Gonad establishment during asexual reproduction in the annelid Pristina leidyi.

Developmental Biology 405 (2015) 123–136

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Evolution of Developmental Control Mechanisms

Gonad establishment during asexual reproduction in the annelid Pristina leidyi B. Duygu Özpolat n,1, Alexandra E. Bely n Department of Biology, University of Maryland, College Park, MD 20742, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 18 November 2014 Received in revised form 15 March 2015 Accepted 2 June 2015 Available online 29 June 2015

Animals that can reproduce by both asexual agametic reproduction and sexual reproduction must transmit or re-establish their germ line post-embryonically. Although such a dual reproductive mode has evolved repeatedly among animals, how asexually produced individuals establish their germ line remains poorly understood in most groups. We investigated germ line development in the annelid Pristina leidyi, a species that typically reproduces asexually by paratomic fission, intercalating a new tail and head in the middle of the body followed by splitting. We found that in fissioning individuals, gonads occur in anterior segments in the anterior-most individual as well as in new heads forming within fission zones. Homologs of the germ line/multipotency genes piwi, vasa, and nanos are expressed in the gonads, as well as in proliferative tissues including the posterior growth zone, fission zone, and regeneration blastema. In fissioning animals, certain cells on the ventral nerve cord express a homolog of piwi, are abundant near fission zones, and sometimes make contact with gonads. Such cells are typically undetectable near the blastema and posterior growth zone. Time-lapse imaging provides direct evidence that cells on the ventral nerve cord migrate preferentially towards fission zones. Our findings indicate that gonads form routinely in fissioning individuals, that a population of piwi-positive cells on the ventral nerve cord is associated with fission and gonads, and that cells resembling these piwi-positive cells migrate along the ventral nerve cord. We suggest that the piwi-positive ventral cells are germ cells that transmit the germ line across asexually produced individuals via migration along the ventral nerve cord. & 2015 Elsevier Inc. All rights reserved.

Keywords: Piwi Vasa Nanos Gonad development Germ line Fission Asexual reproduction Regeneration Annelida

1. Introduction Germ line cells are a key cell type that is the source of gametes and is therefore required for sexual reproduction. Establishing and maintaining germ line is thus critical in the life cycle of most animals. It was long thought that in animals a deep and permament distinction was established early on in embryogenesis between germ line and somatic cell populations (Weismann, 1893). This view was initially supported by findings from model organisms such as fruit flies, C. elegans, zebrafish and mouse in which the germ line is segregated during early embryogenesis and in which deletion of germ line (or its precursors) leads to sterility (Carmell et al., 2007; Cox et al., 2000, 1998; Houwing et al., 2007; Sulston et al., 1983). However, as a wider diversity of organisms have been n

Corresponding authors. Fax: þ 1 301 314 9358. E-mail addresses: [email protected] (B.D. Özpolat), [email protected] (A.E. Bely). URL: http://www.life.umd.edu/biology/bely/belylab/BelyLab/Home.html (B.D. Özpolat). 1 Current address: Evolution & Development of Metazoans Lab, Institut Jacques Monod, Cedex 13, Paris 75005, France. http://dx.doi.org/10.1016/j.ydbio.2015.06.001 0012-1606/& 2015 Elsevier Inc. All rights reserved.

investigated, it has become clear that germ line development is much more variable than work on these models initially suggested (Extavour and Akam, 2003; Juliano et al., 2010). In contrast to findings from some arthropod, nematode, and vertebrate models, studies in diverse invertebrate groups indicate that germ line development may occur across a range of developmental stages and in several developmental contexts. Some animals establish germ line relatively late during embryogenesis (Cho et al., 2014; Extavour and Akam, 2003; Juliano et al., 2010; Kang et al., 2002) and in groups such as flatworms and hydra, the germ line can even be established in the adult phase, from adult stem cells (Bosch and David, 1987; Gustafsson, 1976; Ladurner et al., 2000; Wang et al., 2007). Germ line and/or gonads can also be regenerated in a range of animals, including in hydra, flatworms, ascidians, and annelids (Egger et al., 2006; Giani et al., 2011; Herlant-Meewis, 1946a; Nishimiya-Fujisawa and Sugiyama, 1995; Pfister et al., 2008; Sato et al., 2006; Tadokoro et al., 2006; Takamura et al., 2002). Some animals can also reproduce by agametic asexual reproduction (e.g., fission, budding) and asexually produce offspring that are capable of becoming sexually mature (Brusca and Brusca, 2003; Drewes and Brinkhurst, 1990; Ghiselin, 1987; Gustafsson et al., 2009; Kobayashi et al., 1999; Sköld et al.,

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B.D. Özpolat, A.E. Bely / Developmental Biology 405 (2015) 123–136

2009; Sunanaga et al., 2006). In such animals, germ line must be transmitted over asexual generations (e.g., Stoner and Weissman, 1996) and/or established anew in asexually formed animals. Studies of post-embryonic germ line development remain far more limited than those focusing on embryonic development, however, and additional studies are needed to determine in which groups and by what mechanisms post-embryonic germ line development occurs. Annelids, or segmented worms, are a useful group for studying the establishment and maintenance of germ line during postembryonic development. This group includes many species capable of extensive regeneration, including of gonads (Giani et al., 2011; Herlant-Meewis, 1946a; works by Janda reviewed in Stephenson, 1930; Sugio et al., 2008; Tadokoro et al., 2006). Asexual reproduction by fission has evolved several times within annelids and occurs in numerous species known to also retain the ability to reach sexual maturity (Bely, 1999; Christensen, 1994; Schroeder and Hermans, 1975; Zattara, 2012). Typically, such fissioning annelids undergo many asexual generations interspersed by short bouts of sexual reproduction, often at a characteristic time of year (Dehorne, 1916; Loden, 1981). Although germ line must be maintained and/or re-established during regeneration and asexual reproduction, the processes by which this occurs in annelids remain poorly known. Several genes are characteristically expressed in germ line across the Metazoa and can thus be useful markers for studies of germ line development in a variety of contexts and animal groups. piwi, nanos and vasa are among the most conserved of these, being expressed in germ cell lineages and required for germ line function in diverse animals (Juliano et al., 2010). piwi, an Argonaute gene, encodes a regulatory protein involved in transposon silencing and RNA interference; nanos encodes a zinc-finger protein that acts as a translational repressor; and vasa encodes an ATP-dependent RNA DEAD box helicase that acts as a translational regulator. Functional studies in disparate animal groups indicate that these genes are required for germ line development, germ line stem cell maintenance, and/or gametogenesis (piwi: Cox et al., 2000, 1998; reviewed in Juliano et al., 2011) (nanos: Bhat, 1999; Kobayashi et al., 1996; Sada et al., 2009; Wang and Lin, 2004) (vasa: reviewed in Gustafson and Wessel, 2010; Schupbach and Wieschaus, 1986). Although piwi, nanos, and vasa were initially thought to be strictly restricted to the germ line, more recent studies in diverse animals have shown that they and other “germ line genes” are frequently expressed in pluri-/multipotent cells that may give rise to somatic cells and/or germ line (reviewed in Juliano et al., 2011; Solana, 2013). For example, human hematopoetic stem cells, salamander limb regeneration blastemas, planarian adult stem cells, and hydra budding and regeneration zones all express such “germ line genes” (Mochizuki et al., 2000; Sharma et al., 2001; Solana, 2013; Zhu et al., 2012). These genes are thus better described as being part of a germ line/multipotency program that has a broader role in governing “stemness” (Juliano et al., 2011). Expression of germ line/multipotency genes has been investigated in a few annelids, namely species of the clitellates Enchytraeus, Tubifex, and Helobdella and the polychaetes Capitella, Platynereis, Ophryotrocha, and Myzostoma. In all of these, some combination of piwi, vasa, and nanos homologs are expressed in the germ line, with expression having been detected in embryos, in presumptive germ cell precursors, and/or in juveniles or adults, in developing and mature gonad tissue (Brubacher and Huebner, 2011; Cho et al., 2014; Dill and Seaver, 2008; Gazave et al., 2013; Giani et al., 2011; Kang et al., 2002; Oyama and Shimizu, 2007; Pilon and Weisblat, 1997; Rebscher et al., 2012, 2007; Sugio et al., 2008; Tadokoro et al., 2006; Weigert et al., 2013). Thus, germ line/ multipotency genes are useful for identifying germ line cells in annelids. In addition, in several of these species, these genes have

