Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells Eve Gazave a,1, Julien Béhague a,1, Lucie Laplane a, Aurélien Guillou a, Laetitia Préau a, Adrien Demilly a, Guillaume Balavoine a,n, Michel Vervoort a,b,n a b

Institut Jacques Monod, CNRS, UMR 7592, Univ Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France Institut Universitaire de France, 103, bd Saint-Michel, 75005 Paris, France

art ic l e i nf o

a b s t r a c t

Article history: Received 22 August 2012 Received in revised form 28 June 2013 Accepted 15 July 2013

Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the “segment addition zone” (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral “stemness” program. & 2013 Elsevier Inc. All rights reserved.

Keywords: Stem cells Posterior elongation Segmentation Annelids Stemness genes

Introduction Posterior elongation (posterior growth or posterior addition) is the process by which some animals elongate their anterior– posterior axis in an anterior to posterior temporal progression through the progressive addition of new tissues in the posterior part of their body. Although posterior elongation also occurs in some groups of non-segmented animals, this process has been so far mainly described in segmented bilaterian animals, many of which forming most of their body axis through the sequential posterior addition of segments (reviewed in De Rosa et al. (2005) and Martin and Kimelman (2009)). Posterior elongation is indeed involved in the formation of all the tissues posterior to the head in

n Corresponding authors at: Institut Jacques Monod, CNRS/Université Paris Diderot-Paris 7, 15 rue H. Brion, 75205 Paris cedex 13, France. Fax: +33 1 57 27 80 87. E-mail addresses: [email protected], [email protected] (E. Gazave), [email protected] (J. Béhague), [email protected] (L. Laplane), [email protected] (A. Guillou), [email protected] (L. Préau), [email protected] (A. Demilly), [email protected] (G. Balavoine), [email protected] (M. Vervoort). 1 Equal contribution.

vertebrates, of most of the segments in the vast majority of arthropod (not in long germ-band holometabolous insects such as Drosophila) and annelid groups (Jacobs et al., 2005; Martin and Kimelman, 2009). In these animals, posterior elongation can happen at two distinct developmental periods, either during embryonic development through the addition of segmental units as seen in the somitogenesis of vertebrates or in segment formation processes in short germ insects, or during post-embryonic development, as seen in the juvenile development of many annelid species or the larval development of some crustaceans from a three-segmented nauplius larva. In both cases, posterior elongation relies on the presence of a segment addition zone (SAZ; de Rosa et al., 2005; Janssen et al., 2010), i.e., a localized terminal or subterminal body region where new segments are being produced. The presence in this SAZ of pluripotent stem or progenitor cells whose mitotic activity continuously provides new tissues to the elongating body axis (Martin and Kimelman, 2009) remains however in many cases a contentious issue, especially during embryonic development. In amniotes for instance, lineage tracing experiments suggest that the tail bud of vertebrates contains multipotent stem cells that are the source of both neural tube and mesoderm elongation (e.g., Kanki and Ho, 1997; Davis and Kirschner, 2000; Mathis et al.,

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Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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E. Gazave et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 1. The two studied stages of posterior elongation in Platynereis. Confocal images of Hoechst labeled worms (A) and schematic drawings (B) are shown. In the case of the early stages of posterior elongation (formation of the SAZ and of the 4th segment), 3 days post-fertilization (dpf), 5dpf, and 15dpf worms are shown. The 3dpf and 5dpf worms bear three chaetigerous segments (S1, S2, and S3) and a pygidium (py). The chaetigerous segments are characterized by the presence of pairs of appendages (parapodes) harboring tufts of chaetae. Posterior addition starts at about 7–10dpf and a fourth (still immature) segment (S4) can be observed in 15dpf worms. The white dotted circle indicates the position of the mouth. In the case of post-caudal regeneration posterior elongation, the posterior half of atokous worms were amputated and the worms were allowed to develop for 11 days. A pygidium was regenerated and several segment primordia were produced sequentially. All the segment primordia are still growing and differentiating. Asterisks indicate the last fully grown segment of the worm that was not amputated. On all the images the white dashed lines mark the boundary between the posteriormost segment and the pygidium. ac ¼ Anal cirri, a characteristic feature of the mature pygidium; Mg ¼midgut; S¼ segment. Orange brackets indicate the part of the worm that will be shown in most of the figures of the article. (C) Graph of the number of segment primordia visible over time after caudal amputation. 17 worms at 60–70 segments were amputated at mid-body. Pygidium regeneration is completed and the new SAZ starts to bud new segments on day 5. The pace of segment addition decreases over time, from 2.3 to 0.3 segments per day, close to its normal rate.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

E. Gazave et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

2001; Roszko et al., 2007; Wilson et al., 2009; Tzouanacou et al., 2009). Tail bud stem cells most notably produce the paraxial mesoderm that divides into somites in an anterior to posterior sequential fashion (reviewed in Holley (2007)). These cells are also responsible for the elongation of the posterior half of the spinal cord (e.g., Mathis et al., 2001; Roszko et al., 2007). In arthropods and their close relatives (onychophores and tardigrades), a posterior population of stem cells has not been yet clearly identified, except in malacostracan crustaceans (Peel et al., 2005; Mayer et al., 2010; and references therein). In these latter, the SAZ contains teloblasts, i.e., large stem cells that lie in the posterior part of the body and produce the body axis tissues by dividing asymetrically (Dohle and Scholtz, 1988; Scholtz, 1990; Scholtz, 1993). The SAZ is usually composed of distinct ectoteloblasts and mesoteloblasts that produce ectodermal and mesodermal derivatives, respectively. However, in one malacostracan group, Amphipoda, only mesoteloblasts are found, whereas ectodermal tissues derive from posteriorly aggregating germ disc cells (Dohle and Scholtz, 1988; Scholtz, 1990). In other arthropods, posterior elongation processes have been only barely characterized. In annelids, embryonic posterior elongation has been most thoroughly characterized in the evolutionary derived clitellates (i.e., leeches and oligochaetes). In these annelids, the whole segmental mesoderm and ectoderm arise from a SAZ that consists of five bilateral pairs of teloblasts, one for the mesoderm (mesoteloblasts) and four for the ectoderm (ectoteloblasts; Weisblat and Shankland, 1985; Zhang and Weisblat, 2005). Clitellate teloblasts are embryonic stem cells that undergo iterated asymmetric divisions, generating columns (bandlets) of segment progenitor cells (primary blast cells). Within each bandlet, the first-born blast cells will contribute to more anterior segments than the later-born blast cells. The blast cells produce specific segmental derivatives through stereotypical cell lineages depending on the bandlet they belong to. In most non-clitellate annelids, only the anterior most segments are formed during embryonic and larval development (Fischer et al., 2010). Many segments are added later on during juvenile development from a subterminal posterior SAZ (Seaver et al., 2005; De Rosa et al., 2005; Mayer et al., 2010). While it is assumed that the annelid SAZ contains ecto- and mesoteloblasts, morphologically visible teloblasts or bandlets of blasts cells similar to those found in clitellates have not been identified, making the cellular process of posterior tissue production in non-clitellate annelids a question still open (reviewed in De Rosa et al. (2005)). Despite the diversity of morphogenesis processes at cellular level described above, molecular studies have identified a common core of genes involved in posterior elongation in vertebrates, annelids, and arthropods. These include the homeobox genes caudal/cdx and evenskipped (eve)/evx, the T-box gene brachyury, and the Wnt signaling pathway (e.g., Chawengsaksophak et al., 2004; De Rosa et al., 2005; Shimizu et al., 2005; Martin and Kimelman, 2008, 2009; Bolognesi et al., 2008; McGregor et al., 2008; Janssen et al., 2010; van de Ven et al., 2011; Van Rooijen et al., 2012). These similarities in the genetic network controlling posterior elongation in distantly related animals can be interpreted as homologies, suggesting that this process and its regulation is ancestral to bilaterians (De Rosa et al., 2005; Martin and Kimelman, 2009). Further testing of this hypothesis will require a better knowledge of this process, in particular in annelids and arthropods. Platynereis dumerilii is a useful model to study posterior elongation in annelids (De Rosa et al., 2005; Rebscher et al., 2007; Ferrier, 2012). This process occurs during two different phases in Platynereis. Embryonic and larval development produce, in about three days, a small worm-like larva (also known as nectochaete stage; Fischer et al., 2010), which is composed of a head, three simultaneously formed segments bearing appendages (parapodes), and a terminal piece called the pygidium, which

