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