Introduction

In sexually reproducing animals, development of a viable germ line is essential for reproductive success and survival of the species. In most species, the germ line has a single embryonic origin, the primordial germ cells (PGCs), which are defined as a population of undifferentiated stem cells that will exclusively give rise to germ cells (Extavour and Akam 2003). Two very different mechanisms of PGC specification have been identified among metazoan species. In some animals, including Drosophila (Williamson and Lehmann 1996), C. elegans (Kimble and White 1981), and zebrafish (Yoon et al. 1997), PGCs are specified early in development by maternally inherited cytoplasmic determinants (preformation). In other animals, including mouse (Lawson and Hage 1994; Tam and Zhou 1996) and axolotl (Nieuwkoop 1947), PGCs are specified later in development by inductive signals from neighboring tissues (epigenesis). Among metazoans, epigenesis is more prevalent than preformation and is also the mechanism found in basal metazoans (e.g., cnidarians and sponges; reviewed in Extavour and Akam 2003). For these reasons, epigenesis is proposed to be the ancestral mode of germ cell specification in the Metazoa.

Studies in model organisms indicate that, regardless of whether a species exhibits preformation or epigenesis, at least some aspects of the molecular patterning of germ line development are well conserved. The Vasa and Nanos gene products are expressed in PGCs in nearly every species examined and have been shown to regulate germ line development in a variety of species (reviewed in Extavour and Akam 2003). Vasa-related proteins are adenosine triphosphate-dependent ribonucleic acid (RNA) helicases and members of the DEAD box protein family (Mochizuki et al. 2001). Numerous studies suggest that vasa genes are expressed exclusively in potential germ cell progenitors or other stem cells (Extavour and Akam 2003). However, recent reports for a polychaete (Platynereis dumerilii) have identified vasa expression in the germ line as well as in multiple somatic tissues, including the mesodermal bands, brain, foregut, and posterior growth zone (Rebscher et al. 2007; Zelada-González 2005). Likewise, in the oligochaete annelid, Tubifex tubifex, vasa is broadly expressed during cleavage stages and in the presumptive segmental tissues in addition to the germ line (Oyama and Shimizu 2007).

Nanos proteins contain two highly conserved CCHC zinc fingers in the C terminus with little conservation outside of this domain (Curtis et al. 1997). Nanos family proteins function as translational inhibitors (Curtis et al. 1997) and regulate both somatic and germ line development (reviewed in Extavour and Akam 2003). Among lophotrochozoans, nanos-related genes are expressed in the germ line in Helobdella robusta (a leech; Kang et al. 2002) and in PGCs in P. dumerilii (a polychaete) (Rebscher et al. 2007; Zelada-González 2005). These and other lophotrochozoan studies also report nanos expression in somatic tissues. H. robusta nanos is expressed in ectodermal stem cells (Kang et al. 2002), and P. dumerilii nanos is expressed in multiple somatic tissues including brain, foregut, and mesodermal bands (Zelada-González 2005). The Ilyanassa obsoleta (a snail) nanos gene becomes restricted to the 4d mesoderm lineage, and morpholino knockdown of nanos results in loss of mesodermal and endodermal tissues (Rabinowitz et al. 2008).

This study examines vasa- and nanos-related genes in Capitella sp. I, a member of the phylum Annelida. Capitella sp. I is a benthic marine polychaete with male, female, and hermaphrodite sexes. In females, oocytes develop in paired ovaries attached to the body wall in mid-body segments (Eckelbarger 1984). Sperm development takes place in paired sac-like genital ducts suspended in the lateral coelom between the seventh and eighth segments (Eckelbarger and Grassle 1987a). Oogenesis and spermatogenesis have been studied in young adults (Eckelbarger and Grassle 1983, 1987b; Eckelbarger et al. 1984); however, germ line development has not been examined during embryonic or larval stages. PGCs have not yet been identified, and the mechanism of germ line specification (preformation or epigenesis) is unknown. Furthermore, germ cell differentiation can be labile in Capitella sp. I: (1) Adult males can transform into fertile hermaphrodites (Holbrook and Grassle 1984), and (2) adult females can regenerate segments with ovaries (Hill, personal communication). Although these phenomena suggest an epigenetic mechanism of germ line development, germ line regulation in adults may involve a mechanism distinct from that involved in embryonic or larval development. For example, in the ascidian Ciona intestinalis, PGCs are localized to the tail of the tadpole stage, which migrate into the adult gonad rudiment during metamorphosis. If the tail and PGCs are removed, resulting in adults forming mature sperm, suggesting that in ascidians, there is a compensatory mechanism that regulates germ line formation at a later life history stage (Takamura et al. 2002). To begin to investigate the embryonic origin(s) of PGCs and the primary mode of germ line development in Capitella sp. I, we isolated orthologs of the vasa (CapI-vasa) and nanos (CapI-nanos) gene families and examined their developmental expression patterns by whole-mount in situ hybridization during embryonic, larval, juvenile, and adult stages.