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includes the anus (Fig. 1A and B). Once settled, the juvenile worms will undergo sequential posterior addition of new segments, the fourth segment being visible 12–14 days after fertilization. This posterior addition process will continue for most of the life of the worm. Posterior addition pace varies slightly between individuals and according to environmental conditions (food availability) but does not exceed one segment every two days. When they are 5-segmented, the juvenile worms build the characteristic silk tube in which they spend most of their life. The worms are named “atokous worms” during this stage, continue to grow through posterior addition of segments until they possess around 80 segments and then undergo sexual metamorphosis (Fischer et al., 2010). Sexually mature animals of both sexes (epitoks) leave their silk tubes, engage in mass swarming and die after gamete release. Posterior elongation is also observed after the caudal regeneration process that follows the amputation of the posterior-most part of the body of atokous worms (De Rosa et al., 2005). We will hereafter refer to this accelerated process as “postcaudal regeneration posterior elongation” (Fig. 1). In a previous study, De Rosa et al. (2005) found that orthologs of the caudal/cdx and even-skipped/evx genes are expressed during post-caudal regeneration posterior elongation in a narrow ring of ectodermal cells that lies at the anterior border of the pygidium. As these cdx- and evx-expressing cells incorporate and retain BrdU in pulse and chase experiments, it has been suggested that the two genes are expressed in ectodermal stem cells of the SAZ (De Rosa et al., 2005). Neither evx and cdx expression, nor BrdU labeling were observed in internal cells during post-caudal regeneration posterior elongation, therefore precluding the identification of the putative mesodermal stem cells. The two genes are also expressed in the posterior part of 3-day-old larvae, suggesting that cdx and evx may also been involved in the early stages of SAZ formation (De Rosa et al., 2005). In another study, Rebscher et al. (2007) showed that orthologs of genes (vasa, nanos, piwi and pl10) involved in germ cell development in Drosophila and vertebrates (Johnstone and Lasko, 2001; Extavour and Akam, 2003; Gustafson and Wessel, 2010; Juliano et al., 2010, 2011) are not only expressed in Platynereis germ cells, but also in a group of internal cells whose position suggests that it may corresponds to the mesodermal part of the forming SAZ of nectochaete larvae. No ectodermal expression of these genes was reported and their expression during later stages of posterior elongation was not studied. In this article, we cloned orthologs of several genes expressed in germinal and/or somatic stem cells in other animals and found that most of these genes are expressed during posterior elongation in cells of the SAZ. Combined with EdU incorporation and pulse and chase experiments, we show that teloblast-like stem cells are responsible for posterior elongation in Platynereis (and presumably in most other annelids). As most of the genes expressed in the posterior somatic stem cells are also expressed in primordial germ cells, our study also reinforces the hypothesis of a common genetic program for “stemness” in germinal and somatic stem cells (e.g., Juliano et al., 2010, 2011; Alié et al., 2011; Leclère et al., 2012).

Materials and methods Breeding culture and worm collection P. dumerilii worms were obtained from a breeding culture established in the Institut Jacques Monod (Paris), according to the protocol of Dorresteijn et al. (1993). Early and late nectochaete (3 days post-fertilization (dpf) and 5dpf, respectively) and errant juvenile (15dpf) worms, as well as atokous worms 11 days after caudal amputation were fixed in 4% PFA in 2  PBS+0.1% Tween20

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

E. Gazave et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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and 1  PBS+0.1% Tween20, respectively, and stored at  20 1C in methanol 100%. Cloning of the genes and phylogenetic analyses Platynereis genes were either identified by sequence similarity searches against a large EST collections and genomic sequences (Platynereis resources, 4dx.embl.de/platy/). Large gene fragments were subsequently cloned by PCR using sequence specific primers on cDNAs from mixed larval stages. PCR products were TA cloned into the PCR2.1 vector following the manufacturer's instructions (Invitrogen) and sequenced. Partial cDNA obtained were then used as templates to produce RNA antisense probes for whole mount in situ hybridizations (WMISH) using Roche reagents. Primer sequences are available upon request. Accession numbers for the newly cloned genes are mentioned in Table 1 with the exception of Pdu-PCNA whose accession number is HF935038. Orthology relationships were defined using as criteria sequence similarities, presence of specific domains, and phylogenetic analyses (Fig. S1). The predicted amino-acid sequences of the identified Platynereis gene fragments were aligned with their presumptive orthologs from a selection of animal groups. These genes were either annotated as such in public databases, or found as predicted genes in whole genome BLAST screening. Multiple alignments were performed

with MUSCLE 3.7 (Edgar, 2004) using the Phylogeny.fr web server (www.phylogeny.fr; Dereeper et al., 2008) and were subsequently manually improved. Handling of the multiple alignments was done using SEAVIEW (Gouy et al., 2010). Maximum likelihood analyses were performed using PHYML (Guindon et al., 2005, 2010) with the Le and Gascuel amino acid substitution model (Le and Gascuel, 2008) and two rate categories (one constant and four gamma rates). Statistical support for the different internal branches was assessed by approximate likelihood-ratio test (aLRT; Anisimova and Gascuel, 2006). Trees were handled using TreeDyn (Chevenet et al., 2006). The whole list of sequences used in this study and the multiple alignments are available upon request. Whole-mount in situ hybridizations (WMISH), Immunolabellings, EdU cell proliferation assays, Microscopy and image processing NBT/BCIP WMISH, fluorescent WMISH and immunolabellings were done as described in Tessmar-Raible et al. (2005). Extensive proteinase K incubations were performed for later stages (3 min for 5 days and 4 min for 15 days, 100 mg/ml). EdU proliferation assays were performed using the Click-iT EdU kit 488 or 555 (Molecular Probes), as described in Demilly et al. (2011). Incorporations of EdU (200 mM) were done for 30 min or 1 h directly in

Table 1 Summary of the studied Platynereis genes and their expressions. SAZ¼ segment addition zone; VNC ¼ ventral nerve cord. 7 Indicates an expression in the SAZ not observed at all stages. Details about the expression of the genes in the SAZ can be found in Fig. 9. Genes/gene famillies

Platynereis genes

Reference for Platynereis genes

pumilio/pum

Rebscher et (2007) Rebscher et Pdu-pl10 (2007) Rebscher et Pdu-piwi (2007) Rebscher et Pdu-nanos (2007) Pdu-pumilio This study

puf-A

Pdu-pufA

vasa/DDX4 pl10/DDX3 piwi/miwi nanos/nos

Pdu-vasa

al. al. al. al.

