the Whole mount in situ hybridisation (WMISH) of larvae and worms (15 hpf stem cell genes piwi, and PL10. Germ cell specification is onward) was carried out as described previously (Loosli et al., 1998; Arendt et completed with the emigration of four PGCs from within the al., 2001), except that proteinase K treatment was reduced to 30 s (15–24 hpf), – MPGZ. Based on these findings, and in account of comparative 1 min (24 72 hpf) or 2 min (>4 dpf), respectively. For juveniles and mature worms, proteinase K treatments of 2 to 3 min gave best results. Following data from other basal phyla, we discuss a two-step model of WMISH, some of the worms were embedded in low melting agarose and germ cell specification in Platynereis involving co-specification sectioned on a VT1000S vibratome (Leica). WMISH of embryos (0 to 7 hpf) of germ cells and stem cells. was performed according to Lartillot et al. (2002). N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 601

Fig. 1. Vasa-like genes in Platynereis. (A) Multiple sequence alignment of Pdu-vasa and Pdu-PL10. Only a part of the alignment is shown. The eight conserved domains are boxed in black. Black arrows indicate the position of the degenerated primers. Specific primers are shown in red for Pdu-Vasa and blue for Pdu-PL10. Conserved amino acids are marked as ‘.’. (B) Phylogenetic tree constructed by neighbour-joining method using P68 proteins from Mus musculus (CAA46581) and Saccharomyces cerevisiae (NP014287) as outgroup. Platynereis Vasa (red, AM048812), clusters together with members of the Vasa subfamily, and the two Platynereis PL10 splicing variants a and b (blue, AM048813 and AM048814), cluster together with the PL10 subfamily. Additional proteins shown in this tree: Schistocerca gregaria Vasa (AAO15914), Drosophila melanogaster Vasa (AAF53438), Bombyx mori VLG (BAA19572), Mus musculus Vasa and PL10 (NP_034159 and NP_149068), Danio rerio Vasa and PL10 (NP_571132 and NP_571016), Hydra magnipapillata Vas1 and PL10 (BAB13307 and BAB13306), Ciona intestinalis CIDEAD1a and CIDEAD1b (BAA36711 and BAA36710), Saccharomyces cerevisiae Ded1p (NP_014847), Neurospora crassa DED1 (CAB88635) and Xenopus laevis An3 (CAA40605). (C) domain architecture of Pdu-Vasa (upper row) and Pdu-PL10 (below). Zinc fingers are indicated by grey diamonds, RGG repeats by white circles. The DEAD-box and Helicase C domain are shown with boxes. (D) Structure of the Platynereis vasa genomic locus. Dark grey: coding region, light grey: UTR.

Generation of anti-Vasa antibodies Proteins were transferred for 2 h on a PVDF membrane by semi-dry blotting at 1.5 mA/cm2. Membranes were blocked for 1 h in 2% BSA/PBT, incubated in Peptide antibodies were generated against a Vasa specific region of the primary antibodies (anti-Vasa 1:2500 or anti-Actin 1:5000) in 0.5% BSA/PBT deduced protein by Peptide Speciality Laboratories, Heidelberg. Twenty ml of over night at 4 °C and alkaline phosphatase coupled goat anti-rabbit secondary the polyclonal serum were affinity purified on a HiTrap NHS-activated HP- antibodies (1:15,000 in 0.5% BSA/PBT) for 2 h at room temperature. column (Amersham) coupled to 5 mg of the peptide (GHFSRECPNG). Chromogenic detection was performed using NBT/BCIP according standard Antibodies were desalted by ultrafiltration (Amicon) and adjusted to a procedures. concentration of 2 mg/ml. Immunohistochemistry Immunoblot Larvae and worms were fixed for 10 to 20 min in 4% PFA/2× PBT and stored Larvae or worms were sonicated in 10 volumes of ice cold TRIS 50 mM pH in methanol (MeOH) at −20 °C until use. Fixed specimens were rehydrated 7.5, PMSF 1 mM, Phenantroline 1 mM, and centrifuged 10 min at 4 °C. 5 μg stepwise (75% MeOH/PBT; 50% MeOH/2× PBT; 25% MeOH/PBT; and PBT). protein of each stage were resolved on a 12.5% SDS-polyacrylamide gel. Fertilised eggs, embryos and larvae up to 24 hpf were pre-treated with RNAse A 602 N. Rebscher et al. / Developmental Biology 306 (2007) 599–611