also been found to be expressed in highly proliferative somatic tissues, such as the posterior growth zone (Cho et al., 2014; Gazave et al., 2013; Giani et al., 2011; Sugio et al., 2008). The annelids that have been investigated thus far are incapable of reproducing asexually, with the exception of Enchytraeus japonensis, which can reproduce by fragmentation; however, the study of E. japonensis focused specifically on regeneration, not asexual reproduction (Tadokoro et al., 2006). Thus, there has not yet been an investigation of germ line/multipotency genes during agametic asexual reproduction in annelids, and maintenance and transmission of germ line during asexual reproduction has remained virtually unstudied. Pristina leidyi Smith (Clitellata: Naididae) is a small, freshwater, largely transparent naidid annelid that can grow continuously, can regenerate all body regions, and can reproduce asexually by fission, making it an attractive model for investigating post-embryonic development. Under favorable conditions, P. leidyi adds new segments continuously from a subterminal posterior growth zone and reproduces asexually by paratomic fission (Bely and Wray, 2001; Dehorne, 1916; Zattara and Bely, 2011). During paratomic fission, a new tail and a new head develop within a midbody segment, forming a region of new tissue referred to as a fission zone. The process produces a chain of linked worms, or zooids, that eventually separate. Under high food conditions, multiple fission zones can develop in the same individual, with additional fission zones being initiated in progressively more anterior segments as well as in the mid-body region of the posterior worm. Although P. leidyi reproduces only asexually in the lab (the cues for initiation of sexual reproduction being unknown), in nature individuals of this species do become sexually mature at characteristic times of year (Loden, 1981; Stephenson, 1930). Sexual individuals are hermaphroditic and develop mature gonads in two consecutive segments, a pair of testes developing in segment 6 and a pair of ovaries developing in segment 7 (corresponding, respectively, to segments VII and VIII in classical oligochaete segment notation) (Sperber, 1948). When amputated, P. leidyi rapidly regenerates anteriorly or posteriorly, developing a new head or tail in just 4–5 days (Zattara and Bely, 2011). The goals of this study were to investigate whether asexually reproducing individuals possess gonads, how germ line/multipotency markers are expressed during post-embryonic developmental processes, and how germ line is established or maintained during paratomic fission in the annelid P. leidyi. Our study represents the first investigation in annelids of how germ line might be transmitted during asexual reproduction by paratomic fission, and the first use of time-lapse imaging to investigate the process of fission. We characterize the expression of piwi, nanos, and vasa homologs during regeneration and fission, identify piwi-positive gonads in asexual adults, identify a population of piwi-positive ventral cells we suggest may be migratory germ cells during fission, characterize the spatial relationship between these piwi-positive ventral cells and the regeneration blastema and the fission zone, and use live-imaging to investigate during fission the cell migration behaviors of cells resembling these piwi-positive ventral cells. Finally, we propose a working model for the transmission of germ cell precursors to asexually produced progeny in P. leidyi.

2. Materials and methods 2.1. Animal material Batch cultures of P. leidyi were maintained in 8-in. glass bowls in artificial spring water (purchased sea salt diluted to a concentration equivalent to 1% seawater) with paper towel as a substrate and dried Spirulina powder as food. Animals were fed


approximately once a week with 0.01 g Spirulina. Under these conditions, worms reproduce continuously by fission. To obtain rapidly fissioning individuals that possessed multiple fission zones, batch cultures were fed a 0.07 g pulse of Spirulina five days prior to collection. For certain experiments, worms were maintained individually in wells of a 24-well plate with 1.5 ml spring water and a feeding regime as indicated for the particular experiment (see sections below). To elicit regeneration, worms were anesthetized in 50 μM nicotine in spring water and amputated either anteriorly or posteriorly with a scalpel. Because the presence of fission zones can delay or inhibit regeneration (Zattara and Bely, 2013) worms without visible fission zones were used as regeneration material. Chaetae (segmental bristles) were used as visual landmarks for determining the desired amputation location; cuts were placed between the chaetae of consecutive segments, as noted below. Regenerating worms were maintained in spring water without food until the desired time after amputation. 2.2. Gene cloning and phylogenetic analysis To identify P. leidyi homologs of piwi and vasa, we searched (using tblastn) a P. leidyi fission/regeneration transcriptome (Nyberg et al., 2012) using as queries publicly available sequences of gene homologs from other annelids. We identified from the transcriptome two piwi homologs, PRIle-piwi1 (pISOTIG33900) and PRIle-piwi2 (pISOTIG80454), and one vasa homolog, PRIle-vasa (with sequence from pISOTIG61714 and pISOTIG92288 from the published transcriptome and Contig80724 from an earlier, unpublished transcriptome assembly). Extensive queries with vasa sequence from P. leidyi and other annelids did not yield any other vasa homologs in the transcriptome. PRIle-nanos sequence was cloned in a previous study (Bely and Sikes, 2010). We confirmed and extended the transcriptome sequences of the piwi and vasa homologs using PCR and RACE (primers listed in Suppl. Table 1). 3′ RACE was successfully performed for PRIle-piwi1, PRIle-piwi2, and PRIle-vasa and 5′RACE was successfully performed for PRIle-piwi1. All RACE reactions were carried out using Clonetech's 5′ and 3′ RACE Kit following the manufacturer's protocol. PCR and RACE fragments were gel extracted, sequenced, and, if used for in situ probes, cloned into PGEM-T Easy vector (Promega). Transcriptome, PCR, and RACE sequences were assembled in Geneious (6.1.7). We confirmed the identity of cloned sequences through phylogenetic analysis. Amino acid translations of the three P. leidyi genes were aligned to homologs and outgroups from other species using ClustalW implemented through Geneious. The piwi amino acid alignment included the PRIle-piwi1 amino acid sequence (including the PAZ and PIWI domains), the PRIle-piwi2 amino acid sequence (including the PAZ and most of the PIWI domains), piwi homologs from across the Metazoa, and Argonaute genes as outgroups (Suppl. Table 2). The vasa amino acid alignment included the first 500 amino acids of PRIle-vasa (including the DEAD box and HELICc domains), vasa homologs from across the Metazoa, and PL-10 genes as outgroups (Suppl. Table 3). Phylogenetic relationships were analyzed using MRBAYES (Huelsenbeck and Ronquist, 2001), implemented through Geneious using default parameter settings. 2.3. Whole mount in situ hybridization In situ hybridization was carried out as previously described (Bely and Wray, 2001) except that fixations (4% formaldehyde in 3/ 4 PBS for 30 min) and all steps (except for Pronase E digestion) until prehybridization were performed on ice. The template for the PRIle-piwi1 probe was a 1900 bp 3′RACE fragment containing most of the PIWI domain and 3′UTR (positions 990–2968 in