Materials and methods

Cloning of Capitella sp. I vasa and nanos genes

Several overlapping expressed sequence tag (EST) sequences representing a single nanos homolog (CapI-nanos) and another set representing a single vasa homolog (CapI-vasa) were identified in BLAST searches of Capitella EST libraries (sequenced by the Joint Genome Institute [Department of Energy, Walnut Creek, CA, USA] as part of the Capitella sp. I 8x genome sequencing project). Each set of sequences was aligned and compiled into a single predicted transcript for each gene. The predicted transcripts were submitted to National Center for Biotechnology Information (NCBI) as third-party annotation sequences with the following accession numbers: CapI-nanos (BK006522) and CapI-vasa (BK006523). Two ESTs representing CapI-nanos were recovered from glycerol stocks of a pBluescript SK phagemid (Stratagene) mixed-stage complementary deoxyribonucleic acid (cDNA) library and sequenced (Macrogen, Seoul, South Korea) for verification; clone 4923 contains a 2,067-bp fragment corresponding to base pairs 267–2,334 of the full-length transcript, and clone 16516 contains an 812-bp fragment representing base pairs 800–1,612. Sequence of the CapI-vasa EST was used to design primers (forward: 5′-GTGTGGTGAAGAAGGGCATT-3′; reverse: 5′-GGGAACTCGGACTGAGACAA-3′) to amplify this sequence from Capitella cDNA. The resulting 1,122-bp fragment was ligated into pGEM T-easy vector (Promega) and sequenced.

Phylogenetic analysis

Phylogenetic analyses of the Nanos and Vasa families were performed to determine gene orthology. We assembled sets of Nanos and Vasa protein sequences from multiple species representing diverse metazoan taxa. Amino acid alignments were made with the ClustalW program included in the MacVector package. Analysis of the Nanos protein sequences was carried out with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the “wag” amino acid model and 106 generations sampled every 100 generations with four chains over a single run. Neighbor-joining analysis was performing using PAUP*4.0b10 (Swofford, 2002) with 1,000 replicates. Analysis of the Vasa family was carried out using an amino acid alignment of the DEAD-box domains from the following sequences: H. robusta predicted Vasa-like protein (HroVasa), JGI protein ID: 65423; I. obsoleta Vasa (IoVasa), ABU49329; Capitella sp. I predicted PL10-like protein (CapIPL10, JGI protein ID: 224610; Capitella sp. I Vasa, BK006523; Lottia gigantea predicted Vasa-like protein (LgVasa), JGI protein id: 177322; P. dumerilii PL10a (PdPL10a), CAJ15140; P. dumerilii PL10b (PdPL10b), CAJ15141; P. dumerilii Vasa (PdVasa), CAJ38803; Strongylocentrotus purpuratus Vasa (SpVasa), XP_781494; Ephydatia fluviatilis Vasa (EfVasa), BAB13310; E. fluviatilis PL10-related protein (EfPL10), BAB13309; Mus musculus Ddx4 (MmVasa), EDL18409; M. musculus PL10 (MmPL10), NP_149068; Crepidula fornicata PL10 (CfPL10), ABD59346; Danio rerio PL10 (DrPL10), NP_571016; D. rerio Vasa (DrVasa), NP_571132; Nematostella vectensis PL10 (NvPL10), AAW29072; N. vectensis Vasa2 (NvVasa2), AAW29074; N. vectensis Vasa1 (NvVasa1), AAW29073; C. intestinalis Vasa (CiVasa), BAA36710; Enchytraeus japonensis Vasa (EjVasa), BAF76795; Crassostrea gigas Vasa (CgVasa), AAR37337; Hydra magnipapillata Vasa (HmVasa), BAB13307. Analysis of the Nanos family was carried out using an amino acid alignment of the two CCHC zinc finger domains from the following sequences: I. obsoleta Nanos (IoNos), ABV54788; H. robusta Nanos (HroNos), AAB63111; Podocoryne carnea Nanos (PcNos), AAU11514; H. magnipapillata Nanos2 (HmNos2), BAB01492; M. musculus Nanos (MmNos), NP_848508; N. vectensis Nanos2 (NvNos2), AAW29071; N. vectensis Nanos1 (NvNos1), AAW29070; Apis mellifera Nanos (AmNos), NP_001035321; D. rerio Nanos (DrNos), AAH97090; L. gigantea predicted Nanos protein (LgNos), JGI protein ID: 60286; P. dumerilii Nanos (PdNos), CAJ28985; Capitella sp. I Nanos (CapINos), BK006522; Drosophila melanogaster Nanos (DmNos), AAA28715; Schistocerca americana Nanos (Sa), AAO38523; T. tubifex Nanos (TtNos), BAD90110; Xenopus tropicalis Nanos (XtNos), NP_988857; Drosophila simulans Nanos (DsNos), AAF68510; Caenorhabditis elegans Nanos2 (CeNos2), AAB93424; Caenorhabditis elegans Nanos1 (CeNos1), NP_496358; E. fluviatilis Nanos (EfNos), BAB19253; Musca domestica Nanos (MdNos), AAA87461; Chironomus samoensis Nanos (CsNos), AAA87459; Xenopus laevis Xcat2 (XlXcat2), NP_001081503; Anopheles gambiae Nanos (AgNos), AAW71999; Homo sapiens Nanos1 (HsNos), NP_955631. JGI predicted protein sequences were obtained from the Joint Genome Institute web browsers for each species (http://genome.jgi-psf.org/); all other accession numbers were obtained from NCBI.