This study

Accession numbers of Platynereis genes

Expression in cells of the SAZ in 3, 5 and 15dpf worms

Expression in cells of the SAZ in worms after regeneration

Expression in germinal cells

Other expressions

AM114778

+

+

+



AM048814

+

+

+

Gut

AM076487

+

+

+



AM076486

+

+

+

VNC, brain Gut Gut, mesoderm Gut Gut, VNC, brain

HE971743

+



+

HE971741

+

+

+

puf-B

Pdu-pufB

This study

HE971742

+

+

+

SmB/LsmB

Pdu-SmB

This study

HE971745

+

+

+

This study

HE971734

7





This study

HE971737







Ectoderm

This study

HE971740

+





VNC, brain

This study This study This study

HE971746 HE971747 HE971748 HE971733 KC999055 HE971738

+ + + + + 

+ + + + + 

+ + + + + 

   VNC, brain VNC, gut Ubiquitous VNC, brain, gut Brain, lateral ectoderm

bruno/CELF/CUG-BP1 and Pdu-bruno ETR-3 Like Factors mago nashi/magoh Pdu-magoh Pdumusashi/msi musashi Pdu-tdrd1 tudor/tdrd Pdu-tdrd2 Pdu-tdrd3 brat/TRIM32 Pdu-brat myc Pdu-myc max Pdu-max

This study

Mesoderm

id/emc

Pdu-id

This study

KC999056

+

+



ap2/tfap2

Pdu-ap2

This study

KC999054

+

+



glial cell missing/gcm/ glide

Pdu-gcm

This study

HE971735

+

+



VNC, brain

runt/runx/AML

Pdu-runt

caudal/cdx

Pdu-cdx

even-skipped/eve/evx

Pdu-evx

hox3

Pdu-hox3

hunchback/hb

Pduhunchback

This study De Rosa et al. (2005) De Rosa et al. (2005) Kulakova et al. (2007) Kerner et al. (2006)

KC999057

+

+

+

VNC, brain, gut

DQ188196.3

+

+





DQ188195.1

+

+



VNC

DQ366681.1

+

+



Midline, mesoderm

AM232683.1

+

+



Mesoderm

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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sea water. For fluorescent WMISH, immunodetection and EdU cell proliferation assay, the worms were stained with Hoechst at 1:1000 dilution and mounted in DABCO-Glycerol solution. Bright field images were taken on a Leica microscope. Confocal images were taken with a Leica SP5 confocal microscope. Adjustment of brightness and contrast, Z projections, as well as virtual transverse sections, were performed using ImageJ, Imaris, and Photoshop software. Areas of nucleus, nucleolus and intensity of Hoechst staining were measured and compared on three groups of cells: the putative ectodermal stem cells, the pygidium cells and the segment progenitor cells. The putative ectodermal stem cells localizations were defined by the expression of four stem cells genes (Pdu-piwi, Pdu-nanos, Pdu-ap2, Pdu-hox3) on high-magnification confocal pictures of fluorescent WMISH. Areas and intensity values for each type of cells (n430 in all cases) were measured using Image J measurements tools.

Results Identification of putative “stem cell genes” in P. dumerilii In order to further characterize the production of new tissue during posterior elongation in Platynereis and identify the putative stem cells involved in this process, we studied Platynereis orthologs of several genes known to have roles in the formation, behavior, or maintenance of stems cells in other models. We focused on two categories of genes: (i) genes that are expressed in germinal stem cells in Drosophila and/or vertebrates, as orthologs of some of these genes are expressed in the SAZ of Platynereis (Rebscher et al., 2007) and of another annelid Capitella teleta (Giani et al., 2011; Dill and Seaver, 2008), and in somatic stem cells in other animals such as cnidarians, ctenophores, and platyhelminthes (e.g., Extavour et al., 2005; Juliano et al., 2010; Gustafson and Wessel, 2010; Alié et al., 2011; Leclère et al., 2012); (ii) genes that are expressed in somatic stem cells in Drosophila and vertebrates, in particular neural stem cells, mammalian embryonic stem cells (ES), and hematopoietic stem cells. The expression patterns of these two categories of gene were studied in combination with genes previously shown to be involved in posterior development in various species, including Platynereis. A summary of all the studied genes/gene families with relevant information can be found in Table 2. We newly cloned 17 Platynereis genes (along with 8 previously known) (Table 1). For the newly cloned genes, orthology relationships were defined using as criteria sequence similarities, presence of specific domains, and phylogenetic analyses (Fig. S1). In both Tables 1 and 2, genes/gene families were grouped according to the nature of their product (mostly RNA-binding proteins and transcription factors). Single Platynereis genes were found for all families, but Tudor for which three paralogous genes were identified. We studied gene expressions at three different developmental times, three and five days post-fertilization (3dpf and 5dpf) when the pygidium is taking shape and the SAZ is presumably put in place but no new segment is yet visible, and 15dpf, when the 4th segment is visible but still developing. The systematic study of juvenile posterior elongation beyond this time point is technically difficult (segment addition is slow and WMISH are hampered by the thickening epidermal cuticle). To circumvent these difficulties, we also studied post-caudal regeneration posterior elongation as a proxy to normal posterior elongation. A regeneration blastema is formed upon the wound and gives rise to a new pygidium in around 4 days. Posterior segment addition then starts again from a regenerated SAZ. The overall morphology of the posterior part of the body after caudal regeneration is similar to the posterior part of a normally growing worm, both externally and internally (Prud'homme et al., 2003; De Rosa et al. 2005). The segment