(0.25 μg/ml) for 1 h at 37 °C in order to break up the ribonucleotide particles. goat-anti-rabbit, Sigma, 1:250 in blocking solution) were added. After 90 min at Permeabilisation was carried out in 0.1 mg proteinase K/ml PBS for 30 s (larvae) room temperature, the samples were washed five times with PBT and mounted or 1 min (worms), respectively. Proteinase K digestion was stopped by two in DABCO-Glycerol. washes with glycine 2 mg/ml and five washes with PBT. Samples were incubated in blocking solution (5% heat inactivated sheep serum in PBT) for 1 h Cryosections before primary antibodies were added (anti-Vasa 1:250 in blocking solution). Incubation was performed for 2 h at room temperature on a rocking platform. Worms were fixed for 20 min with PFA 4%/2× PBT, incubated for 2 h in Samples were washed five times with PBT before secondary antibodies (FITC- 15% sucrose/PBS, then over night in 30% sucrose/PBS. Specimens were

Fig. 2. Pdu-vasa transcript in embryos, larvae, and young worms. (A) Pdu-vasa is uniformly distributed at the four cell stage, animal view. Dotted lines demark the blastomeres A–D. (B) At 15 hpf, Pdu-vasa is confined to the ventral plate and the mesodermal bands. Postero-ventral view, optical section. (C) Corresponding stage, schematic representation modified after (Wilson, 1892). At the vegetal pole, the thickened ectoderm of the ventral plate is overlaid by the mesodermal bands (m) which extend antero-dorsaly. The inner mass of the embryo is occupied by four yolk-rich macromeres, the entoblasts (A–D) containing lipid droplets (ld). The ciliary band of the prototroch (p) starts to form. (D) 24 hpf trochophora larva, exhibiting strong staining in the future mesodermal posterior growth zone (MPGZ, arrow). The dotted line indicates the prototroch. Dorsal view, optical section. (E) At 48 hpf, Pdu-vasa is almost exclusively localised to the MPGZ (arrow). The anlagen of the three larval segments (C, I, and II) are detectable in the trunk region. (F) Corresponding stage, schematic representation modified after (Wilson, 1892). The stomodaeum (s) has formed. The cephalic segment C will be incorporated into the head during later development. Apical to the prototroch (p), larval eyes (e) are distinguishable. The MPGZ has been pushed to the posterior by the elongation of the ventral plate. (G) 72 hpf larva showing Pdu-vasa accumulation in the MPGZ (arrow). (H) Pdu-vasa is expressed in the MPGZ (arrow) of this 5 dpf worm. Chaetiferous segments are numbered C, I and II. (I) Corresponding stage, schematic representation modified after (Wilson, 1892). The stomodaeum (s) has fused with the lipid droplet (ld) containing entoblasts to form the digestive tract (g). Adult eyes (e) appear apical to the prototroch (p). The anlagen of the segments C, I and II have evaginated and the setae become visible. (J–M) Pdu-vasa, Pdu-nanos, Pdu-piwi, Pdu-PL10 in the MPGZ of 72 hpf larvae. Scale bar in 2M corresponds to 50 μm(A–I) and 25 μm(J–M), respectively. N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 603 mounted in Tissue-Tek (Sakura), frozen at −20 °C and sectioned (40 μm) on a Pdu-vasa mRNA is ubiquitously expressed in oocytes and Leica cryostat. Immunohistochemistry was performed on slides as described embryos, and becomes restricted to the mesodermal posterior above starting with the blocking reaction. growth zone during larval development DiI-labelling In order to determine the temporal and spatial course of Pdu-

Worms of 20–60 segments were anaesthetised in MgCl2 3.75%/50% ASW vasa expression, we performed WMISH on Platynereis larvae for 10 min followed by pressure injection with 0.1 mg/ml DiI in ethanol (cell and worms at different developmental stages. tracker, C-7000, Molecular Probes) using a glass capillary in a micromanipu- WMISH revealed that Pdu-vasa mRNA is uniformly dis- lator. Worms were washed twice with ASW and kept in the dark for up to tributed in early cleavage stages (Fig. 2A), and is progressively 2 weeks. restricted to the postero-ventral region and the mesodermal BrdU staining bands during gastrulation (Figs. 2B, C). At 24 hpf, Pdu-vasa is most abundant in the future pygidial area of the larva (Fig. 2D). Larvae were incubated in BrdU (Sigma, 30 mg/100 ml ASW) for 3 h, This region has long-time been supposed to harbour the meso- washed and fixed as mentioned above. Prior to antibody staining, the fixed dermal posterior growth zone (MPGZ) and thus the budding larvae were rehydrated and pre-treated for 5 min with 0.1 N HCl at room zone of somatic mesoderm (Anderson, 1973). Only faint stai- temperature, followed by 1 h with 2 N HCl at 37 °C. Incorporated BrdU was detected using monoclonal anti-BrdU antibodies (Sigma) in a 1:250 ning remains in the parapodial area and in the stomodaeum (not dilution. TRITC-coupled secondary antibodies (Sigma) were used in a 1:500 shown). From 48 hpf onward, Pdu-vasa is localised to the dilution. MPGZ, with strongest expression at 5 days (Figs. 2E–I). We observed a similar progressive restriction to the MPGZ for the Results mRNAs of Pdu-nanos, Pdu-piwi, and pdu-PL10 (Figs. 2J–M). In juveniles II (>20 segments), vasa expression is becoming Cloning of Platynereis germ cell-related genes very strong in groups of cells forming a dorsal, transverse stripe