Animal husbandry and in situ hybridization

A Capitella sp. I colony was maintained in the laboratory using published culture methods (Grassle and Grassle 1976) and broods were recovered as described previously (Seaver et al. 2005). Embryos and larvae were dissected from brood tubes, fixed in 3.7% formaldehyde in filtered sea water at 4°C for 16–24 h and then processed for whole-mount in situ hybridization according to published protocols (Seaver and Kaneshige 2006; Seaver et al. 2001). Juveniles and adults were treated with the same conditions as embryos and larvae with the exception that proteinase K treatment was increased from 3 to 10 (juveniles) or 20 min (adults), and for adult stage in situ experiments, the volume of all washes and hybridizations was increased from 0.5 to 1 ml. Digoxigenin-labeled riboprobes were generated with the MEGAscript kit (Ambion); following hybridization, probes were detecting using nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate color substrate. Riboprobes were generated for two ESTs representing fragments of CapI-nanos (16516 and 4923) and one clone representing a fragment of CapI-vasa (see above). The two nanos probes gave identical results at all stages examined; embryos shown in Fig. 2 were hybridized with riboprobe generated from EST 16516.

Results and discussion

Cloning and phylogenetic analyses of Capitella sp. I nanos and vasa genes

Searches of the Capitella sp. I genome identified a single nanos-related gene and ESTs representing this nanos-related gene were identified with BLASTX searches of sequenced EST libraries (see “Materials and methods”). The full-length CapI-nanos transcript is predicted to encode a 327-amino acid protein, which includes two CCHC zinc finger domains that display a high degree of amino acid identity with other metazoan Nanos proteins. Phylogenetic analysis based on an amino acid alignment of the two CCHC zinc finger domains of Nanos proteins from a diverse representation of metazoan species confirms CapI-nanos as a member of the Nanos gene family (Fig. 1a). CapI-Nos groups with other lophotrochozoan Nanos proteins and is more closely related to vertebrate, cnidarian, and arthropod sequences than to the C. elegans Nanos proteins (Fig. 1a), which form an outgroup in this and previous analyses of the Nanos family (Extavour et al. 2005). No additional nanos-related genes were identified in our searches of the Capitella sp. I genome, consistent with the presence of a single Capitella nanos gene.

Fig. 1
figure 1

Phylogenetic analysis of Capitella sp. I vasa and nanos genes. Color-coding for both trees is as follows: red = lophotrochozoan, violet = arthropod, green = cnidarian and sponge, brown = chordate, black = nematode, and blue = echinoderm. a Bayesian consensus tree of the CCHC zinc finger domains of metazoan nanos genes. Numbers above branches indicate posterior probabilities. b Neighbor-joining consensus tree depicting the relationship among DEAD-box helicase domains of metazoan vasa and PL10 genes. Numbers above the horizontal branches indicate bootstrap values for neighbor-joining analysis. Species abbreviations are as indicated in “Materials and methods”

Full size image

Sequence searches of the Capitella sp. I genome identified a single vasa gene, CapI-vasa. The CapI-vasa transcript is predicted to encode a protein of 515 amino acid residues (CapI-Vasa) containing a conserved DEAD-box helicase domain. This domain is characteristic of genes in the DEAD-box protein family, which includes the closely related Vasa and PL10 subfamilies (Mochizuki et al. 2001). The amino acid sequence of CapI-Vasa is most closely related to other Vasa-related proteins. We performed a phylogenetic analysis of the helicase domains from Capitella vasa and PL10 genes to determine the orthology of CapI-vasa. The analysis confirms an orthology assignment of CapI-vasa as a member the Vasa family (Fig. 1b). CapI-vasa clusters with vasa genes from other species with a strong bootstrap support value (100), while CapI-PL10, an additional DEAD-box gene identified in Capitella sp. I genomic sequences, clusters with the PL10 genes (Fig. 1b). Within the Vasa cluster, CapI-Vasa is most similar to a sequence from an oligochaete (EjVasa), and also groups with Vasa orthologues from other lophotrochozoans (Fig. 1b).

CapI-nanos mRNA embryonic, larval, and juvenile expression patterns

CapI-nanos transcript is detected in the yolk-free cytoplasm surrounding the nucleus in uncleaved zygotes (Fig. 2a). This pattern indicates maternal expression but does not suggest specific asymmetric localization. Through early cleavage stages, CapI-nanos is broadly expressed in most if not all cells (including all four embryonic quadrants) and co-localizes with the nuclei (Fig. 2b,c). At the 56-cell stage (fifth to sixth cleavage), expression is enriched in 4d daughter cells (Fig. 2c, arrows), although it is also detected at lower levels throughout the embryo. This pattern contrasts with the restricted messenger RNA (mRNA) expression for a nanos gene at a similar stage in the snail, I. obsoleta (Rabinowitz et al. 2008). Although nanos is expressed broadly during early cleavage in this lophotochozoan species, after fourth-quartet micromeres are born, expression becomes highly restricted to the 4d lineage (Rabinowitz et al. 2008). In the Helobdella embryo (leech), Nanos protein is robustly expressed in ectodermal precursor cells during cleavage with lower expression levels in mesodermal precursors. Thus, leech nanos also lacks restricted expression in the 4d lineage (Pilon and Weisblat 1997).