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addition process after caudal regeneration is strictly sequential, similar to normal juvenile elongation. The rate of segment addition is at first much faster than during normal growth as about two segments per day are formed, but decreases back to normal after a few days and when a chain of about 12–15 new segments has been produced (Fig. 1C). While we cannot exclude that small differences may initially exist between post-caudal regeneration posterior elongation and normal posterior elongation, these differences will necessarily fade away as time goes because the two processes are in temporal continuity with each other. We therefore performed WMISH on worms 11 days after posterior amputation when posterior addition is most similar to normal elongation, but posterior parts have a thinner cuticle more amenable to molecular experimentation. The overall morphology of the worms at the different studied stages is shown in Fig. 1A and B. One of the analyzed genes, Pdu-magoh is not expressed in the posterior part of the worms at any stages, and one other, Pdu-max, only show weak and ubiquitous expressions at all studied stages (not shown). These genes will not be further described. Table 1 gives a summary of the expression patterns we have found at different stages for each gene, including some expression patterns in developing organs such as the gut and the nervous system (brain and ventral nerve cord) that we will not discuss further in this article. Platynereis putative “stem cell genes” are expressed in migrating germinal stem cells As a majority of the newly identified genes are expressed in germinal stem cells in other model animals, it was interesting to monitor their expression in the germ line of Platynereis. In the previous studies, Rebscher et al. (2007, 2012) described the origin, migration and proliferation of these cells, based on EdU labeling and the expressions of four RBP-encoding genes, Pdu-piwi, Pdu-vasa, Pdupl10, and Pdu-nanos. Four primordial germ cells (PGCs) are presumably produced early in embryogenesis by the first divisions of the “primary mesoblasts” of the 4d lineage, which are also at the origin of the whole trunk mesoderm of the worm later on in embryogenesis (Fischer and Arendt, 2013). These four PGCs remain mitotically quiescent and clustered near the vegetal pole of the trochophora larva and then in the vicinity of the forming pygidium. At 4dpf, PGCs start migrating anteriorly to join a region posterior to the pharynx called the “primary gonad”, where these cells will start to proliferate in older juvenile worms (420 segments). The migration routes of the PGCs towards this primary gonad are variable because PGC labeling with anti-Vasa antibody in 3- and 4-segmented young worms reveals various configurations of positions, with cells grouped in pairs or isolated beneath the epidermis of the trunk, often near the base of the parapodia, in chaetigerous segments 2 and 3 (Rebscher et al., 2007). The expression of a majority of the genes we studied in migrating PGCs was most obvious in 15dpf worms (Fig. 2). In addition to the four RBPs already characterized by Rebscher et al. (2007), Pdu-pumilio, -pufA, -puf-B, -SmB, -tdrd1, -tdrd2, -tdrd3, -brat, -myc and -runx are found expressed in isolated cells clearly similar to the migrating PGCs described above. Platynereis putative “stem cell genes” are expressed in posterior teloblast-like cells Rebscher et al. (2007, 2012) showed the existence at 3dpf of a prospective “mesodermal posterior growth zone” as a group of internal cells located at the border between the 3rd chaetigerous segment and the forming pygidium, expressing Pdu-piwi, Pdu-vasa, Pdu-pl10, and Pdu-nanos. These cells are thought to be derived from the primary mesoblasts of the 4d lineage after they had produced the PGCs and the precursor cells of the larval trunk mesoderm. They incorporate BrdU at 3dpf, showing that they are

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Table 2 Summary of the studied genes/gene families and their expressions/functions in stem and germinal cells. RBP ¼RNA-binding protein; TF¼ transcription factor; Prot-Prot inter¼Protein-protein interaction. puf-B, although not known so far for a stem cell related function, was studied because of its close evolutionary relationship with pumilio and puf-A genes. Genes/gene famillies Type of encoded proteins

Domain(s)

Expression(s)/function(s) in germ cells/somatic stem cells/posterior elongation

References

vasa/DDX4

RBP

DEAD box

Extavour et al. (2005), Rebscher et al. (2007), Juliano et al. (2010), Dill and Seaver (2008), Gustavson and Wessel (2010), Alié et al. (2011), and Leclère et al. (2012)

pl10/DDX3

RBP

DEAD box

piwi/miwi

RBP

PAZ+Piwi

nanos/nos

RBP

Zf-Nanos

pumilio/pum

RBP

PUF

puf-A puf-B SmB/LsmB

RBP RBP RBP

PUF PUF Sm

Germ cells in many vertebrates and non-vertebrates (e.g., Drosophila, annelids, cnidarians, Caenorhbaditis and a ctenophore); somatic stem cells in various nonvertebrates (e.g., cnidarians, a ctenophore, annelids, and Platyhelminthes) Germ cells in many vertebrates and non-vertebrates (e.g., Drosophila, annelids, cnidarians, Caenorhbaditis and a ctenophore); somatic stem cells in various nonvertebrates (e.g., cnidarians, a ctenophore, annelids, and platyhelminthes) Germ cells in many vertebrates and non-vertebrates (e.g., Drosophila, annelids, cnidarians and a ctenophore); somatic stem cells in various non-vertebrates (e.g., cnidarians, a ctenophore, annelids, and platyhelminthes) Germ cells in many vertebrates and non-vertebrates (e.g., Drosophila, annelids, and Caenorhbaditis); somatic stem cells in various non-vertebrates (e.g., cnidarians, annelids, and platyhelminthes) Germ cells in Drosophila, Caenorhabditis and various vertebrates Germ cells in zebrafish – Germ cells in Drosophila; neoblasts in a platyhelminthe Germ cells in Drosophila and a ctenophore; neoblasts in a platyhelminthe and adult somatic stem cells in a ctenophore Germ cells in Drosophila

bruno/CELF/CUG-BP1 RBP and ETR-3 Like Factors mago nashi/magoh RBP

RRM

musashi/msi

RBP

RRM

tudor/tdrd

Tudor

myc

Prot-Prot inter. Prot-Prot inter. TF

bHLH-Zip

max id/emc

TF TF

bHLH-Zip HLH

ap2/tfap2 glial cell missing/ gcm/glide runt/runx/AML

TF TF

TF-AP2 GCM

TF

Runt

caudal/cdx

TF

brat/TRIM32

even-skipped/eve/evx TF

Mago-nashi

NHL+TRIM

Neural stem cells in Drosophila and mouse, epithelial stem cells in mouse Germ cells in Drosophila and various vertebrates (e.g., mouse) Germ cells in Drosophila; neural stem cells in Drosophila and mouse Germ cells in Drosophila; ES and iPS cells in mammals, various somatic stem cells in Drosophila and vertebrates, stem cells in Hydra Stem cells in Hydra Hematopoietic and neural stem cells, ES cells in mammals Neural crest cells in vertebrates Neural stem cells in mouse

Hematopoietic, neural, and intestine stem cells in vertebrates; stem cells (seam cells) in Caenorhabditis Homeodomain Tailbud of vertebrates and SAZ of various arthropods and annelids

hox3

TF

Homeodomain Tailbud of vertebrates and SAZ of various arthropods and annelids Homeodomain SAZ in annelids

hunchback/hb

TF

Zf-H2C2

SAZ in insects and Platynereis

proliferative at this early stage. We observed that 20 of the studied genes were expressed in one or a few cells with this same location (Fig. 3, orange arrows), reinforcing the view that this group of cells corresponds to the prospective SAZ (or at least a part of it). 15 of these 20 genes are also expressed in more anterior cells whose position suggests they belong to the developing midgut (Fig. 3, yellow arrows). 6 of the studied genes are expressed in specific cells of the forming pygidium (Fig. 3, light blue arrows).