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GenBank KM078049). Despite considerable efforts and performing several probe synthesis reactions, we were unable to obtain in situ hybridization signal with the PRIle-piwi2 sequence. The template for the PRIle-vasa probe was a 2250 bp PCR product containing the DEADBox, HELICc domain, and 3′UTR (positions 229–2481 in GenBank KM078051). The template for the PRIle-nanos probe was 1450 bp containing the Nanos RNA-binding domain (GenBank GQ369728.1). Processed samples were mounted in 25% PBS/75% glycerol and imaged using a Zeiss Axioplan2 microscope, a Zeiss AxioCam HRc camera, and AxioVision (v4.8.2) software. PPVC scoring: We analyzed the number of piwi-positive ventral cells (PPVCs) along the total length of the body in 10 worms from a batch culture that was maintained in high food conditions (0.1 mg/ week Spirulina). For these scorings, we divided the body into six regions: the head (prostomium and 4 anterior-most segments), gonad region (next 3 segments), anterior trunk (next 4–7 segments), fission zone(s), posterior trunk (8–11 segments), and posterior growth zone and pygidium. To standardize the number of positive cells in body regions of different sizes, the number of positive cells was divided by the number of segments in the body region, with the fission zone considered as equivalent to 1–3 segments (depending on the number of fission zones) and the posterior growth zone and pygidium being considered, together, as equivalent to 1 segment. 2.4. Methyl green-pyronin staining Whole worms were fixed in 70% EtOH overnight at room temperature, hydrated to PBSt (PBS þ1% Tween 20), stained in methyl green-pyronin (MGP) (Sigma, #HT70) for 1–2 h at room temperature, rinsed once in 90% EtOH and twice in 100% EtOH, transferred to EtOH/xylenes (1:1), and finally transferred to xylenes. Samples were mounted in Permount (Fisher) and imaged as described for in situ hybridization samples. 2.5. Statistical analysis Wilcoxon rank sum tests were performed in R statistical computing environment (R Development Core Team, 2011) and exact tests of independence and goodness of fit were calculated using online spreadsheets (www.biostathandbook.com) from McDonald (2014). Error bars in all graphs represent standard errors. 2.6. Time-lapse imaging The migration of cells on the ventral nerve cord, where PPVCs are located, was documented during early fission by time-lapse imaging. To obtain animals with very early fission zones and high transparency (little gut pigmentation), we imaged recent offspring of rapidly growing worms. Sets of worms were cultured individually in 24 well plates with high food (0.1 mg/week, administered over two feedings). Posterior offspring of these worms typically had a very early fission zone themselves and were collected for imaging. Time-lapse imaging was carried out according to the methods of Zattara (2012). Briefly, worms were paralyzed by incubation in 3–10 mM tetrodotoxin overnight at room temperature, washed in spring water, mounted in 1% low-melting agarose (SeaPlaque), and covered with a cover glass whose edges were sealed with 146 halo-carbon oil (FLY-70007, LabScientific) to prevent drying. A z-stack with 5–10 steps (centered on the best view of the ventral nerve cord) with 2 μm intervals was acquired every 3 min under DIC optics using the same imaging setup described for in situ hybridization. Time-lapse movies were made of nine early fission zones (from nine different individuals), each for at least 6 h. We analyzed the first 6–13 h of each movie, since imaged animals typically look

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healthy through at least the first 12–24 h. Three movies were centered on the fission zone (FZ), three movies were centered on the region immediately anterior to the fission zone (A), and three movies were centered on the region immediately posterior to the fission zone (P), though movies varied in how much of the imaged area and which body regions were scorable. From these movies, we recorded the movements of cells that had features characteristic of PPVCs, specifically, cells that resided on the dorsal surface of the ventral nerve cord and had a spindle-shape, with large surface area contacting the ventral nerve cord. We scored any such cell that could be clearly discerned that was in the fission zone region or within two segments immediately anterior or posterior to it, following cells across z-planes if needed. Cells that moved faster than 1 μm/min (4 cells out of 60 cells scored) were difficult to track confidently across frames and therefore excluded from the final scoring. Each cell that met our criteria was classified as one of the following (scoring method 1): moving anteriorly (if that cell moved only anteriorly during the movie or migrated a longer distance anteriorly than posteriorly), moving posteriorly (if that cell moved only posteriorly or migrated a longer distance posteriorly than anteriorly), showing non-directional movement (if the cell moved both anteriorly and posteriorly and did so about the same distance in each direction, thus without a clear preference in direction), or not moving during the movie. We also analyzed the same data using an alternative scoring method (scoring method 2), in which any cell that moved both anteriorly and posteriorly was scored as having non-directional movement,

regardless of whether or not it showed a directional preference.

3. Results 3.1. Identification of piwi and vasa homologs in P. leidyi We identified in P. leidyi two homologs of piwi, PRIle-piwi1 (GenBank accession KM078049) and PRIle-piwi2 (GenBank accession KM078050), and one homolog of vasa, PRIle-vasa (GenBank accession KM078051); an earlier study had already identified a homolog of nanos (PRIle-nanos) (Bely and Sikes, 2010). For PRIlepiwi1, we obtained 2968 bp of sequence including the PAZ domain (from aa138 to aa250), the PIWI domain (aa259–aa704), and 3′ UTR. For PRIle-piwi2, we obtained 1510 bp of sequence including the PAZ domain (aa26–aa154) and most of the PIWI domain (aa162–aa503). For PRIle-vasa, we obtained 3113 bp of sequence including part of the DEAD box helicase domain (aa1–aa199), the HELICc domain (aa210–aa309), and 3′UTR. Phylogenetic analysis confirms that PRIle-piwi1 and PRIle-piwi2 are in the piwi gene family and that PRIle-vasa is in the vasa gene family (Suppl. Figs. 1 and 2). Several annelids have two piwi paralogs identified; although orthology relationships among annelid genes are not fully resolved, PRIle-piwi1 is in a subclade that includes Capitella piwi1 and a Platynereis piwi homolog and PRIle-piwi2 appears to be an ortholog of Capitella piwi2 and Helobdella piwi2. For vasa, only single gene copies have been identified from annelids investigated

Fig. 1. Expression of PRIle-piwi1, PRIle-nanos and PRIle-vasa in areas of growth. PRIle-piwi1 (A–C), PRIle-nanos (D–F), and PRIle-vasa (G–I) are each expressed in the fission zone, posterior growth zone (PGZ) and regeneration blastema. PRIle-nanos is also expressed at the base of the proboscis (J), as well as in cells of the proximal part of the prostomium (J'). Arrows in B, E, and H indicate the pygidium, in which no expression is detected. Arrowheads in H' indicate internal patches of labeled tissue in young segments. Cartoon of worm (K) illustrates location of proboscis/prostomium, fission zone, and PGZ areas (boxes), as well as the location of the stomach (swelling in segments 7 and 8) and gonads (shading in segments 6 and 7). In this and in all subsequent figures, anterior is to the left and views are lateral unless otherwise indicated. Gray bars indicate the extent of new tissue made in fission zones and blastemas. Scale bar (black): 100 mm.


thus far; PRIle-vasa forms a well-supported clade with the other annelid vasa genes, suggesting direct orthology to these. 3.2. Expression of piwi, nanos and vasa homologs in zones of growth PRIle-piwi1, PRIle-nanos, and PRIle-vasa are strongly expressed in highly proliferative zones in adults, most notably in the posterior growth zone, the fission zone, and the regeneration blastema (Fig. 1). In growing adults, all three genes are expressed broadly in the posterior growth zone. Expression is strongest in the most posterior part of the growth zone and fades anteriorly, in progressively older segments (Fig. 1B, B', E, E', H, H'). None of the genes are detected in the pygidium, the posterior asegmental cap of tissue (arrows in Fig. 1B, E, and H). In addition to the broad diffuse staining in the posterior growth zone, PRIle-piwi1 and PRIle-vasa are expressed in internal (likely mesodermal), ventrolateral patches of cells (arrowheads in Fig. 1H'), as well as in the epidermis (Suppl. Fig. 3) in young segments. PRIle-nanos, but not the other genes, is also detected in a ring at the base of the asegmental,