Fig. 2
figure 2

CapI-nanos RNA expression patterns during embryonic, larval, and juvenile development. The image in panel q was captured using a 100× objective, all other images were taken with a 40× objective. Except for cleavage stages (a–c), all animals are oriented anterior to the left. In lateral views, ventral is down. An asterisk marks the position of the blastopore (stage 3) or stomodeum (stages 4–9), t indicates the telotroch, and br indicates the brain in all panels. a In the uncleaved zygote, CapI-nanos RNA is detected in the yolk-free cytoplasm. b Animal view at the 56-cell stage shows nuclear expression of CapI-nanos throughout the embryo. c An equatorial view (vegetal down) facing the D-quadrant at the 56-cell stage shows CapI-nanos expression in all nuclei in view; expression appears enriched in the 4d daughter cells (arrows). d Ventral view of a late gastrula embryo (stage 3). CapI-nanos expression colocalizes with the nucleus in cells around the closing blastopore (asterisk) and in two ventro-lateral cell clusters (arrowheads), in the position of 4d derived mesodermal cells. e shows a lateral close-up of CapI-nanos expression in the mesoderm stem cells (black arrowheads, refers to region denoted by black arrowheads in d). White arrowhead points to one of the dorsal cells shown in g. f Dorsal view shows CapI-nanos expression in the presumptive brain and in the mesoderm stem cells (arrowheads). The position of the presumptive telotroch is marked by vertical lines. g Enlarged view in a more surface focal plane of the boxed region shown in f highlighting two additional CapI-nanos-positive cells flanking the dorsal midline. h, i, j CapI-nanos expression in the brain (h, j), foregut (staining lateral to the stomodeum in i), and in the mesodermal bands (i, arrowheads) in ventral (h, i) and lateral (j) views. Boxed area in i denotes position of view depicted in (h) at a different focal plane. j, k Same stage 4 embryo with a DIC image (j) and a Hoechst 33342 stain (k) showing CapI-nanos-positive ectodermal cells (arrows) positioned in a circumferential row immediately anterior to the telotroch (t). l Ventral and n lateral views of a stage 6 larva showing continued CapI-nanos expression in the brain (br) and foregut (staining lateral and dorsal to the stomodeum), expanded expression in the body segments (between the stomodeum and telotroch), and in a small domain in the hindgut (hg). m, n Two ventro-lateral clusters of CapI-nanos-expressing cells are located in segment 4 (arrows), positioned between the yolky endoderm and the ventral nerve cord (putative primary germ cells, PGCs). Image in m is a cropped view of a ventral focal plane centered around region depicted by box in l. Lateral (o) and ventral (p) views of stage 8 larvae show strong CapI-nanos expression in the PGCs (arrows) and posterior growth zone (staining anterior to the telotroch); low levels of expression are also detected in the brain, foregut, and body segments. Box in p depicts region shown in q, r, and s. q The PGCs in p were viewed at 100× magnification to highlight the perinuclear pattern of CapI-nanos staining in these cells. Three distinct patterns of CapI-nanos PGC expression are observed among stage 8 larvae: two bilaterally symmetrical cell clusters (p, q), two clusters positioned asymmetrically at or near the midline (r), and a single cluster centered on the midline (s) (ventral views). t By stage 9, CapI-nanos expression is limited to a single PGC cluster at the midline (arrow) and mesoderm and ectoderm of the posterior growth zone. u, v During early juvenile stages (juv), CapI-nanos is expressed exclusively in the ectoderm and mesoderm of the posterior growth zone (arrows; posterior end, u, lateral view; v, ventral view). Note mesodermal expression that extends posterior into the pygidium. Scale bar, 50 μm for all panels except g, q, r, and s. Scale bar in g, 20 μm; scale bar in q, 10 μm; scale bar in r and s, 25 μm

Full size image

At the end of gastrulation (stage 3), CapI-nanos expression transitions to a more discrete pattern with four distinct domains (Fig. 2d–g) that includes the presumptive brain (Fig. 2f), the closing blastopore (Fig. 2d), a pair of cells straddling the dorsal midline (Fig. 2g), and mesodermal cells (Fig. 2d,e). In the following section, we elaborate on the details of expression for each of these domains individually as they progress through larval development.

CapI-nanos is expressed in two bilateral patches of anterior ectoderm during stages 3 and 4 (Fig. 2f,h). This expression domain corresponds to the presumptive brain (Meyer and Seaver, in preparation). By stage 6, when the brain is morphologically distinct from the overlying ectoderm, CapI-nanos is expressed in the brain (Fig. 2l,n). Expression in the brain is reduced by stage 8 (Fig. 2o) and is not detected at later developmental stages.