Rebscher et al. (2007), Juliano et al. (2010), Gustavson and Wessel (2010), Alié et al. (2011), and Leclère et al. (2012)

Rebscher et al. (2007), Juliano et al. (2010), Alié et al. (2011), Giani et al. (2011), Juliano et al. (2011), and Leclère et al. (2012)

Extavour et al. (2005), Rebscher et al. (2007), Dill and Seaver (2008), Juliano et al. (2010), and Leclère et al. (2012) Parisi and Lin (2000), Spassov and Jurecic (2003), and Kerner et al. (2011) Kuo et al. (2009) and Kerner et al. (2011) Kerner et al. (2011) Tharun (2009), Anne (2010), Gonsalvez et al. (2010), and Fernandéz-Taboada et al. (2010) Barreau et al. (2006), Chekulaeva et al. (2006), Guo et al. (2006), and Alié et al. (2011) Kataoka et al. (2001), Boswell et al. (1991), and Parma et al. (2007) Potten et al. (2003) and Okano et al. (2005) Thomson and Lasko (2005) and Siomi et al. (2010) Bowman et al. (2008), Schwamborn et al. (2009), and Harris et al. (2011) Knoepfler (2008), Laurenti et al. (2009), and Hartl et al. (2010) Hartl et al. (2010) Massari and Murre (2000), Iavarone and Lasorella (2006), Kee (2009), and Romero-Lanman et al. (2011) Eckert et al. (2005) and Wang et al. (2011) Hitoshi et al. (2011) Nimmo et al. (2005), Appleford and Woollard (2009), and Wang et al. (2010) Chawengsaksophak et al. (2004), De Rosa et al. (2005), Martin and Kimelman (2009), and van Rooijen et al. (2012) Beck and Slack (1999), Song et al. (2002), Copf et al. (2003), and De Rosa et al. (2005) Kulakova et al. (2007), Fröbius et al. (2008), and Pfeifer et al. (2012) Wolff et al. (1995), Mito et al. (2005), and Kerner et al. (2006)

At 5dpf, we found that most of the studied genes are expressed in a few internal cells located around the posterior part of the developing gut and anterior to the pygidium (Figs. 4A–A′ and 5). These cells are found at two slightly different positions as exemplified in the case of Pdu-piwi (Fig. 4A–A′). Some of these cells are located along the cone-shaped midgut that becomes clearly visible at this stage (Fischer et al., 2010; Fig. 4A, pink arrows) – expression of Pdu-piwi is strong in cells close to the posteriormost part of the gut and much weaker in

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Fig. 2. Expression of the studied genes in germinal cells in 15 days post-fertilization (dpf) worms. Whole-mount in situ hybridizations (WMISH) for the genes whose name is indicated on each panel are shown. All panels are dorsal views (anterior is up). Arrows point to the migrating primordial germinal cells. These cells are located in variable anterior-posterior and left/right positions, because their routes are variable between individuals (Rebscher et al., 2007). The purple dotted brackets indicate the posterior part of the worms which are shown in Fig. S2. O: high magnification view of Pdu-piwi expression in both migrating germinal cells and cells of the SAZ.

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Fig. 3. Expression of the studied genes in the posterior part of 3dpf worms. WMISH for the genes whose name is indicated on each panel are shown. All panels are dorsal views (anterior is up) and only the posteriormost part of the worms is shown. Yellow arrows indicate an expression in the developing gut, red arrows an expression in the putative internal posterior stem cells, and light blue arrows an expression in the pygidium. In this and the following figures, the dashed lines mark the boundary between the 3rd segment and the pygidium. White asterisks denote strong expressions of Pdu-bruno and Pdu-ap2 in the trunk mesoderm and the ectoderm of the parapods, respectively.

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Fig. 4. Expression of Pdu-piwi in 5dpf and 15dpf worms. WMISH for the Pdu-piwi gene are shown. All panels but (F′) are dorsal views (anterior is up) and only the posteriormost part of the worms is shown. (A) and (A′) correspond to two different focal planes of the same 5dpf worm, (A) being slightly more dorsal than (A′). (B–E) are superficial dorsal views of 15dpf worms, (B′–E′) correspond to a focal plane at the level of the gut of the same worms, and (B″–E″) correspond to a more ventral focal plane of the same worms. I to IV correspond to four sub-stages that can be defined at the 15dpf stage (see main text for details). Position of the gut is shown by a yellow asterisk – Pdu-piwi is expressed in some gut cells at 5dpf but not at 15dpf. Black asterisks indicate the position of the chaetae in the developing 4th segment. Purple arrows point to superficial Pdu-piwi-expressing cells, pink arrows/arrowheads to Pdu-piwi-expressing cells located around the posterior part of the midgut, and red/arrowheads arrows to Pdu-piwi-expressing cells located ventrally with respect to the midgut. In 15dpf worms, pink and red arrows point to the posterior cells that strongly express Pdu-piwi and the arrowheads point to the anteriormost expressing cells. F and F′ are fluorescent WMISH (green) counterstained with Hoechst (blue). F′ is a virtual cross-section (dorsal up) of F. Cross-section has been done approximately at the level of the red arrows. The ventralmost part of the worm, including ventral ectoderm cannot be seen on this crosssection. The weaker expression of Pdu-piwi in the dorsal ectoderm observed with NBT/BCIP WMISH is not detectable by fluorescent WMISH.

more anterior ones. These Pdu-piwi-expressing cells probably correspond to mesodermal cells located very close to the gut cells and therefore much likely belong to the visceral mesoderm. The other Pdupiwi-expressing cells are slightly more ventral than the gut and only located posterior to it, at the border with the pygidium (Fig. 4A′, red arrows). Based on their location, these latter cells likely also belong to the mesoderm, probably to the somatic mesoderm. Several other genes are also strongly expressed in the ventral posterior mesodermal

cells (Fig. 5A–L and Q; red arrows) and weakly or not expressed in the cells located along the gut (Fig. 5A–L and Q). In contrast, Pdu-myc, Pduid and Pdu-ap2 are strongly expressed in the cells that are located along the gut (Fig. 5M–O; pink arrows), but weakly or not expressed in the posterior ventral mesodermal cells (Fig. 5M–O). Pdu-gcm is only expressed in two bilateral groups of cells in the pygidium (Fig. 5P, light blue arrows), probably at the base of the forming tentacular cirri. Pducdx is expressed both in mesodermal cells in the prepygidial zone

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Fig. 5. Expression of the studied genes in the posterior part of 5dpf worms. WMISH for the genes whose name is indicated on each panel are shown. All panels are dorsal views (anterior is up) and only the posteriormost part of the worms is shown. Position of the gut is shown by a yellow asterisk. Pink arrows point to an expression in cells located around the posterior part of the midgut, red arrows to an expression in cells located ventrally to the midgut, dark blue arrows to an expression in superficial cells immediately anterior to the pygidium, and light blue arrows to an expression in pygidial cells.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