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elongated proboscis, in a region previously shown to be an anterior growth zone (Zattara and Bely, 2013), as well as in scattered epidermal cells of the proximal (non-elongated) part of the prostomium (Fig. 1J and J'). During fission, all three genes are expressed in the fission zone, in both the developing head and tail regions, and in both epidermis and internal tissues (Fig. 1A, A', D, D', G, and G'). Expression is mostly broad and even in the fission zone, except that the posterior border of the new developing tail (where a new posterior growth zone is established) shows stronger expression (Fig. 1A', D', and G'). This pattern is most evident in PRIle-nanos and PRIle-vasa (Fig. 1D', and G'), in which posterior-most expression is very strong and expression in the rest of the fission zone is considerably weaker; by comparison, the relative expression of PRIlepiwi in the posterior border and the rest of the fission zone is much more similar (Fig. 1A'). During regeneration, all three genes are highly expressed in both anterior and posterior blastemas (anterior blastemas shown in Fig. 1C, F, and I). Expression is strongest in internal (mesenchymal) cells of the blastema, with weaker expression also detected

Fig. 2. Expression of PRIle-piwi1, PRIle-nanos and PRIle-vasa in gonads. (A and A') PRIle-piwi1 is expressed in prospective testes and ovaries (arrowheads) in segments 6 and 7, respectively. (B) In the developing head of a fission zone, PRIle-piwi1-positive cell clusters in segments 6 and 7, presumed to be developing gonads, have a flat anterior border and contact the new septa between segments 5 and 6 and between segments 6 and 7. (C and C') PRIle-piwi1 is sometimes detected in cell clusters anterior to segments 6 and 7 (arrows); these cells are presumed to be supernumerary germ cell clusters. In panel C, positive cell clusters occur between chaetal bundles of segments 1/2, 3/4, 4/5 (arrows) in addition to cell clusters between chaetal bundles of segments 5/6 and 6/7 (arrowheads). (D and E) PRIle-nanos and PRIle-vasa are expressed in prospective testes and ovaries but staining appears weaker and more diffuse than for PRIle-piwi1. (F and F') MGP staining reveals compact ventrolateral clusters of cells (arrowhead in F') in the same location as PRIle-piwi1, PRIle-nanos, and PRIle-vasa expression. F' is a magnified view of F. t-testis; o-ovary; s-stomach. Scale bars: 100 mm.

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in the overlying ectoderm. 3.3. Asexual P. leidyi possess gonads that express piwi, nanos and vasa homologs Although P. leidyi reproduces only asexually under our lab conditions, we found gonad structures positive for germ line/ multipotency genes in asexually reproducing individuals. Published descriptions of sexually mature Pristina and close relatives indicate that paired, ventrolateral testes and ovaries occur in segments 6 and 7, respectively, and that these gonad structures extend posteriorly from the anterior septum of their respective segments (Brinkhurst and Jamieson, 1971; Sperber, 1948). We found that fissioning P. leidyi from rapidly growing cultures strongly express PRIle-piwi1 in ventrolateral clusters of cells in segments 6 and 7 (Fig. 2A, and A'). Furthermore, within fission zones, where new head segments are developing, these cell clusters develop with a flat anterior border where they are in contact with the anterior septum (Fig. 2B). Although P. leidyi is reported as forming only segments 1–6 of the head during fission (and not segment 7, in which we find gonad structures), our careful analysis of fixed material at a range of stages indicates that the septum between segments 6 and 7 is made within the fission zone, as is the septum between segments 5 and 6. Thus, both the 5/6 septum and the 6/7 septum, to which the cell clusters are connected, are newly formed during fission. The cell clusters are transparent and typically small and can be difficult to see in unstained individuals, even when viewed under high magnification with DIC optics. However, staining with methyl-green pyronin (MGP), which stains DNA blue-green and RNA (cytoplasm) red (Kurnick, 1950), reveals these cell clusters in segments 6 and 7 (Fig. 2F and F'). We interpret these findings as indicating that asexually reproducing P. leidyi develop gonads that express PRIle-piwi1 (and other germ line/multipotency gene markers, see below) and that gonads form during fission in association with the posterior face of newly formed anterior septa of segments 6 and 7. Although most actively fissioning individuals have PRIle-piwi1 expression in paired cell clusters presumed to be gonads in segments 6 and 7 (that is, in 4 clusters), some individuals show fewer (0–3) positive clusters and we also very occasionally find individuals that have more than 4 clusters and/or clusters anterior to segment 6 (Fig. 2C, and C'). These extra clusters are typically ventrolateral (like the gonads in segments 6 and 7) but sometimes more dorsal in position (Fig. 2C', arrow). In one batch of worms, the frequency of individuals with PRIle-piwi1-positive clusters anterior to segment 6 was 3/21, but it is usually much lower. PRIle-nanos and PRIle-vasa are also expressed in prospective testes and ovaries (Fig. 2D, D', E, and E'), although this expression is more diffuse, with less distinct borders, than PRIle-piwi1 expression. Clusters of positive cells anterior to segment 6 were very occasionally seen for PRIle-vasa, but were even less common than for PRIle-piwi1. Processing subsamples of worms from the same batch culture in parallel for either PRIle-piwi1, PRIle-nanos, or PRIle-vasa indicates that PRIle-nanos and PRIle-vasa staining is weaker and less consistently detected in gonads than is PRIle-piwi1 staining. In one batch, expression in prospective testes and ovaries was detectable in all (9/9) individuals processed for PRIle-piwi1 but detectable in less than half (3/8) of individuals processed for PRIlevasa (3/8 had only prospective testes expression; 2/8 completely lacked gonad expression), and detectable in only one quarter (3/12) of individuals processed for PRIle-nanos. A similar pattern was found in two additional batches of worms processed in this way (data not shown). Because we found PRIle-piwi1 to be the most robust marker of gonads, we focused primarily on PRIlepiwi1 for the rest of our studies.