We cloned the full coding region of the Platynereis DEAD- box genes, Pdu-vasa and Pdu-PL10. The Pdu-vasa transcript encodes a protein of 712 amino acids. Two variants of Pdu- PL10 were isolated, encoding proteins of 881 and 772 amino acids, respectively. Multiple sequence alignment (Fig. 1A) and phylogenetic analysis (Fig. 1B) confirmed orthology of both Pdu-vasa and Pdu-PL10. Pdu-Vasa exhibits the eight conserved domains characteristic for Vasa proteins (Liang et al., 1994; Komiya et al., 1994)(Figs. 1A, C): the four sites involved in ATP binding and cleavage (AXXXXGKT, PTRELA, GG, TPGR, DEAD,) the RNA unwinding motifs (SAT, HRIGR), and the helicase C domain (unwinding: ARGXD, RNA binding: HRIGR). Thus, Pdu-Vasa most likely constitutes an ATP-dependant RNA helicase. Additionally Pdu-Vasa has three CCHC-type zinc fingers (C–X2–C–X4–H–X4–C) as well as three RGG motifs in the N-terminal region (Fig. 1C). Pdu-PL10 shares the same eight conserved domains found in Pdu-Vasa, but the zinc fingers as well as the RGG motifs are missing (Fig. 1C). Next, we used genomic data to investigate the Platynereis vasa gene locus (Fig. 1D). Almost the complete transcript could be mapped to a single clone (CH305_59A6) isolated from a genomic BAC library derived from Platynereis sperm DNA. The very 5′ end of the sequence, including the 5′UTR, was not found on this BAC. This could be due to a large intron reaching Fig. 3. Pdu-vasa mRNA in young worms and females. (A) Pdu-vasa expression beyond the end of the BAC, strong sequence polymorphisms or in gonial clusters in the primary gonad of a juvenile II, dorsal view, anterior is to the presence of an unresolved sequence region in the current the left. The retractable pharynx harbours the prominent jaws (j). Anterior BAC sequence. The mapped transcript covers a locus of about chaetiferous segments are numbered I–IV. The cephalic segment (C) has 25 kb and is distributed over 15 exons, with the last exon transformed into a pair of peristomial cirrhi. (B) Pdu-vasa expression in a gonial covering the full 3′UTR. Following a general trend for Platy- cluster from the parapodial area of a juvenile II, transversal section. (C) Pdu-vasa expressing PGCs (asterisk) in the parapodial area of a juvenile II. (D) Pdu-vasa nereis genes (Raible et al., 2005), intron numbers of the Pla- expression in oocytes (asterisks) in the coelomic cavity and the parapodial area, tynereis vasa gene are higher than in other protostome vasa transversal section. (E, F) Schemes corresponding to panels C and D, genes. respectively. Abbreviations: m=muscles, g=gut, a=aciculae, p=parapodia. 604 N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 behind the jaws (Fig. 3A). These cells are gonial clusters Pdu-Vasa is also found in juveniles (not shown) and mature (Fischer, 1975). Later, Pdu-vasa expression is also detectable in females, but not in mature males. The protein is absent from gonial clusters (Fig. 3B) as well as in distinct cells (Figs. 3C, D) somatic tissue of spawned individuals, indicating that it is germ in the parapodial area of the more posterior segments. In fe- cell-specific. males, Pdu-vasa is expressed in growing oocytes (Figs. 3D, F). No transcript is found in males approaching maturity (not Pdu-Vasa is a component of the yolk free cytoplasm and shown). These results were confirmed by northern blot and RT- segregates into the founder cells of the mesodermal posterior PCR (Fig. 1, supplementary material). growth zone