CapI-nanos transcript is detected in the foregut at very early stages of foregut development (stage 4; Fig. 2i) in cells that likely correspond to at least a subset of the CapI-nanos-positive cells around the closing blastopore at stage 3 (Fig. 2d). Robust foregut expression is detected at mid-larval stages (e.g., stage 6, Fig. 2l, n) and at low levels at stage 8 (Fig. 2o). CapI-nanos is also briefly expressed in the hindgut during stages 5–6 (Fig. 2l,n, and data not shown).

A line of CapI-nanos-positive cells are also apparent immediately anterior to the telotroch (Fig. 2j, k)—the posterior larval ciliary band—at stage 4, in a similar position to the dorsal CapI-nanos-expressing cells at stage 3 (Fig. 2g). These CapI-nanos-positive cells are large ectodermal cells that form a band around most of the circumference of the larva, although no expressing cells span the ventral midline. The position of this band of cells corresponds with the future posterior growth zone, and these cells may represent ectoteloblasts. Within each cell, the transcript is localized to the nucleus (Fig. 2k), and the most prominent expression is restricted to a small spot within the nucleus. There are no unlabeled ectodermal cells interspersed between the CapI-nanos-positive cells that make up this band (Fig. 2k), and expression of CapI-nanos in these cells is not detected at any later stages of development.

In spiralians (including molluscs and annelids), the germ line is assumed to be of mesodermal origin arising from the 4d lineage (Extavour and Akam 2003). CapI-nanos expression in the 4d descendants (Fig. 2d,e) may encompass cells that will give rise to the germ line; however, it is clearly not exclusive to the germ line, as the major derivative of these cells is segmental mesoderm, which includes body wall and visceral muscle and coelomic cavity linings. The mesoderm precursor cells (and their derivatives) continue to divide during stage 3 such that by stage 4, they have formed two bilateral bands (the mesodermal bands), which expand circumferentially around the larva, generating the mesodermal component of the first ten segments (Seaver et al. 2005). CapI-nanos is expressed throughout the mesodermal bands at stage 4 (Fig. 2i,j). At stage 6, CapI-nanos is broadly expressed in the segmental region (between the prototroch and telotroch) with strongest expression in the posterior-most segments (Fig. 2l,n). In lateral view, it is apparent that the segmental CapI-nanos expression encompasses both mesodermal and ectodermal tissues; expression abuts the yolky endoderm and extends outward to the surface of the ectoderm (Fig. 2n). After stage 6, expression diminishes in the formed segments, and robust expression is maintained in the posterior growth zone at late larval (Fig. 2o,p,t) and juvenile stages (Fig. 2u,v).

At stage 6, an additional CapI-nanos expression domain appears, in two bilateral ventro-lateral clusters of cells in segment 4 (Fig. 2m). This expression domain is more easily detected at stage 8, when the overlying mesoderm and ectoderm expression has diminished (Fig. 2o,p,q). There are no previous descriptions of any structure(s) that develop at the position of these CapI-nanos-positive cells. Because of the morphology of these cells and the fact that CapI-vasa is also expressed in this region (see below), we consider them as putative PGCs. Within segment 4, three distinct patterns of CapI-nanos PGC expression are observed among stage 8 larvae: (1) two bilaterally symmetric cell clusters (Fig. 2p,q, n = 6), (2) two asymmetrically positioned clusters at or near the midline (Fig. 2r, n = 8), and (3) a single cluster centered on the midline (Fig. 2s, n = 14). These three different patterns of CapI-nanos PGC expression may indicate that the two ventro-lateral clusters migrate to the midline during stage 8. Alternatively, one of the clusters may be developmentally lost. From our expression data, it is not possible to distinguish between these two alternatives. Viewing the PGCs at 100× magnification shows perinuclear localization of the CapI-nanos transcript and large nuclear to cytoplasmic ratio (Fig. 2q). By stage 9, CapI-nanos expression is dramatically reduced and is only detected in a single PGC cluster at the midline and in the mesoderm and ectoderm of the posterior growth zone (Fig. 2t). There is no evidence of CapI-nanos expression in PGCs in juvenile stages.

CapI-vasa mRNA embryonic, larval, and juvenile expression patterns

With a few exceptions, CapI-vasa mRNA expression overlaps with that of CapI-nanos throughout all stages examined. Like CapI-nanos, CapI-vasa transcript is maternally expressed and appears in the yolk-free cytoplasm surrounding the nucleus in the uncleaved zygote (Fig. 3a). Expression is detected throughout cleavage in most if not all cells (Fig. 3b,c). Although we could not find any CapI-vasa-negative cells, it is possible that a nonexpressing cell went undetected. At 56-cell stage, CapI-vasa expression appears in the cytoplasm and the nuclei of most cells, but in some cells, it appears to be localized to the nucleus (Fig. 3b,c). In contrast to CapI-nanos expression at the 56-cell stage (Fig. 2c), CapI-vasa expression does not appear to be enriched in 4d derivatives (Fig. 3c). A study from I. obsoleta reports vasa transcript expression limited to a subset of 4d derivatives during cleavage and no expression detected at any later stage (Swartz et al., 2008). Thus, CapI-vasa expression does not correlate with the expression reported in this mollusc.