E. Gazave et al. / Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

(Fig. 5R, red arrows) and in the whole pygidium (Fig. 5R, light blue arrow). Pdu-evx is expressed in the midgut (Fig. 5S, yellow asterisk), in ectodermal prepygidial cells (Fig. 5S′, dark blue arrow), and pygidial cells (Fig. 5S′, light blue arrow). Pdu-hox3 displays a complex expression: it is expressed in most or all cells of the ventral surface of the pygidium (Fig. 5T, light blue arrows), in an incomplete ring of superficial cells immediately anterior to the pygidium (Fig. 5T′ and T″; dark blue arrows), and in a few internal cells located close to the gut (Fig. 5T ′; red arrows), probably corresponding to some of the posteriormost cells that also express genes such as Pdu-piwi (Fig. 4A′). Finally, Pduhunchback is only expressed in the developing midgut (Fig. 5U) in which several other genes, such as Pdu-piwi, are also expressed (Figs. 4A and A′; 5A, B, D–G, N, R, S, and U). At 15dpf, most of the studied genes are expressed in the developing 4th segment in complex spatio-temporal patterns (Figs. 4B–F′ and S2). There is no more developmental synchrony between the worms of a same batch – based on morphological criteria, we defined 4 successive stages that can be observed (Fig. 4B–E″). Stage I corresponds to worms in which there is no recognizable 4th segment and that are therefore morphologically similar to 5dpf worms. In stage II worms, a small 4th segment can be observed but the parapodes of this segment have yet not developed. At stages III and IV, larger and more mature 4th segments are observed – the 4th segment bears parapodes and chaetae can be observed (asterisk in Fig. 4D′–E″). Stage IV differs from stage III by the presence of more mature parapodes as seen for example by the presence of two sets of chaetae per hemisegment (corresponding to the two lobes of mature parapodes) instead of one seen in stage III worms. Most of the studied genes show differential expressions at these different stages and follow a similar temporal dynamics that can be exemplified with the expression of Pdu-piwi (Fig. 4B–F″). At the four stages, Pdu-piwi is expressed in three bilateral cell populations with distinct positions along the dorsoventral axis: dorsolateral superficial (therefore probably ectodermal) cells (Fig. 4B–E; purple arrows), dorsolateral internal cells located close to the gut (Fig. 4B′–E′; pink arrows/arrowheads), and ventral internal cells (Fig. 4B”-E”; red arrows/arrowheads). The two latter Pdu-piwi-expressing cell populations much probably correspond to those observed at 5dpf – however at 15dpf there is no clear delimitation between these two populations of cells (Fig. 4F′). The temporal dynamic of expression is similar for the three Pdupiwi-expressing cell populations: in stage I worms, expression is found in a few posterior cells located immediately anterior to the boundary with the pygidium (Fig. 4B–B″). During stages II and III, there is a marked increase in the number of Pdu-piwi-expressing cells throughout the anteroposterior axis of the developing 4th segment (Fig. 4C–D″), although in the mesoderm and around the gut, the expression of Pdu-piwi is much stronger in posterior cells than in more anterior ones (Fig. 4C′, C″, D′, and D″). At stage IV, the expression of Pdu-piwi in the anterior part of the 4th segment significantly decreases and strong expression is only maintained in a few cells immediately anterior to the pygidium (Fig. 4E–E″). Most other genes (with the exception of Pdu-cdx, Pdu-evx, Pdu-hox3, and Pdu-hunchback) show the same kind of expression profile and expression dynamics than Pdu-piwi (stage II/III worms in Fig. S2A–P″; not shown for the other stages). At the four stages observed in 15dpf worms, Pdu-cdx is expressed in the developing gut and in a few internal cells located posteriorly in the 4th segment and in the pygidium (Fig. S2Q). Pdu-evx and Pdu-hox3 are both expressed in a large dorsal patch of superficial cells in the 4th segment as well as in some gut cells (Fig. S2R–S′) – the number of superficial cells expressing these genes increases from stage I to III and then decreases in stage IV worms (not shown). Pdu-hunchback displays a weak expression in a few posterior mesodermal cells throughout the four stages (Fig. S2T and not shown). Finally, we were unable to detect any expression of Pdu-gcm at 15dpf.

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The study of the expressions of the same genes during postcaudal regeneration posterior elongation revealed two very prominent groups of cells in the SAZ. We found that 12 of the genes are expressed in a ring-like group of posterior mesodermal cells located immediately anterior to the pygidium boundary (Fig. 6, red arrows). In continuity with this strong ring-like expression, most of these genes are also expressed in a decreasing gradient in more anterior mesodermal cells belonging to recently produced segments (Fig. 6). These patterns suggest that gene expressions start in a ring-like region of mesodermal progenitor or stem cells in front of the pygidium, and are maintained in some of their daughter cells in the segment primordia that are produced by their proliferation. We also observed that 14 of the studied genes (among which 6 that are also expressed in mesodermal cells) are prominently expressed in a 1- to 3/4-cell wide ring of ectodermal cells immediately anterior to the pygidium (Fig. 7, dark blue arrows). These patterns are very similar to those already described in ectodermal teloblast-like cells for Pdu-cdx and Pdu-evx (De Rosa et al., 2005 and Fig. 7L and M). The ring, most clearly visible on the dorsal side of the worms extends to their ventral face but is in most cases interrupted in the ventralmost part of the ectoderm (not shown). The average anterior–posterior extension of the ringlike expression varies between the genes. Some of the genes (Pdunanos, Pdu-myc and Pdu-ap2) are expressed in a very narrow (1–2cell wide) ring (Fig. 7D, H, and J). Some other genes (Pdu-id, Pducdx, Pdu-evx, Pdu-hox3) are expressed in a wider ring (3/4-cell wide) with a posterior to anterior decreasing gradient shape (Fig. 7I, L–N), suggesting that their expression is maintained not only in the stem cells, but also in some of their anterior daughters that are going to be incorporated into segment primordia. By contrast with the mesodermal expression however, most of the genes are not expressed in the epidermis of the growing segment primordia. One exception is Pdu-SmB that is also expressed in a decreasing gradient-like fashion in more anterior segmental epidermal cells (Fig. 7F). Pdu-evx and Pdu-cdx are also expressed in pygidial cells (Fig. 7L and M; light blue arrows). Pdu-myc and Pdugcm expression is also found at the basis of anal cirri (Fig. 7H and K; green asterisks). Many of the studied genes were also expressed in the central nervous system of the developing segments (not shown). Finally, we were unable to detect any expression of Pdumusashi in the worms 11 days after posterior amputation. The localized ring-like expressions of most of the genes in the ectoderm and in the mesoderm suggest that these genes are expressed by putative teloblast-like stem cells. In order to detect cytological properties that would make these stem cells distinct from the neighboring non-stem pygidial and segmental precursor cells, we carried out higher magnification confocal imaging of fluorescent WMISH for some of the most strongly expressed genes (Pdu-piwi, Pdu-nanos, Pdu-ap2 and Pdu-hox3) combined with a staining with the nuclear dye Hoechst. At the level of the ectoderm, nuclear labeling clearly reveals a continuous 1–2 cell wide ring of cells that have large nuclei and nucleoli, as well as less densely labeled nuclei, as compared to the more anterior segment progenitor cells and more posterior pygidial cells (Fig. 8A–E). These cells with high nuclear/cytoplasmic ratio are those that express all four genes. Pdu-piwi and Pdu-hox3 are also expressed, although at lesser levels, in a few rows of more anterior segmental progenitor cells. At the level of the mesoderm, we could follow the labeling for Pdupiwi and with an anti-Vasa antibody (Fig. 8F and F′). We observed cells with similar high nuclear/cytoplasmic ratio, different from the neighboring pygidial cells on the ventral side of the SAZ. However, similar nuclei were not observed in more lateral or dorsal Piwi+/Vasa+ cells, suggesting that mesodermal cells with stem properties are restricted to the ventral side of the SAZ and not organized in a ring-shape as the ectodermal teloblasts are. This would imply that the ring of Piwi+/Vasa+ mesodermal cells of the

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Fig. 6. Expression of the studied genes in mesodermal cells during post-caudal regeneration posterior elongation. WMISH for the genes whose name is indicated on each panel are shown. All panels are ventral views (anterior is up) and only the posteriormost part of the worms is shown. The red arrows point to the posterior mesodermal expression domain that likely includes the mesodermal stem cells.