3.4. PRIle-piwi1 þ ventral cells are distributed along the ventral nerve cord and are associated with gonads during fission In addition to being expressed in zones of growth and in prospective gonads, we found that in actively fissioning animals PRIlepiwi1 is expressed in a population of cells on the ventral nerve cord (Fig. 3). We refer to this population of cells as piwi-positive ventral cells, or PPVCs. These cells are typically spindle-shaped with a large nucleus and occur along the ventral midline, contacting the dorsal surface of the ventral nerve cord (Fig. 3A, A', and C). They are found along most of the length of the body (Fig. 3C), although infrequently in fully formed heads, and usually occur as a thin line of cells one cell wide, although, when the density of cells is high, cells are occasionally seen making a line up to two cells wide (Fig. 3F, inset). Occasionally, positive cells resembling these PPVCs are also detected lateral to the line, especially in young posterior segments (Fig. 3C and G). At early stages of fission, PPVCs are often numerous within and near the fission zone (Fig. 3B); early signs of gonad expression are evident in some individuals at these early stages as well (as early as Stage B, Zattara and Bely, 2011) (Fig. 3B). In well-fed, rapidly fissioning animals, some PRIlepiwi1 þ cells are occasionally found forming a line or an arc connecting the gonads (usually the testes, but sometimes the ovaries) to the line of PPVCs on the ventral nerve cord (Fig. 3D–F), suggesting an association between the cells on the nerve cord and the gonads. This configuration of cells was found in the gonadal segments of adult (anterior) individuals (Fig. 3D and E) as well as within developing head segments of mid- to late-stage fission zones (Fig. 3F). Even though the fission zone has tissues positive for PRIle-piwi1 in addition to newly-forming gonads, PPVCs were never observed to form a line or an arc associated with non-gonadal tissues. Given the distribution and associations of these cells, we hypothesize that PPVCs are germ cells or germ cell precursors that can stream into or out of gonads and migrate along the ventral nerve cord. 3.5. PRIle-piwi1 þ ventral cells are associated with head development during fission but not regeneration Although PPVCs have an irregular distribution along the ventral nerve cord, certain body regions tend to have more or fewer of these cells. In order to describe these patterns quantitatively, 10 well-fed worms with multiple fission zones were fixed, processed for PRIle-piwi1 in situ hybridization, and scored for PPVCs along the length of the body. The number of PPVCs was determined for each of six regions of the body (together comprising the entire worm): the head, the gonad region, the anterior trunk, the fission zone region, the posterior trunk, and the tip of the tail (Fig. 4A and B). We found that the average number of PPVCs/segment was lowest in the head and the tip of the tail (0.08 and 0 PPVCs/segment, respectively), intermediate in the gonad region and anterior trunk (0.8 and 1.37 PPVCs/segment), and highest in the fission zone region and posterior trunk (2.26 and 2.37 PPVCs/segment). Because the posterior trunk had the highest concentration of PPVCs, we analyzed the distribution of PPVCs in posterior zooids more finely (Fig. 4C and D; individual scorings provided in Suppl. Fig. 3). Specifically, we were interested in determining whether PPVCs tend to be associated with the anterior half of the zooid, close to where gonads are developing in the fission zone, or with the posterior half of this zooid, where the posterior growth zone is located. Proximity to developing gonads and the posterior growth zone was of particular interest because we speculated that these two regions were two likely sources or destinations of PPVCs. We found that the number of PPVCs was significantly higher (p o0.05) in the anterior half of the posterior trunk than in the posterior half, with approximately five times as many PPVCs in the anterior half


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Fig. 3. PRIle-piwi1 expression in cells on the ventral nerve cord and associated with gonads during fission. (A–G) Lateral views (A and A') and ventral views (B–G) of PRIle-piwi1 positive ventral cells (PPVCs) along the ventral nerve cord and in association with gonads in fissioning worms. Open arrowheads indicate some of the many PPVCs on the dorsal surface of the ventral nerve cord. A' is a magnified view of the cell surrounded by a dashed box in A, showing strong in situ signal in the anterior and posterior cytoplasm of the cell but signal being excluded from the large nucleus. Broad expression is associated with early- to mid-stage fission zones in the center of A, the left side of B, the middle of the central panel of C, and the left side of F. Expression in gonads is evident in B (developing head of a posterior zooid; angled arrows point to gonad expression just posterior to the early fission zone), C (left panel, adult head of anterior zooid), D (adult head of anterior zooid), E (adult head of anterior zooid) and F (developing head of posterior zooid; mid-stage fission zone); in these panels, t and o indicate prospective testes and ovaries, respectively. Note that the left-most positive cell cluster in D is presumed to be a supernumerary gonad cluster; this ventral view is of the same individual shown in Fig. 2C in lateral view. In C–F, PPVCs appear to form a line (C, D, F) or an arc (E) to the gonads. The three panels of C are anterior, middle, and posterior regions of the same worm. Inset in F is a different focal plane of that body region, superimposed in order to show the PPVCs along the ventral midline. Scale bars: 100 mm.

than in the posterior half (Fig. 4C). PPVCs were not only uncommon in the posterior half of zooids but they were almost never found in the posterior-most segments. In 9/10 individuals, the posterior zooids had 4 or more segments in front of the posterior growth zone that were devoid of PPVCs and, across the 10 individuals, there was an average of about five PPVC-free segments between the posterior-most PPVC and the posterior growth zone (Fig. 4D). Based on our dataset of 10 individuals, we found no obvious relationship between PPVC number and fission zone stage (Fig. 4E) or total worm length (Suppl. Fig. 3). Together, our data indicate that PPVCs are associated with the anterior part of the newly forming posterior zooids, where gonads are forming, and not with the posterior growth zone. Because PPVCs appear to be associated with head development during fission, we asked whether these cells are also associated with head development during regeneration. We were particularly interested in determining whether PPVCs or cells of the gonads could be the source of new PRIle-piwi1 positive cells of the regeneration blastema. To investigate this, we amputated worms (i) anteriorly, removing the anterior 2–4 segments (and thus leaving the gonad segments and at least 2 segments anterior to the gonads), or (ii) posteriorly, removing the posterior-most 4–8 segments. In both cases, we found similar results: we found no evidence suggesting that PPVCs or other PRIle-piwi1 positive cells are associated with blastema formation (Fig. 5). For both types of amputations, we performed a detailed time course of PRIle-piwi1 expression during regeneration, scoring 5–10 worms for each of 9 time points during regeneration spanning from 1 h post amputation (hpa) (well before the initiation of PRIle-piwi1 blastema expression) to 4 days post amputation (dpa) (after blastema expression fades). In these samples, we never found PPVCs in the

region between the gonads and the blastema in the anteriorly cut animals and only very rarely found PPVCs close to the blastema in the posteriorly cut animals. Thus, PRIle-piwi1 expression in the anterior and posterior blastemas appears to arise de novo. We also assayed PRIle-nanos and PRIle-vasa expression during anterior and posterior regeneration across the same 9 timepoints during regeneration (from 1 hpa to 4 dpa). As we found for PRIlepiwi1, we found that both markers are expressed in anterior and posterior blastemas but found no evidence suggesting an association between trunk cells positive for these markers and the development of the blastema (Suppl. Fig. 4). Thus, during both anterior and posterior regeneration, blastema expression of PRIlenanos and PRIle-vasa appears to arise de novo, as it does for PRIlepiwi1. 3.6. Time-lapse imaging reveals cells migrating along the ventral nerve cord towards fission zones Because our inference that PPVCs are migratory during fission was based on interpreting static in situ hybridization samples, we sought to obtain more direct evidence of cell movements. We therefore used a newly developed technique to perform timelapse imaging in P. leidyi (Zattara, 2012) to investigate the cell movement behaviors of cells in and around early fission zones. We imaged very early fission zones (early Stage A; Zattara and Bely, 2011), prior to the earliest stage at which we have detected gonads (early Stage B). We imaged such fission zones from 9 different worms, analyzing 6–13 h of continuous imaging from each animal (see Materials and methods for details). We then scored for cell movement within the fission zone and in segments immediately anterior and posterior to it, focusing specifically on cells that

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Fig. 4. Distribution of PRIle-piwi1 positive ventral cells (PPVCs) along the body. (A) Representative individual labeled for PRIle-piwi1 expression. Asterisks indicate the axial position of individual PPVCs in this individual. (B) Average number of PPVCs per segment in the six body regions along the body, based on ten scored individuals. Body regions are diagrammed in the cartoon above. Note that PPVCs are most common in the trunk of posterior zooids. (C) Number of PPVCs in three regions of the posterior zooid: the developing head, anterior half of the trunk, and posterior half of the trunk. The number of PPVCs in the anterior half of the posterior zooid is significantly greater than that in the posterior half (Wilcoxon paired test V ¼55, p ¼0.0057, n¼ 10). (D) Boxplot of the number of PPVC-free segments immediately anterior to the posterior growth zone. (E) PPVCs in the posterior zooid across stages of fission. The number of PPVCs in the newly forming head region (within the fission zone) and in the segments immediately posterior to it (segments 7–11) is plotted for each of the 10 individuals scored (see Suppl. Fig. 5), divided according to their stage of fission (stages B–D correspond approximately to early, mid-, and late fission; see Zattara and Bely (2011)).