Vasa protein is present throughout the life cycle of Platynereis, In many species such as Drosophila, Xenopus, and C. except for mature males elegans, maternal Vasa protein is localised to specific regions of oocytes and early embryos, while the mRNA is initially Pdu-Vasa antibodies detect a protein of approximately uniformly distributed (Hay et al., 1988; Komiya et al., 1994; 80 kDa in extracts of worms of various stages (Fig. 1C, sup- Gruidl et al., 1996). We therefore investigated Vasa localisation plementary material). An identical band was obtained with anat the protein level by immunohistochemistry. Pdu-Vasa is tibodies raised against Vasa proteins from Drosophila found in a perinuclear ring in the unfertilised egg (Fig. 4A, for (Tomancak et al., 1998) and Schistocerca gregaria (Chang et control see Fig. 2A, supplementary material). Upon sperm entry al., 2002), not shown. The size of the protein is close to the and germinal vesicle breakdown, ooplasmic segregation yields estimated molecular weight of 75 kDa for Pdu-Vasa as cal- an area of yolk free cytoplasm at the animal pole (Wilson, 1892; culated from the deduced amino acid composition. We therefore Dorresteijn, 1990). Pdu-Vasa is localised to the yolk free assume that the 80 kDa band corresponds to Pdu-Vasa protein. cytoplasm in the fertilised egg (Fig. 4B) and during cleavage High levels of protein are present in eggs, embryos, and larvae. stages (Fig. 4C). The bulk of the yolk free cytoplasm is known

Fig. 4. Pdu-Vasa during embryonic and larval development. (A) Mature oocyte showing a ring of Pdu-Vasa protein (arrow) around the nucleus (n). (B) Zygote during ooplasmic segregation, lateral view. Pdu-Vasa is localised to the forming yolk free cytoplasm (yfc) at the animal pole (arrow). (C) Four-cell stage, animal view. Pdu-Vasa in the yolk free cytoplasm of all blastomeres. Nuclei are counterstained with Hoechst. (D) 38-cell stage, lateral view, optical section. Pdu-Vasa is found in the micromeres, including the mesoblast 4d (arrow), but is absent from the entoblasts A and D. (E) 10 hpf embryo, Pdu-Vasa is specifically enriched in the secondary mesoblast cells (arrow). (F) Pdu-Vasa is expressed in the mesodermal bands of the 15 hpf larva. Note the stronger staining in the cells at the basis of the mesodermal bands (arrow). (G) Group of cells in the MPGZ of a 48 hpf larva, lateral view, showing a faint Vasa-positive immunoreaction (arrow). Dotted line indicates the prototroch. (H) At 72 hpf, individual Vasa positive cells are distinguishable in the MPGZ (arrow). Segments are numbered C, I, and II. The scale bar for figures A–H corresponds to 50 μm. N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 605 to segregate into the micromeres, while only a small portion remains in the yolk rich macromeres. High Pdu-Vasa levels are accordingly detectable in the micromeres at the 38 cell-stage (Fig. 4D). One of these vasa-positive micromeres is the mesoblast 4d, the founder cell of both the MPGZ and the mesodermal bands (Wilson, 1892; Dorresteijn, 1990). At 10 hpf, a group of small, roundish 4d descendants (‘secondary mesoblast cells’) are strongly stained (Fig. 4E). Following gastrulation, Pdu-Vasa is detected at low levels in the mesodermal bands as well as in the ectoderm, with slightly higher concentrations in the area of the future MPGZ (Fig. 4F). Faint Pdu-Vasa expression in the MPGZ is detectable at 48 hpf (Fig. 4G). Similar results have been obtained with an antibody raised against insect Vasa proteins (Chang et al., 2002), data not shown. The MPGZ-specific staining is clearly evident at 72 hpf (Fig. 4H).