Fig. 3
figure 3

CapI-vasa RNA expression patterns in embryos, larvae, and juveniles. The image in n was captured with a 100× objective; all other images were captured with a 40× objective. Except for cleavage stages, animals are oriented anterior to the left with ventral facing down in lateral views. An asterisk marks the position of the blastopore (stage 3) or stomodeum (stages 4–9), t indicates the position of the telotroch, and br indicates the brain in panels where these structures are in view. a In the uncleaved zygote (unc), CapI-vasa RNA is detected in the yolk-free cytoplasm. b In animal view at 56-cell stage (fifth to sixth cleavages), cytoplasmic and nuclear staining is detected in most if not all micromeres. c An equatorial view (vegetal down) shows CapI-vasa staining in vegetal macromeres and in first through fourth quartet micromeres. d A ventral view of a late gastrula (stage 3) embryo shows CapI-vasa expression around the closing blastopore and at the position of the mesoderm 4d descendants (arrowheads). The out of focus staining posterior to the 4d descendants is transcript localized to the nucleus in a small number of unknown cells. e shows a close-up lateral view of CapI-vasa expression in the mesoderm cells (area depicted by arrowhead in d. f A dorsally mounted stage 3 embryo shows clear presumptive brain expression. Arrowheads point to CapI-vasa expression in the mesoderm cells. Stage 4 ventral (g, h) and lateral (i) views show CapI-vasa expression in brain, foregut (staining lateral to the stomodeum), and mesodermal bands (arrowheads). Region shown in g is depicted by boxed areas in h. j, k Ventral (j) and lateral (k) view of stage 6 larvae showing CapI-vasa expression in the foregut (staining lateral and dorsal to the stomodeum), brain, mesoderm (me) of the body segments (region between the stomodeum and telotroch, inset in j), and hindgut (hg). Arrows in j and k point to two patches of CapI-vasa-expressing cells (putative primordial germ cells, PGCs) in segment 4. Lateral view (k) shows the PGCs positioned between the endoderm and the ventral nerve cord. Ec Ectoderm. Lateral (l) and ventral (m) views of stage 8 larvae show strong expression in the PGCs (arrows) and posterior growth zone (staining anterior of the telotroch). n The PGCs in l were viewed at 100× magnification to show the perinuclear pattern of CapI-vasa expression. (n, o, p ventral views, cropped views of boxed region in m). Three distinct patterns of CapI-vasa PGC expression are observed among stage 8 larvae: (1) two bilaterally symmetric cell clusters (m, n), (2) two clusters positioned asymmetrically near or at the midline (o), and (3) one cluster at the midline (p). q At stage 9, CapI-vasa is expressed in the posterior growth zone and in the PGCs (arrow; ventral view). Posterior end of a juvenile worm (juv) in lateral (r) and ventral (s) view shows robust CapI-vasa expression in the mesoderm and ectoderm of the posterior growth zone (arrows) and weak expression in the ventral nerve cord ganglia (arrowheads). Scale bar, 50 μm for all panels except n, o, and p. Scale bar in n, 10 μm, scale bar in o and p, 25 μm

Full size image

At stage 3, CapI-vasa is detected in three of the four CapI-nanos expression domains: in the presumptive brain (Fig. 3f), around the closing blastopore (Fig. 3d), and in the region of 4d mesodermal descendants (Fig. 3d–f). In contrast to the nuclear localization of CapI-nanos in these three domains at stage 3 (Fig. 2d–g), the CapI-vasa transcript is present in the cytoplasm. Posterior to the mesodermal expression domain, a few surface cells show nuclear CapI-vasa expression (Fig. 3d). These cells do not show any particular arrangement and may represent residual expression from cleavage stages. At stage 4, CapI-vasa is detected in the brain (Fig. 3g), in the presumptive foregut (Fig. 3h), and in the mesodermal bands (Fig. 3h, i). In contrast to CapI-nanos (Fig. 2j, k), no CapI-vasa expression was detected in the row of ectodermal cells anterior to the telotroch (Fig. 3i). Throughout the rest of larval development, CapI-vasa expression is nearly indistinguishable from CapI-nanos expression. CapI-vasa mRNA is detected in the developing brain (Fig. 3j–l), foregut (Fig. 3j,k,m), hindgut (Fig. 3k), and in the segmental mesoderm (Fig. 3j,k,m). At stage 6, expression is detected throughout the segmental mesoderm (Fig. 3j inset) with strong expression in both the ectoderm and mesoderm of posterior segments (Fig. 3k). Segmental expression begins to diminish at stage 7 and becomes highly reduced except for strong expression in the posterior growth zone at stages 8 (Fig. 3l, m) and 9 (Fig. 3q). In juveniles, CapI-vasa is expressed in the ectoderm and mesoderm of the posterior growth zone and weakly in the ventral nerve cord (Fig. 3r,s).