possessed the characteristic of PPVCs (spindle-shaped cells residing on the dorsal surface of the ventral nerve cord and with a large surface area contacting the nerve cord). Time-lapse imaging movies revealed a range of behaviors of such focal cells on the ventral nerve cord (Fig. 6; Suppl. Figs. 5–7; Suppl. Movies A–C). We observed such cells moving anteriorly, cells moving posteriorly, cells changing direction, cells intermittently moving and stopping, as well as cells not moving at all during the course of imaging (Fig. 6A–A'''; Suppl. Figs. 5 and 6). Although focal cells exhibited a range of migration behaviors, we found that they moved preferentially toward the fission zone (Fig. 6C): in segments immediately anterior to the fission zone the

most common behavior (exhibited by 54% of focal cells) was posterior migration, in segments immediately posterior to the fission zone the most common behavior (exhibited by 42% of focal cells) was anterior migration, and at the fission zone the most common behavior was no movement (exhibited by 64% of focal cells). Using an alternative scoring method for categorizing cell movements (scoring method 2, see Materials and methods) showed a similar pattern in the data (Suppl. Fig. 7). We statistically analyzed the behavior of cells that migrated directionally in the region anterior to the fission zone and the region posterior to the fission zone. Since the proportion of directionally migrating cells that moved toward the fission zone (rather than away from the


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Fig. 5. Expression of PRIle-piwi1 during anterior and posterior regeneration. PRIle-piwi1 expression is shown throughout the course of regeneration, from 1 hpa to 4 dpa, following either anterior or posterior amputation. Red lines in top diagrams indicate the approximate amputation planes. Note that no expression is detected in the segments closest to the developing blastema. Gray bars mark the detectable blastema (new tissue); green bars mark PRIle-piwi1 expression at the wound site and in the blastema; orange bars mark segments adjacent to the blastema that have no detectable PRIle-piwi1 expression. The small to large spot of expression in the middle of most of the anterior regeneration panels is expression in the (original) gonads. During both anterior and posterior regeneration, PRIle-piwi1 expression becomes detectable shortly before the blastema becomes histologically evident. Specimens shown are representative of samples at that time point; at least five individuals per time point were assayed. Scale bar (black): 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Migration behaviors of PPVC-like cells near fission zones revealed by time-lapse imaging. (A–A''') Four frames of a representative time-lapse movie showing one migrating cell (arrowhead) and one stationary cell (asterisk) on the ventral nerve cord. Frames are from Suppl. Movie C and span approximately 15 min in total. (B) Cartoon of worm with multiple fission zones used as source material for time-lapse movies. Recently-detached posterior zooids with a very early fission zone were selected for imaging. The dotted box indicates the region imaged. (C) Migration behavior of cells on the ventral nerve cord near early fission zones. Data are from 9 time-lapse movies (from 9 different worms) and represent a total of 56 cell traces. Cell behaviors are presented for three regions: (1) “Anterior”: 1–2 segments anterior to the fission zone; (2) “Fission Zone (FZ)”: the fission zone itself; and (3) “Posterior”: 1–2 segments posterior to the fission zone. For each of the three regions, the percentage of cells in that region that moved posteriorly, did not move, moved anteriorly, or moved both anteriorly and posteriorly is shown, using scoring method 1 (see Materials and methods); see Suppl. Fig. 6 for the same data presented using scoring method 2. The size of the bars and arrows is proportional to the fraction of cells with that movement behavior. Note that cells on the ventral nerve cord that are anterior or posterior to the fission zone tend to move toward the fission zone, while such cells within the fission zone tend to not move. Scale bar: 50 mm.

fission zone) was not statistically different between the two regions (p ¼0.678; Fisher's exact test of independence, two-tailed), we pooled the data on directionally migrating cells for these two regions. We found that there were significantly more directionally migrating cells that migrated toward the fission zone (22 cells) than away from the fission zone (8 cells) (p ¼0.016; exact test of goodness of fit, two tailed). Therefore, although we do not yet have the tools in P. leidyi to track individual PRIle-piwi1 positive cells in real time, our time-lapse studies provide direct evidence that cells possessing PPVC-like characteristics migrate and do so preferentially toward the fission zone, where new gonads are forming.

4. Discussion 4.1. Asexually reproducing P. leidyi routinely develop gonads In animals capable of both asexual agametic reproduction and sexual reproduction, individuals typically undergo only one of these reproductive modes at a time (Adiyodi and Adiyodi, 1994, 1993; Hughes, 1989). A common pattern is that asexual

reproduction occurs in individuals that are not yet sexually mature, and that such individuals halt asexual reproduction before reproducing sexually. Several life history models similarly suggest that either sexual or asexual reproduction (not both) is favored under any one set of conditions (Hughes, 1989). For these reasons, sexual and asexual reproduction are often thought to be largely mutually exclusive of one another. In the lab, the annelid Pristina leidyi reproduces only asexually by paratomic fission. Gonads have been well described in sexually mature individuals of this species and other fissioning close relatives (Brinkhurst and Jamieson, 1971; Loden, 1981; Sperber, 1948). However, little is known about the presence or establishment of gonads in asexually reproducing individuals, beyond some early observations in some naidids that gonads and “reproductive cells” are sometimes detectable histologically in fissioning worms (Dehorne, 1916; Stephenson, 1930). We found that germ line/ multipotency gene expression and methyl-green pyronin staining reveal gonad structures in asexual P. leidyi. Gonads form in the ventrolateral region of segments 6 and 7, corresponding to where testes and ovaries (respectively) form in sexually mature individuals (Brinkhurst and Jamieson, 1971). These gonad structures


form routinely in our lab stocks, even while individuals fission, and even though the P. leidyi worms in our cultures do not become sexually mature. We infer that the lineage of P. leidyi we work on can maintain and/or re-establish germ line over a prolonged asexual period. The P. leidyi cultures in our lab have been propagated asexually for nearly 20 years (since ca. 1995) and they themselves are derived from asexual stocks of the Carolina Biological Supply Company that are thought to date back to the 1950’s, and possibly even earlier (S. Binkley from Carolina Biological Supply Company, pers. comm.). Therefore, our P. leidyi cultures have probably been reproducing by fission for over 60 years, during which we estimate they have undergone on the order of 1000–3000 rounds of fission. We thus conclude that our P. leidyi cultures have been establishing gonads over thousands of asexual generations. Several other agametically reproducing species have been shown to be capable of becoming sexually mature after a prolonged agametic asexual phase. For example, lab cultures of the planarian Dugesia ryukyuensis were induced to become sexually mature (by being fed sexual individuals of another flatworm species) after 15 years of continuous asexual propagation by fission (Kobayashi et al., 1999; Nakagawa et al., 2012). Although we have not been able to induce sexuality in our P. leidyi lab cultures, most species of fissioning naidids can become sexual (Loden, 1981; Stephenson, 1930) and some lab cultures of other naidid species occasionally produce some sexually mature individuals (Özpolat, Zattara, and Bely, unpublished data); future studies in such species should be helpful for testing whether the gonads and reproductive cells produced by asexual animals are functional in this group. 4.2. Germ line expression of germ line/multipotency genes in P. leidyi and other annelids Consistent with findings from other annelids, we found that in P. leidyi adults, homologs of piwi, vasa, and nanos are expressed in gonads, in both prospective testes and ovaries (Fig. 2) (Cho et al., 2014; Dill and Seaver, 2008; Gazave et al., 2013; Giani et al., 2011; Pilon and Weisblat, 1997; Rebscher et al., 2012; Sugio et al., 2008; Tadokoro et al., 2006). Germ line expression of these genes has thus been found in all adult annelids investigated to date and is likely an ancestral feature for annelids. However, the relative timing of expression of these genes and the specific germ line tissues in which these genes are expressed appear to be somewhat variable across annelid species and/or across developmental stages or processes. In P. leidyi, we found that the piwi homolog was expressed in gonads considerably more strongly and consistently than homologs of nanos and vasa, suggesting that expression of the piwi homolog may occur in a broader range of stages and/or tissues during gonad development than the nanos and vasa homologs, although finer scale and quantitative studies are needed to confirm this. However, in the embryos of the leech Helobdella robusta, the female primordial germ cells only express piwi and vasa while male primordial germ cells only express nanos (Cho et al., 2014; Kang et al., 2002); in the embryos of the polychaete Capitella teleta, homologs of piwi, vasa and nanos appear to be coexpressed in primordial germ cells (Dill and Seaver, 2008; Giani et al., 2011); and in juveniles of the polychaete Platynereis dumerilii, although homologs of both vasa and piwi are expressed in the primary gonad, vasa is expressed in developing oocytes of juveniles but not in males approaching maturity (Rebscher et al., 2012). Together, available data suggest some differences in the expression of piwi, vasa, and nanos may exist during germ line development across annelids. Broader sampling of annelid species, targeted comparisons across developmental modes (i.e., embryonic vs. post-embryonic), and resolution of orthology across gene homologs being studied in different species are all needed to help