In the mesodermal posterior growth zone Vasa is expressed in highly proliferative descendants of the mesoblast 4d

Vasa is found in the cells of the MPGZ in the 72 hpf larva, posterior to the chaetiferous segments I and II (Fig. 4H). The same cells are labelled after injection of the lineage tracer TRITC-Dextran into the mesoblast 4d, as shown by Ackermann et al., 2005 (Fig. 5A). This posterior region of the larva corresponds to the so-called ‘pigmented area’ (Fig. 2I), which is Fig. 5. The origin of the mesodermal growth zone. (A) Lineage tracer found in the MPGZ (arrow) of this 72 hpf larva after dye injection into the mesoblast 4d. supposed to descend from the mesoblast 4d in Nereis spec Picture taken from Ackermann et al., 2005, with permission from the authors. (Wilson, 1892). The lineage tracing experiments support (B–D) Proliferating cells as revealed by BrdU staining co-localise with Vasa Wilson's initial assumption that the MPGZ forms from the protein in the MPGZ (arrow) in 72 hpf larvae. (B) BrdU staining, (C) Vasa secondary mesoblast cells which bud of from the mesoblast 4d immunfluorescence, (D) overlay. Segments are numbered C, I, and II. Scale bar μ (see above). In order to verify the co-localisation of Vasa corresponds to 50 m. expression with the MPGZ, BrdU pulse labelling experiments were performed. Proliferating cells are numerous in the ‘neck’ region of segment III posterior to the pharynx in worms ectodermal posterior growth zone as well as in the MPGZ and of around 10 segments (Fig. 7A, for control see Fig. 2C, the outgrowing mesodermal bands (Fig. 5B). Vasa is found in the supplementary material). We refer to this transitory resident MPGZ as well as in the cells of the mesodermal bands, from the place of the PGCs as ‘primary gonad’ (see Discussion). With the latter it disappears soon after they left the MPGZ (Figs. 5C, D). further outgrowth of the jaws, the primary gonad is progressively pushed backwards (Figs. 7B–D, and Fig. 3B, supple- Four primordial germ cells emerge from the MPGZ at the end mentary material) until it reaches segment V. The relative of larval development position of the PGCs with respect to the jaws remains un- changed. In all worms <20 segments (juveniles I), exactly four At 4 dpf, four small, roundish cells emerge from the centre of PGCs were found (n=64). Once the worms exceed 20 segments the strongly stained MPGZ and migrate anteriorly (Figs. 6A, B, (juveniles II), gametogenesis is initiated and the PGCs in the for control see Fig. 2B, supplementary material). The cells are primary gonad start to proliferate, yielding numerous clusters of found in the chaetiferous segments I and II in young worms of gametogonia (Figs. 7B–D). In these clusters, Vasa protein is co- up to eight segments (Figs. 6C–F and Fig. 3A, supplementary expressed with Pdu-vasa (Fig. 3A) and Pdu-piwi (Figs. 7E–G) material). Notably, these cells never enter the cephalic segment transcripts, corroborating the gamete nature of these cells. (C), which will later be integrated into the head (Hauenschild and Fischer, 1969). In order to verify the nature of these four PGC migration and gametogenesis cells, we combined anti-Vasa immunohistochemistry with WMISH for the germ cell markers Pdu-vasa (Figs. 6G, H), In juveniles II, masses of migrating PGCs are distinguishable and Pdu-nanos (Figs. 6I, J), respectively. These double- in the parapodial area. These cells (Figs. 8A, B) are labelling experiments revealed that both transcripts are co- characterised by their elongated shape, a large nucleus, localized with Vasa protein in the MPGZ as well as in the four, filopodia, and by Vasa-positive granules. Injections of DiI roundish cells, corroborating their identity as PGCs. into the primary gonad yielded labelled cells in the more The inferred migration route of the PGCs apparently varies, posterior segments at the base of the parapodia after 3 days, but they end up in a transverse stripe below the epidermis in the supporting the notion that the PGCs migrate away from the 606 N. Rebscher et al. / Developmental Biology 306 (2007) 599–611