SAZ is mostly formed of non-self-renewing mesodermal precursors, some of which are presumably migrating toward and populating the dorsal side. We have however no other data supporting this interpretation and further studies will be required to clarify this point in the future. In conclusion, we found that most of the 25 studied genes show restricted expression patterns in the posterior part of the growing worms during the early phase of posterior elongation that leads to the formation of the 4th segment and during post-caudal regeneration posterior elongation (Table 1). Expression patterns at the different stages are summarized in Fig. 9, highlighting the fact that most of these genes are expressed in defined sets of cells in the SAZ (see Discussion), with separate ectodermal and mesodermal components. To get further evidence of the stem nature of some of the cells of the SAZ, we next studied cell proliferation during posterior elongation. Cell proliferation profiles during posterior elongation in P. dumerilii To identify proliferating cells, we made EdU incorporations (Sphase marker) in 3dpf to 15dpf worms as well as in worms 11 days after posterior amputation. We also studied by WMISH the expression of three previously cloned cyclin B genes (Demilly et al., 2013), which encode mitotic Cyclins and are therefore markers of the Mphase of the cell cycle, as well as a newly cloned PCNA gene, a marker of proliferating cells (Fig. S1). We focused on proliferation in the posterior part of the worms – cell divisions also occur in other regions of the worms (e.g., brain and ventral nerve cord), but this

will not be described here. In 3dpf worms (Fig. 10A), EdU positive cells are mainly found in cells in the posterior part of the 3rd segment as well as in the pygidium. Based on their location, the former probably include some midgut cells and some of the posterior cells that express most of the stem cell genes we studied (Fig. 3). We also observed an expression of the cyclin and PCNA genes in these two populations of cells (Fig. S3A–D), further indicating that these cells are in a proliferating state. At 4dpf (Fig. 10B), the EdU incorporation profile is similar to that observed at 3dpf. At 5, 6 and 7dpf (Fig. 10C and not shown), EdU positive cells are found in the developing gut as well as in a few cells that are located close to it (these cells probably correspond to those that expressed stem cell genes such as Pdu-piwi and Pdu-myc; see Figs. 4A and 5). Only very few EdU positive cells are observed in the pygidium in contrast to the previous stages. The expression patterns of the cyclin and PCNA genes are similar to the EdU incorporation experiment at 5dpf (Fig. S3E–H). At 10dpf and 15dpf (Fig. 10D–G′), a large number of EdU positive cells are observed throughout the developing 4th segment including both the mesoderm and the ectoderm. Fluorescent WMISH combined with EdU labeling indicates that many EdU positive cells are Pdupiwi-expressing cells and vice-versa (Fig. 10H–I″). At 15dpf, cyclin and PCNA genes are also widely expressed in the mesoderm and the ectoderm of the developing 4th segment (Fig. S3I–J″ and not shown). At least some of the EdU positive cells observed in the posterior part of the 3dpf to 7dpf worms likely belong to the SAZ. To define how these cells contribute to the formation of the 4th segment, we

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Fig. 7. Expression of the studied genes in putative ectodermal stem cells during post-caudal regeneration posterior elongation. WMISH for the genes whose name is indicated on each panel are shown. All panels are dorsal views (anterior is up) and only the posteriormost part of the worms is shown. The dark blue arrows point to a ring of ectodermal expression immediately anterior to the pygidium, the light blue arrows to an expression in pygidial cells and the green asterisks to an expression in anal cirri. Diffuse staining in nascent segments just in front of the putative ectodermal stem cells is also observed for several genes.

performed EdU pulse and chase experiments (Fig. 10J–L). One hour EdU pulses were followed by chases in natural sea water until 21– 24dpf, at which stage the majority of the worms shows a well developed 4th segment. When the EdU pulse is done at 3dpf, the 4th segment remains almost entirely unlabeled. Only a few EdU internal positive cells were observed at the level of the 4th segment (Fig. 10J), suggesting that the EdU incorporating cells at 3dpf do not contribute much to the formation of the 4th segment. In contrast, EdU pulses performed at 4, 5, 6 or 7dpf lead to 21dpf worms whose 4th segment contains many EdU positive cells (Fig. 10K–L and not shown). Given the small number of EdUincorporating cells in the posterior part of 4dpf to 7dpf worms (Fig. 10B, C and not shown) and the weak and punctuated EdU labeling observed after the chases in 21dpf worms (Fig. 10K), it is much likely that the 4th segment is produced from a small population of stem cells that divide after 3dpf. It has been broadly observed that stem cells, because they divide slowly compared to their non-stem daughters, are able to retain proliferation label

within their nucleus over long chase periods, as shown for some well characterized stem cells in other systems (see for example Alié et al., 2011). In our experiment, indirect evidence for the presence of functional stem cells in the posterior part in early nectochaete larvae is provided by some of the worms labeled with EdU at 4dpf (Fig. 10L). These worms have a 4th segment weakly labeled, a mostly unlabeled pygidium and, in between, a more strongly labeled ring-like region (purple arrows) appears. Given the width of this region, it is likely that these worms have started to produce a fifth segment but the fact that EdU is retained strongly in ectodermal and mesodermal cells directly abutting the pygidium shows the posterior stem cells have divided around 4dpf and that these few divisions gave all the ectodermal and mesodermal precursors of the 4th segment. We next performed EdU incorporations in worms 11 days after caudal amputation. Cell proliferation is observed both in the mesoderm and the ectoderm (Fig. 11). The patterns of proliferation are clearly distinct at the level of the SAZ and in the growing segment

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Fig. 8. High-magnification views of the putative ectodermal and mesodermal stem cells during post-caudal regeneration posterior elongation. (A–D′, F, F′) Hoechst labeling (cyan) and fluorescent WMISH (red) for the genes whose name is indicated on the right panels are shown. In (F, F′) an anti-VASA immunolabelling (green) is shown in addition to Hoechst labeling and fluorescent WMISH. A–D′ panels are dorsal views and F, F′ panels are ventral views (anterior is up in all cases), only a small posterior part of the worms is shown. A–D′, the dotted lines delineate a row of cells that appear distinct from the more anterior (segment anlagen) and posterior cells (pygidial ectoderm) based on Hoechst labeling. These cells correspond to the posteriormost cells expressing Pdu-piwi, Pdu-nanos, and Pdu-hox3 and the only cells that express Pdu-ap2. (E) We quantified three cell properties (nucleus size, nucleolus size, and intensity of the Hoechst labeling) in three populations of cells, those delineated by the dotted lines (“putative stem cells”; see main text), more anterior cells (“segment progenitor cells”), and more posterior ones (“pygidial cells”). Values indicate the mean7the standard deviation (SD). The putative stem cells significantly differ from the two other cell populations for the three cell properties (double asterisks, Student's t-test, p40.01%). F–F′, dotted lines delineate a central pool of cells that appear distinct from the posterior cells (pygidial mesoderm) and lateral cells, based on Hoechst labeling. These cells correspond to the cells expressing Pdu-piwi and whose cytoplasm contains Pdu-Vasa protein. These cells therefore likely are mesodermal stem cells.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Fig. 9. Summary of the expression of the studied genes during posterior elongation. Schematic drawings of the posterior part of 3, 5, and 15dpf worms (dorsal views) as well as of worms 11 days after posterior amputation (ventral views) are shown. For 15dpf worms, the represented expression patterns are those found in stage II–III worms, i.e. worms in which the 4th segment has been produced but is still growing. For 15dpf worms, a transverse section at the level of the dashed line is also shown. Drawings and pictures of the entire worms with anatomical information can be found in Fig. 1. ac ¼ Anal cirri; S¼ segment; py¼ pygidium.