At stages 6 and 7, CapI-vasa expression is detected in two ventro-lateral clusters of cells in segment 4, the PGCs (Fig. 3j, k, and not shown for St. 7). As described for CapI-nanos, three different patterns of CapI-vasa PGC are observed in stage 8 larvae: (1) two bilaterally symmetric cell clusters (Fig. 3m,n, n = 8), (2) two asymmetrically positioned clusters at or near the midline (Fig. 3o, n = 11), and (3) one cluster centered on the midline (Fig. 3p, n = 14). CapI-vasa PGC expression appears to be perinuclear when viewed at 100× magnification (Fig. 3n). A single PGC cluster is visible in late stage larvae (stage 9; Fig. 3q).

CapI-vasa and CapI-nanos mRNA adult expression patterns

Expression of CapI-vasa and CapI-nanos was also examined in young adults 1 and 2 months after metamorphosis at different stages of sexual maturity. Generally, animals are reproductive 8–10 weeks after metamorphosis when raised at 15°C. Both CapI-vasa and CapI-nanos expression are observed in three distinct domains in adults: in the PGCs, the gonads and in the posterior growth zone (Fig. 4). Additionally, CapI-nanos is expressed in a small number of segmentally repeated cells in the ganglia of the ventral nerve cord and in two lateral, ectodermal patches immediately anterior to the nuchal organs (not shown). Within the thoracic region (setigers 1–9) of males, females, and hermaphrodites, CapI-vasa and CapI-nanos are expressed in a discrete nonsegmental structure containing more than 50 cells, located at the ventral midline between the ventral nerve cord and the gut (Fig. 4a–d,l). This structure is clearly distinct from surrounding tissues, does not show obvious signs of cellular differentiation, and is suspended in the coelomic cavity by mesenteries (Fig. 4a,b). It is consistently localized to segment 5, usually extending into segment 6 (Fig. 4a,b). The CapI-vasa and CapI-nanos-positive cells in this structure have large nuclei and do not show overt signs of differentiation (Fig. 4d). We observed this structure in sexually immature males and females (1 month postmetamorphosis) as well as in sexually reproductive adults that contain mature gametes. To our knowledge, this structure has not been previously described in Capitella sp. I adults. Because of the coexpression of both CapI-vasa and CapI-nanos in cells of this midline mesodermal structure, the morphology of the cells, and its similar position to the position of the CapI-vasa and CapI-nanos-expressing PGCs in segment 4 of stage 8 larvae, we propose that this structure represents the adult PGC niche. Furthermore, we hypothesize that germ cells move from this structure into the gonads several segments posterior, where they undergo gametogenesis (Fig. 4l).

Fig. 4
figure 4

Adult expression of CapI-vasa and CapI-nanos. For all panels, animals are oriented anterior to the left with ventral facing down in lateral views. a Close-up lateral view of segments 4–6 (s4–s6) showing CapI-nanos expression in a structure within the coelomic cavity (arrow), between the gut (gt) and ganglia (ga) of the ventral nerve cord in segments 5 and 6. Arrowhead points to mesentery attached to the gut and CapI-nanos-expressing structure. b CapI-vasa is expressed in cells of a mesodermal structure (arrow), positioned between the ganglia of the ventral nerve cord (ga) and the gut (gt) in segments 5 and 6 (lateral view). c Enlarged dorsal view showing CapI-vasa expression in the mesodermal structure positioned at the midline of segment 5 (arrow). In this specimen, the midline of the dorsal body wall was cut and the gut removed for better imaging. d Enlarged view of region of c (100×) showing cellular detail of CapI-vasa-positive cells, which have large nuclei as determined by the nuclear stain Hoechst (not shown). Arrows point to three CapI-vasa-positive cells. e Within the ovaries, CapI-nanos is expressed in immature oocytes (arrowheads) but is absent from mature oocytes (arrows), ventral view. f Ventral view of ovaries showing cytoplasmic expression of CapI-vasa in immature oocytes (arrowheads). Arrows point to mature oocytes that lack vasa expression. g Young adult males show CapI-nanos expression in two bilaterally symmetric mesodermal clusters (arrows) in a ventro-lateral position at the junction between segments 7 and 8 (s7, s8) and segments 8 and 9 (s8, s9). Ventral view. h Arrows point to CapI-nanos-positive genital ducts between segments 7 and 8 (s7, s8) in males. Weak CapI-nanos mesodermal expression is at the junction between segments 8 and 9 (arrowheads), ventral view. i CapI-nanos is expressed in both the ectoderm (ec) and mesoderm (me) in the posterior growth zone (ventral view). Mesodermal expression extends posteriorly along the medial edge of a longitudinal muscle (lm) into the pygidium (arrows). j Surface ventral view showing CapI-nanos expression in segmentally iterated ectodermal cell clusters immediately lateral to the ganglia of the ventral nerve cord in newly forming segments at the posterior end of the animal (vertical arrows). Diagonal arrows point to mesodermal expression in the pygidium. k CapI-vasa is expressed in the mesoderm of the posterior growth zone (diagonal arrows) and in newly formed segments, ventral view. Vertical arrows point to segmentally iterated ectodermal cell clusters positioned lateral to the ventral ganglia. l Schematic of adult hermaphrodite showing position of structures highlighted in a–k, ventral view. The distinct thoracic (Th) and abdominal (Abd) body regions are marked. mo Mouth, pgc putative primordial germ cells, gd genital ducts, ov ovary, gz growth zone, Nos nanos, vas vasa

Full size image

CapI-vasa and CapI-nanos are expressed within the ovaries of adult females. The ovaries of Capitella sp. I are paired ventral structures within the coelomic cavity that straddle the lateral edges of the accessory intestine and span many mid-body segments, beginning in the first abdominal segment, segment 10 (Fig. 4l; Eckelbarger et al. 1984). Each ovary contains oocytes at different stages of development (Eckelbarger et al. 1984). When examined in detail, it is clear that CapI-vasa and CapI-nanos transcripts are present in the cytoplasm of immature oocytes but are not detected in mature oocytes within the same ovary (Fig. 4e,f). Mature clusters of oocytes examined in ovaries of other animals do not express CapI-vasa (not shown).