Fig. 6. Emergence of the PGCs from the MPGZ and migration into the primary gonad. (A, B) Four PGCs (asterisks) leave the MPGZ (arrow) at 4 dpf. (C, D) At 5 dpf, the PGCs (asterisks) migrate into the chaetiferous segments I and II, while other Vasa-positive cells remain in the MPGZ (arrow). PGCs are never found in the cephalic segment (C). (E) In this four segmented young worm, the PGCs are found in the chaetiferous segments I and II (asterisks), ventral view. Additional Vasa-staining is found in the MPGZ (arrow). Note orange autofluorescence of algae (a) in the gut. (F) Juvenile I (five segments), dorsal view, with four Vasa immunopositive cells in the primary gonad (asterisks). The gut is filled with a lgae (a) showing red autofluorescence. Two pairs of eyes (e) are located anterior of the jaws (j). The MPGZ is strongly stained (arrow). (G, H) Vasa protein and transcript are co-localised in the four migrating PGCs (asterisks) and the MPGZ (arrow) in this 6 dpf young worm. Optical section, anti-Vasa antibody-labelling was performed following the WMISH procedure. (I–J) Vasa protein is also co-localised with Pdu-nanos transcripts in the four migrating PGCs (asterisks) and the MPGZ (arrow) in this 7 dpf young worm. primary gonad towards the more posterior segments (Figs. 8C, germinal granules of the germ plasm (Eddy, 1975; Komiya et D). With an anterior–posterior progression, gonial clusters al., 1994; Gruidl et al., 1996; Knaut et al., 2000; Tsunekawa et appear in the parapodial segments (Fig. 8E). These clusters al., 2000, Shirae-Kurabayashi et al., 2006). The germinal comprise 8–64 gonia each and are found in both sexes (Fig. 8F). granules originate from nuage material, ribonucleotide particles During oogenesis, Pdu-Vasa is detected in the cytoplasm of that accumulate during oogenesis around the oocyte nucleus growing oocytes now floating in the coelomic cavity (Fig. 8G). (Saffman and Lasko, 1999). Vasa is known to be a compound of No staining is visible in sperms of mature males (not shown). both nuage and germinal granules (Hay et al., 1988; Toyooka et al., 2000; Carré et al., 2002). Repetitive RGG motifs in the Discussion protein are required for the subcellular localisation of Vasa in nuage structures in both zebrafish (Wolke et al., 2002) and Maternal Vasa protein as a compound of the yolk-free Drosophila (Liang et al., 1994). In Platynereis, Vasa exhibits cytoplasm three of these RGG motifs (Fig. 1C) and the protein is indeed localised to a perinuclear ring in oocytes (Fig. 4A). Here, it co- In many species ranging from nematodes, insects to chor- localises with nuage material previously described for this dates, the precursors of the germ cells are specified by the species (Fischer, 1975), indicating that Vasa protein is a com- inheritance of maternal cytoplasmic determinants located in the pound of nuage also in the polychaete. N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 607

Fig. 7. The onset of gametogenesis. Four PGCs in the primary gonad in segment III of a juvenile I, dorsal view. No staining is detectable in the MPGZ at that stage any longer (not shown). Note autofluorescence in the jaws (j), eyes (e) and gland cells (g). (B, C) Juvenile II, with proliferating PGCs in the primary gonad, segment IV. (D) Juvenile II, 36 segments. Proliferating PGCs have formed numerous gonial clusters. The primary gonad has moved backwards to segment V during the enlargement of the pharynx and the jaws (j). (E–G) The gonial clusters in the primary gonad co-express Pdu-Vasa protein with Pdu-piwi. (E) Immunohistochemistry for Pdu-Vasa, (F) WMISH for Pdu-piwi, (G) overlay. The scale bar corresponds to 200 μm(A–D), and 50 μm(E–G), respectively.

Upon fertilisation, Vasa protein is propagated into the animal Finally, the 4d origin of the MPGZ has been confirmed by yolk-free cytoplasm of the zygote, as part of which it is se- lineage tracing experiments in Platynereis (Fig. 5Aand gregated into the embryonic micromeres by asymmetric Ackermann et al., 2005). cleavage (Figs. 4B–D and 9). The micromeres give rise to ec- Importantly, the founder cells of the MPGZ and thus the todermal and mesodermal tissues during later development, and germ cells are characterised by the presence of specific cyto- additionally to the MPGZ (see below). Vasa is thus rapidly plasmic granules in polychaetes: In Nereis the secondary excluded from the large, yolk-rich endoderm precursors, mesoblast cells are characterised by ‘bluish-black granules’ representing almost 2/3 of the total embryo volume at the 38- (Wilson, 1892). In Salmacina, the putative PGCs are equipped cell stage (Fig. 4D). with conspicuous ‘cytoplasmic spheres’ (Malaquin, 1925). These granules resemble the ribonucleotide particles of the The MPGZ derives from Vasa containing 4d descendants, the germplasm. Stored in these ribonucleotide granules, Vasa secondary mesoblasts protein as well as different mRNAs might be protected from degradation, as it is reported from other species (see below). Our data indicate that the PGCs emerge from the MPGZ, a group of undifferentiated, mitotically active cells (Figs. 5 and 6 Progressive restriction of germline specific transcripts to the and Fig. 3A, supplementary material). Wilson (1892) has studied MPGZ the embryonic origin of this area in detail in the closely related species Nereis. He found that a group of small cells called the We have observed that during Platynereis development, ‘secondary mesoblasts’ bud off from the 4d lineage prior to the vasa transcripts are initially broadly distributed but become formation of the mesodermal bands. These cells later end up in progressively restricted from most of the somatic tissue until the so-called ‘pigmented area’ (Fig. 2F), which we identify as the they remain only in the MPGZ and the future germ cells (Figs. MPGZ. In Platynereis, Vasa protein is specifically enriched in 2A–H). A similar progressive restriction of vasa mRNA to the the secondary mesoblast cells at 10 hpf (Fig. 4E) and later in the germline has recently been reported for the sea anemone Ne- MPGZ (Fig. 4H). In the serpulid polychaete Salmacina dysteri, matostella (Extavour et al., 2005). In the embryo of the medaka two small cells similar to the Platynereis secondary mesoblast fish, maternal vasa mRNA is initially ubiquitously distributed cells are reported to derive from the mesoblast by unequal and localises gradually to the PGCs during late gastrulation cleavage and end up in the posterior region of the trochophore. stages (Shinomiya et al., 2000). These cells have been proposed to give rise not only to the germ The progressive clearance of vasa from somatic cells sug- cells but also to mesodermal tissue (Malaquin, 1925, 1934). gests a mechanism which protects germline specific transcripts 608 N. Rebscher et al. / Developmental Biology 306 (2007) 599–611