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clarify how germ line development and expression have evolved within this group. In three of these annelid species, isolated cells inferred to be migratory germ line cells have been described and also shown to express some of these same genes. In P. leidyi adults, we found that these cells (located along the ventral nerve cord) strongly express a piwi homolog; we refer to these cells as PRIle-piwi1 positive ventral cells, or PPVCs (Fig. 3). However, nanos and vasa homologs are undetectable by in situ hybridization in these cells. The absence of detectable vasa expression in PPVCs is unexpected if these cells are indeed germ line precursors. In future studies, it will be worthwhile to determine whether these cells express vasa but at levels below detectability by in situ hybridization, whether they might express another homolog of vasa, and whether they express vasa protein. In E. japonensis adults, cells very similar to the PPVCs in P. leidyi are positive for piwi but also express vasa homologs (Sugio et al., 2008; Tadokoro et al., 2006). Finally, in P. dumerilii juveniles, the migratory primordial germ cells (four in number) express vasa (transcript and protein) as well as piwi and nanos (Gazave et al., 2013; Rebscher et al., 2007). Although additional studies are needed, the data available suggest the possibility that migratory germ line cells may express different complements of these genes in different species. In addition to finding germ line/multipotency expression in gonad structures within gonadal segments, our study also finds evidence for expression of the piwi homolog in putative supernumerary gonad clusters. In P. leidyi, the piwi homolog was typically detected in gonad regions of only the two gonadal segments (segments 6 and 7, in which testes and ovaries form, respectively) (Fig. 2). However, we also very occasionally detected positive cell clusters in more anterior segments. In several other annelids, supernumerary gonads or primordial germ cell clusters that express germ line markers have also been reported. In embryos of the clitellate Tubifex tubifex, vasa-positive cell clusters initially form in more segments than will form permanent gonads; as development proceeds, these extra vasa-positive cell clusters disappear and only the gonadal segments retain them (Oyama and Shimizu, 2007). Similarly, in embryos of the leech Helobdella robusta, nanos-expressing cell clusters, representing presumptive male primordial germ cells, initially form in 11 segments even though the adult will only develop 5–6 pairs of testisacs (Kang et al., 2002). Pristina, Tubifex, and Helobdella are all clitellate annelids, in which gonads are restricted to a few anterior segments. However, given that most polychaetes have gonads or germ cell primordia in many consecutive segments along the body, it is expected that the clitellate condition (gonads in restricted anterior segments) is derived from one in which germ cell primordia/gonads formed in many consecutive segments. The “supernumerary” primordial germ cell clusters in clitellates may therefore be a remnant of this ancestral condition. Future studies are needed to determine the function, if any, of these cell clusters in clitellates. 4.3. Germ line/multipotency genes are expressed in proliferative somatic tissue in addition to germ line in P. leidyi Recent work has made it clear that genes that were originally considered to be germ line-specific are often expressed more broadly, including in pluripotent cells and/or proliferative zones (Alié et al., 2011; Mochizuki et al., 2000; Sharma et al., 2001; Solana, 2013; Zhu et al., 2012). Consistent with this, we found that in P. leidyi, homologs of piwi, nanos, and vasa are expressed during post-embryonic development in proliferative somatic tissues, in addition to being expressed in the gonads (Fig. 1). Specifically, we found that homologs of all three of these genes are expressed broadly within the posterior growth zone, fission zones, and anterior and posterior blastemas, all regions in which new tissues

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are being formed (Zattara and Bely 2011, 2013). In addition, we found that the nanos homolog (though not the piwi or vasa homologs) is detectable at the base of the proboscis, corresponding to the location of an anterior growth zone that provides cells for the continually replaced proboscis (Zattara and Bely, 2013). The function of these genes in these proliferative regions has yet to be elucidated in annelids, but based on their spatial domains of expression, it is possible that they are involved in supporting cell proliferation, conferring multipotency of cell fates, or both. In other annelids, expression of germ line/multipotency genes has been reported in some of these same highly proliferative somatic tissues, which are thought to include multi-/pluripotent cell populations. Expression of piwi, vasa, and/or nanos homologs has been detected in the posterior growth zone in juveniles and/or adults of Capitella teleta (piwi, nanos and vasa homologs), Platynereis dumerilii (many germ line/multipotency genes, including piwi, vasa, and nanos homologs), and Enchytraeus japonensis (two vasa homologs) (Dill and Seaver, 2008; Gazave et al., 2013; Giani et al., 2011; Rebscher et al., 2012; Sugio et al., 2008) suggesting that expression of germ line/multipotency genes in the posterior growth zone is an ancestral feature for annelids. Notably, in annelid species or developmental stages lacking a posterior growth zone, these genes are not expressed at the posterior end (Cho et al., 2014; Weigert et al., 2013), further supporting the correlation between expression of these genes and tissue multipotency in this body region. With respect to regeneration, germ line/multipotency genes have been shown to be expressed in the posterior regeneration blastema of C. teleta and P. dumerilii (Gazave et al., 2013; Giani et al., 2011), as in P. leidyi. Our study is the first, however, to investigate expression of these genes during anterior regeneration or during fission in annelids; we find that these genes are indeed expressed in the anterior blastema as well as in fission zones, and also find that blastema expression appears to arise de novo, without any apparent migration of cells positive for these markers towards the wound site. Our work thus shows that germ line/multipotency genes are consistently expressed in regions of post-embryonic development, whether that development occurs by posterior growth, regeneration, or asexual reproduction by fission. 4.4. Transmission of germ line across asexual generations in P. leidyi Our study demonstrates that asexually reproducing P. leidyi routinely form gonads, but how does germ line form during the fission process? We found that one of the three germ line/multipotency genes we investigated, PRIle-piwi1, provides insight into this process. PRIle-piwi1 is expressed in isolated spindle-shaped cells on the dorsal surface of the ventral nerve cord and the distribution and morphology of these cells suggest they migrate along this structure (Figs. 3 and 4). Furthermore, these cells sometimes make direct contact with the (PRIle-piwi1 positive) gonads, taking on a configuration suggesting they are migrating into or out of the gonad. These PRIle-piwi1 positive cells are abundant during fission, especially in the region where new heads are developing; however, we find that they are rarely detected near anterior and posterior regeneration blastemas, and are not associated with the posterior growth zone. Such cells are also almost never detected during regeneration following more severe anterior amputations that elicit regeneration of gonad segments (Ozpolat, Sloane, and Bely, unpublished data). Thus, at least when actively expressing PRIle-piwi1, these cells appear to be associated specifically with the fission process. Germ cell migration during development is widespread among animals, and the morphology and distribution of the ventral cells we identified in P. leidyi are consistent with those of migratory germ cells identified in other animals (Anderson et al., 2000; Jaglarz and Howard, 1995; Soto-