In young adult males, CapI-nanos and CapI-vasa are expressed in bilateral cell clusters in a ventro-lateral position within the coelom at the junction between segments 7 and 8 and 8 and 9 (Fig. 4g and data not shown). These clusters are attached to the peritoneum, and their nuclei appear to occupy most of the area of the cell. Although Capitella sp. I lacks a well-defined testis, specialized proliferative zones of cells attached to the body wall between the seventh and eighth segments and in the eighth segment have been described where spermatogonia are released into the coelom (Eckelbarger and Grassle 1987b). We hypothesize that these CapI-nanos and CapI-vasa-positive cell clusters represent male gamete precursors. In more mature males (manifest by the presence of dorsal genital spines), paired genital ducts are visible at the junction of segments 7 and 8 (Eckelbarger and Grassle 1987b; Fig. 4h,l). The genital ducts are positioned dorsal to the CapI-nanos and CapI-vasa-positive cell clusters visible at the junction between segments 7 and 8 (Fig. 4h). Although the genital ducts store mature sperm, we could not unambiguously determine whether the expression we observed in this structure was in the somatic tissue of the coelomoduct or in sperm stored within the coelomoduct. Capitella sp. I also exhibits hermaphroditism (Petraitis 1985), and in these animals, CapI-nanos and CapI-vasa is expressed in maturing oocytes as well as in the ventro-lateral clusters at the junction of segments 7 and 8 and 8 and 9 (not shown). Expression of CapI-nanos and CapI-vasa in both male and female gametes contrasts with expression of these genes in Platynereis, in which expression of vasa is only observed in female gametes (Rebscher et al. 2007).

Expression of CapI-nanos and CapI-vasa in the posterior end of adults is very similar for both genes and includes ectodermal and mesodermal cells of the posterior growth zone (Fig. 4i) and ectodermal cell clusters lateral to the ganglia of the ventral nerve cord in newly formed posterior segments (Fig. 4j,k).

Comparisons of CapI-vasa and CapI-nanos expression patterns

Our whole-mount in situ hybridization studies demonstrate that CapI-nanos and CapI-vasa show largely overlapping expression patterns during embryonic, larval, juvenile, and adult stages, although CapI-nanos has a broader expression profile at several life history stages. Within the same cells, nuclear localization of CapI-nanos during cleavage and gastrulation stages is quite distinct from cytoplasmic distribution of CapI-vasa. CapI-vasa and CapI-nanos exhibit both somatic and germ line expression; thus, in Capitella sp. I, neither transcript is restricted to the germ line. The expression patterns observed at earlier stages are quite broad and include many tissues, although by late larval stages, robust expression of CapI-nanos and CapI-vasa is restricted to the PGCs and the posterior growth zone. A similar progression of expression patterns (broad somatic expression followed by more restricted expression in PGCs) has recently been described for a vasa orthologue in P. dumerilii (Rebscher et al. 2007; Zelada-González 2005), and in the oligochaete T. tubifex (Oyama and Shimizu 2007).

Overlapping perinuclear expression of both CapI-nanos and CapI-vasa in ventro-lateral clusters of cells at larval stages 6–8 identifies a population of putative PGCs. Although early expression of CapI-nanos and CapI-vasa in the PGCs is downregulated long before there is evidence of germ line differentiation, re-expression in the PGCs at the same location and in the gonads is apparent in young adults. Analysis of CapI-nanos and CapI-vasa expression in the PGCs and gonads has led us to the following testable hypothesis concerning the origin and development of PGCs in Capitella sp. I. The PGCs arise from the mesodermal precursor cell, 4d, and are visible in segment 4 as bilateral clusters of three to six cells each at larval stage 6. These two clusters converge at the ventral midline to become a single PGC cluster during late larval stages (stage 8). In young adults, the PGC clusters proliferate, resulting in a large cluster of more than 50 cells in sexually mature adults. From larval to adult stages, the PGCs move posterior by one segment, from segment 4 to 5. PGCs migrate from this large cluster to the appropriate gonad where they undergo gametogenesis. Capitella sp. I maintains a population of PGCs in sexually mature adults, which can respond to environmental stimuli to induce hermaphroditism. Maintenance of PGCs in sexually mature adults also serves as a store for the generation of multiple broods per individual or to populate regenerating ovaries. Future cell lineage studies will be needed to unambiguously determine if the cells we have identified as PGCs arise from 4d and eventually give rise to germ cells.