Fig. 8. Migration and growths of the germ cells. (A) PGCs (asterisk) in the parapodial area adjacent to the aciculae (a), ventral view. For a low magnification image, see panel E, showing a comparable area in the worm at a later stage of gametogenesis. Note Vasa positive granules in the cytoplasm. (B) Migrating PGC with filopodium (arrow). (C) Juvenile II injected with DiI into the primary gonad, dorsal view, anterior is to the left. Segments are numbered. Note autofluorescence of the jaws (j). (D) DiI labelled PGCs (asterisk) in the parapodial area after 1 week incubation. (E) Lateral view of the parapodial region of a juvenile II (40 segments). Gonial clusters are located adjacent to the aciculae (a). (F) Gonial cluster at higher magnification. (G) Female during oogenesis, transversal section. Pdu-Vasa is found in the cytoplasm of the oocytes. and/or proteins in the MPGZ, while promoting degradation in 2006). Our data are fully compatible with the notion that a the differentiating somatic tissues, similar to the posttranscrip- similar protection/degradation mechanism is also acting in the tional degradation–protection mechanism described for the polychaetes. zebrafish (Wolke et al., 2002). Here, motifs in the 3′UTR of vasa mRNA as well as in the encoded protein trigger rapid Singling out the four definitive PGCs from the MPGZ degradation in somatic cells and a stabilisation in the PGCs (Shinomiya et al., 2000; Knaut et al., 2002; Weidinger et al., The previous steps of germline specification seem to involve 2002 Wolke et al., 2002). Stabilisation may also involve incor- the asymmetric inheritance of cytoplasmic determinants—and poration into ribonucleotide particles (Wolke et al., 2002). In the thus ‘preformation’. However the subsequent selection of the ascidia Ciona, Vasa protein and mRNA are associated with a four definite PGCs from the centre of the MPGZ (Figs. 6A–D) putative germinal granule-like structure, the centrosome- occurs late in development (at 4 dpf), and from within a mor- attracting body (CAB), until the formation of the PGC founder phologically and molecularly homogenous mass of cells. This cells by asymmetric cleavage during gastrulation. After release would be consistent with an epigenetic type of germ cell deter- in the cytoplasm, Vasa protein is degraded in the somatic mination as found in for example in the mouse. Here, signalling daughter cells, while it is forming nuage-like perinuclear gra- molecules from the BMP family are known to predispose cells nules in the PGC founder cells (Shirae-Kurabayashi et al., in the epiblast to become primordial PGCs (McLaren, 2003). N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 609

Fig. 9. Two-step mode of germ cell determination. Cytoplasmic determinants containing Vasa (green) are present in the fertilised egg (A) and segregate together with the yolk-free cytoplasm (yfc) into the micromeres (B), embryo at 8 hpf, lateral view, asterisk indicates the closing blastoporus). One of the micromeres, the mesoblast 4d (green), subsequently gives rise to the secondary mesoblast cells (m, dark green) by asymmetric cleavage, prior to the formation of the larval mesodermal bands. The secondary mesoblasts form the undifferentiated, pluripotent cells of the MPGZ in the 72 hpf larva (C). In a second step, putative inductive signals (red arrow) determine the four PGCs from within the MPGZ cell population.