Suazo and Zorn, 2005). We therefore propose that the PRIlepiwi1 þ ventral cells, which we refer to as PPVCs, are migratory germ line cells or pluripotent cells that can give rise to the germ line. Given that asexually reproducing P. leidyi routinely form gonads, the germ line of new asexual individuals must either be established anew or be inherited from the asexual parent. During fission, the distribution and configuration of PPVCs along the nerve cord and near gonads suggests that PPVCs (and thus presumed germ line cells) are transmitted across asexual generations, although new gonads forming from piwi-negative tissues during fission remains a possibility. To begin to test the possibility of PPVC transmission, we investigated cell migration along the ventral nerve cord during fission using a method for live-time lapse imaging recently developed in P. leidyi (Zattara, 2012). Our study is the second to use this powerful approach to investigate postembryonic development in annelids (after Zattara, 2012), and the first to use time-lapse imaging to investigate the cellular dynamics of fission. Using this approach we found direct evidence for the migration of cells resembling PPVCs along the ventral nerve cord in and around early fission zones (Fig. 6, Suppl. Fig. 5, Suppl. Movies A–C). Rather than finding that migration was exclusively posterior or anterior in direction, we found that cells anterior to a fission zone migrate predominantly posteriorly, while cells posterior to a fission zone migrate predominantly anteriorly. Therefore, cells resembling PPVCs tend to migrate toward early fission zones from both sides of the fission zone. Of note, an early histological study in another fissioning naidid (a species of Nais) also described a population of cells associated with the nerve cord that was found to be more numerous in the posterior zooid as compared to the anterior zooid and most numerous around the fission zone (Herlant-Meewis, 1946b). Although our efforts to record cell migration into or out of the gonads were unsuccessful, and although additional studies are needed to confirm that some or all of the migrating cells seen along the nerve cord in P. leidyi are indeed PRIle-piwi1 positive, our data provide the first direct evidence of cell migration during fission, demonstrate preferential migration towards fission zones, and are consistent with PPVC migration toward fission zones. We propose a model in which PPVCs are germ cells that transmit the germ line across asexually produced individuals via migration along the ventral nerve cord and in which PPVCs are a source of gonad cells and/or are derived from gonad cells. Bidirectional migration of PPVCs on the ventral nerve cord could allow for dynamic redistribution of putative germ line cells along the body axis as well as the potential for these cells to respond to cues from fission zones both anterior and posterior to their current position. The source of PPVCs could be the existing gonads of the anterior zooid and/or the newly forming fission zone, as suggested by the configuration of PRIle-piwi1 þ cells connecting the gonads to the PPVCs we sometimes observed. If, instead, new PPVCs arise from the posterior growth zone or some other posterior tissues, then their source would have to be PRIlepiwi1-negative tissue or a tissue without detectable PRIle-piwi1 transcription, since we found a consistent pattern that PPVCs are virtually never detected in the segments closest to the posterior growth zone. Bidirectional migration of PPVCs along the nerve cord is consistent with our time-lapse data as well as data from a polychaete (Platynereis dumerilii) that suggests both anterior and posterior migration occur during the establishment of gonads (Rebscher et al., 2007). Our time-lapse studies have provided the first window into understanding in vivo cell dynamics during annelid fission, yet many questions remain regarding germ line development and transmission during fission. In particular, it will be important to determine whether more than one cell type is moving along the ventral nerve cord in P. leidyi, what the ultimate


source and destination of PPVCs are, and what ultimate cues trigger the development of gonads during fission and during a transition to sexual maturity. Only a few annelid studies have investigated expression of germ line/multipotency genes during regeneration and none has previously investigated these genes during asexual reproduction. However, our findings and conclusions for fission in P. leidyi are similar in several important respects to those of Tadokoro et al. (2006) for anterior regeneration in another clitellate annelid, Enchytraeus japonensis. In both studies, isolated cells positive for a piwi homolog are detected on the dorsal surface of the ventral nerve cord, are inferred to be migratory, are associated with gonads, and are proposed to be germ line cells or their precursors that establish or are otherwise associated with development of piwi-positive gonads in anterior segments. In E. japonensis, following anterior amputation eliciting regeneration of the anteriorly-located gonad-bearing segments, piwi-positive cells from the trunk appear to accumulate near the anterior blastema and ultimately invade it, initially forming lateroventral masses of isolated cells and ultimately appearing to coalesce into discrete gonad clusters. In P. leidyi the piwi-positive cells do not form large masses of isolated cells; they occur primarily as a medial line with, occasionally, a thin line of cells contacting the ventrolateral gonads. Furthermore, in E. japonensis, the piwi-positive cells reside on the dorsolateral sides of the ventral nerve cord while in P. leidyi they are medial on the ventral nerve cord (unless connecting to the slightly lateral gonads); and in E. japonensis the piwi homolog investigated is not evident in the blastema (other than in the isolated piwi-positive cells and regenerated gonad), while in P. leidyi the piwi homolog we investigated is expressed broadly throughout the early fission zone and blastema. However, despite these differences, the data and conclusions from our study and the Tadokoro et al. (2006) study are striking, suggesting that a common mechanism for post-embryonic gonad establishment may occur in these two species, and possibly among clitellates more broadly. The transfer of germ line cells from parent to agametically produced offspring, as we propose occurs in fissioning P. leidyi, has been indicated in several other asexually reproducing groups, including cnidarians and colonial ascidians. In hydra, males and females have self-renewing germ line stem cells and when an individual reproduces asexually, some of these parental germ line stem cells are transferred to the new polyp formed by budding (Bosch and David, 1986, 1987). In the colonial ascidian Botryllus primigenus, germ line is derived from a piwi-expressing sub-population of hemoblasts, which require piwi for proper germ line function (Sunanaga et al., 2006). When a new individual is formed asexually, some of these piwi-expressing cells are transferred to the new zooid, where they are inferred to be the only source of germ line (Sunanaga et al., 2010). Because asexual, agametic reproduction has evolved numerous times independently among animals (Brusca and Brusca, 2003), mechanisms of germ line maintenance across asexual generations may not be homologous across groups. Investigating germ line establishment in a wider array of asexual groups promises to bring to light the diversity of mechanisms of germ line transfer and establishment in asexual animals, and may also reveal common, convergent features in this process.

Author contributions BDO and AEB designed the experiments; BDO carried out the experiments; BDO analyzed the data; and BDO and AEB wrote the paper.

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Acknowledgments We thank Jamie Kostyun for cloning the vasa homolog and doing preliminary expression assays for this gene; Eduardo Zattara for tracing the culture history of Pristina leidyi; and Ryan Null, Eduardo Zattara, Leo Shapiro, and two anonymous reviewers for helpful discussions and comments on this project and this paper. This research was supported by National Science Foundation (United States) Grant IOS‐0920502 and a University of Maryland Division of Research Tier 1 Seed Grant to A.E.B.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2015.06.001.

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