BMPs along with eFGFs are also reported to induce PGC 2004; Stebler et al., 2004). In Drosophila, Wunen and Wunen 2 formation in isolated animal caps of axolotl embryos (Johnson act as repellents for PGCs (Zhang et al., 1997; Hanyu-Naka- et al., 2003a,b). Notably, a Platynereis FGF receptor specifi- mura et al., 2004;). With Vasa protein demarcating migrating cally co-expresses with vasa in the MPGZ (P. Steinmetz, D. A., PGCs it will be now possible to investigate possible guidance unpublished data). It will be interesting to investigate the role of cues also in Platynereis. BMPs and other signalling molecules known to play a role in PGC induction in other species in the Platynereis MPGZ in The evolution of germ cell development in Metazoa: a stem cell order to find out whether epigenetic mechanisms also play a role connection in the specification of germ cells in the polychaete. Our observations suggest a two-step process of germ cell Transitory formation of an anterior ‘primary gonad’ specification in Platynereis. This hypothesis is summarised in Fig. 9: First, a population of undifferentiated cells is established At 4 dpf, the PGCs detach from the MPGZ (Figs. 6A–D) and in the MPGZ that is excluded from somatic differentiation and migrate anteriorly to form a transverse cluster in the ‘neck’ has the potential to form both mesoderm and PGCs. This step region in juveniles I (Figs. 6F and 7A, and Fig. 3A, sup- appears to involve the inheritance of cytoplasmic determinants plementary material). In the eunicid polychaete Diopatra from the yolk free cytoplasm (preformation). Second, four cuprea, similar transitory PGC niches have been described as PGCs are singled out from within this population, which subse- ‘primary ovaries’ (Lieber, 1931). This discovery in Platynereis quently proliferate and differentiate into gametes. As reasoned was highly unexpected since nereidid polychaetes were so far above, this step may involve inductive processes (epigenesis). reported to lack somatic gonads (Anderson, 1973; Eckelbarger, Comparative data suggest that such a two-step mode may be 1984; Andries, 2001; Fischer and Dorresteijn, 2004). Later, the ancestral for metazoan germ cell specification. Pluripotent stem gonial clusters enter the coelomic cavity, where the germ cell cells that give rise to both PGCs and somatic tissues are precursors differentiate into gametes. commonly found in basal phyla. These stem cells are often The conspicuous clustering of the PGCs in the primary characterised by cytoplasmic structures, resembling germinal gonads (Fig. 7) suggests that this area constitutes a target region granules in ultrastructure and composition. In the cnidarian towards which the PGCs are attracted. In other species, the Pelmatohydra robusta, germinale granules are present not only migration of the PGCs is known to depend on attractive or in PGCs but also in somatic i-cells (Noda and Kanai, 1977). repelling signalling molecules, and/or on extracellular matrix Neoblasts, the totipotent mesenchymal stem cells of flatworms, cues. In vertebrates, the chemokine SDF-1 has recently been are reported to contain a cytoplasmic structure called the “chro- identified as a chemical attractant (Doitsidou et al., 2002; Raz, matoid body” (Shibata et al., 1999). It is likely that in these 610 N. Rebscher et al. / Developmental Biology 306 (2007) 599–611 ribonucleotide particles factors reside that prevent somatic human Vasa gene is specifically expressed in the germ cell lineage. Proc. – differentiation. Germ cell formation from similar undifferen- Natl. Acad. Sci. U. S. A. 97, 9585 9590. Chang, C., Dearden, P., Akam, M., 2002. Germ line development in the tiated, mesenchymal cells has been reported also for other basal grasshopper Schistocerca gregaria: vasa as a marker. Dev. Biol. 252, metazoans such as sponges and acoel flatworms (Tuzet et al., 100–118. 1970; Gaino et al., 1984; Gschwentner et al., 2001). Cox, D.N., Chao, A., Baker, J., Chang, L., Qiao, D., Lin, H., 1998. A novel class The dual potential of MPGZ cells in Platynereis, i-cells in of evolutionarily conserved genes defined by piwi are essential for stem cell – Hydra, and neoblasts in flatworms, is also reflected on the self-renewal. Genes Dev. 12, 3715 3727. Deshpande, G., Calhoun, G., Yanowitz, J.L., Schedl, P.D., 1999. Novel molecular level. We could show that the MPGZ expresses the functions of nanos in downregulating mitosis and transcription during the germ cell markers vasa and nanos along with piwi and PL10, development of the Drosophila germline. 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