primordia. In the mesoderm, the EdU labeling appears in a U-shaped pattern with a ventral posterior unpaired domain, immediately anterior to the pygidium, and two bilateral longitudinal columns that extends from the lateral part of the posterior domain towards more anterior regions and encompasses the newly formed segments (Fig. 11B, B′, and E). The posterior-most mesodermal population of EdU positive cells corresponds to cells that contain Pdu-Vasa protein (as seen with an immunolabelling with a specific antibody; Rebscher et al., 2007), strongly express Pdu-piwi (Fig. 11A–G) and correspond to the stem cell-like large nuclei described above (Fig. 8). Numerous EdU positive cells are also observed in the ectoderm including the developing central nervous system (not shown), which renders interpretation complex. On the dorsal side of the worms however, a salt and pepper pattern of EdU positive cells is observed in the developing segments, as well as a more or less complete ring of EdU positive cells immediately anterior to the pygidium (Fig. 11I and L). These latter EdU positive cells correspond to the cells with stem-cell cytological properties described above (Fig. 8). These cells also express Pdu-ap2 and Pdu-pl10 (Fig. 11H–M). The cyclin and PCNA genes show expression patterns that are very similar to the EdU labellings (Fig. S3K–N′), highlighting both the mesodermal and the ectodermal stem cells of the SAZ.

Discussion In many segmented animals such as vertebrates, most arthropods and annelids, segments are added sequentially through a process known as posterior elongation and that relies on the presence of a segment addition zone (SAZ; De Rosa et al., 2005; Martin and

Kimelman, 2009; Janssen et al., 2010). This region producing new cells and tissues theoretically harbors one or more populations of cells with stem-cell like properties, i.e., capable of differentiating into all cell types present in mature segments and capable of cell renewal for maintaining posterior elongation until all body segments are produced. In vertebrates, the SAZ is found in the tail bud, whereas in clitellate annelid embryos such as leeches (Sedentaria), the SAZ is formed by ten ecto- and mesoteloblasts (see Introduction for details and references). It is generally believed that a similar situation holds true for non-clitellate annelids (Errantia) such as Platynereis. These animals grow during most of their life and add dozens of segments – it is hardly conceivable to have such an extensive and continuous growth without the presence of stem cells that allow both the formation of a new segment at a given time and the capacity to form additional ones later on. However, experimental evidence in favor of the existence of these stem cells and data about their characterization were still lacking. In a recent work focusing on post-caudal regeneration posterior elongation in Perinereis nuntia, Niwa et al. (in press) did not detect evidence of cytologically distinct teloblast-like stem cells and left open the possibility that the posterior addition of new tissues is made through coordinate recruitment of independently proliferating pygidial cells. In Platynereis however, through the combined use of EdU pulse/chase labellings and high magnification confocal images of proliferating cells, we provide in this study unequivocal evidence of the existence of posterior stem cells. In the ectoderm and the mesoderm of Platynereis SAZ, cells with stem-like cytological properties and elaborate molecular signatures of “stem cell genes” divide in a coordinated fashion and are at the origin of the tissues of the nascent segments.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Fig. 10. Cell proliferation during early stages of posterior elongation. (A–G′) and (J–L): EdU (red) and Hoechst (blue) labellings; (H–I″) EdU (red) labeling and fluorescent WMISH for Pdu-piwi (green). (D, D′, D″) and (E, E′, E″) correspond to three different regions along the dorsoventral axis (from ventral to dorsal) of the same worm at 10dpf and 15dpf, respectively. (F, G, G′) and (I–I″) are virtual cross-sections approximately made in the middle of the developing 4th segment. In 15dpf worms, separate ventral (G) and dorsal (G′) side virtual sections of the worms are shown because of tissue thickness. The dashed line indicates the anterior boundary of the pygidium. The yellow asterisks indicate the position of the developing gut. White arrows point to posterior EdU positive cells in the 3rd segment or of the developing 4th segment depending on the stage. In L, purple arrows point to cells of the SAZ that retain EdU in the pulse and chase experiments.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Fig. 11. Cell proliferation during post-caudal regeneration posterior elongation. (A–G) are ventral views focusing on the mesoderm, whereas (H–M) are dorsal views focusing on the ectoderm. EdU labeling is in red, Hoechst staining in blue, fluorescent WMISH in green, and anti-VASA immunolabelling in cyan. (A′, B′, B″) and (A″, B″, C″) are transverse and longitudinal sections, respectively, made at the positions indicated on the (A, B, C) panels. The white arrows point to medial EdU positive cells and whose cytoplasm contains Pdu-Vasa protein. The white asterisks indicate the position of an incomplete posterior ring of ectodermal EdU positive cells that express Pdu-pl10 and Pdu-ap2.

Platynereis orthologs of “stem cell genes” are expressed in both the segment addition zone (SAZ) and the developing segments during posterior elongation The SAZ of annelids has been described to be located in a subterminal position, i.e., posterior to the lastly formed segment but anterior to the pygidium, the annelid's terminal body region that bears the anus (reviewed in De Rosa et al. (2005)). While a few genes were previously shown to be probably expressed in the

Platynereis SAZ (De Rosa et al., 2005; Rebscher et al., 2007), further characterization of its organization and function requires the isolation of additional molecular makers. To this purpose, we have analyzed the expression during posterior elongation of Platynereis orthologs of many genes known to be expressed in germinal and/ or somatic stem cells in other animals (Table 2). Several of these genes (such as pumilio, pufA/B or tudor) were only studied so far in model organisms such as Drosophila, vertebrates and Caenorhabditis, and mainly shown to be involved in germ cell development

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in these species. Here we show that most of these genes are expressed during posterior elongation in overlapping patterns in the posterior part of the developing worms (Fig. 9), in addition to be expressed in the primordial germ cells (PGCs; Fig. 2). At 3dpf, i.e., at the end of embryonic/larval development and several days before posterior elongation starts, almost all the studied genes are expressed in a small group of internal cells located in the posterior part of the 3rd segment and anterior to the pygidium (Fig. 9). An expression of some “germ cell genes” (such as vasa and piwi) in this group of cells has been previously shown (Rebscher et al., 2007) and a recent study suggested that this group of cells contain both 4 PGCs as well as some somatic cells likely belonging to the prospective SAZ (Rebscher et al., 2012). We observed EdU incorporation (Fig. 10A), cyclin B and PCNA gene expression (Fig. S3A–D), in cells with a similar position than those expressing the studied “stem cell genes”, suggesting that at least some of these cells are proliferative. At 5dpf, i.e., shortly before posterior elongation starts, most of the studied genes are expressed in the posterior part of the 3rd segment in one or two small populations of internal cells close to the hindgut (Fig. 9). At this stage, the PGCs have moved to more anterior positions (Rebscher et al., 2007) and therefore the expression we observed in the posterior part of the 3rd segment is thus in somatic cells only. For many of the genes, we also observed an expression in large isolated more anterior cells that likely are the migrating PGCs (not shown for 5dpf worms; Fig. 2 for 15dpf worms). Some of the posterior cells that express the “stem cell genes” at 5dpf are probably proliferative based on EdU incorporation, cyclin B and PCNA expression patterns (Figs. 10C and S3E–H). We can therefore conclude that most of the “stem cell genes” we studied are expressed at 3dpf and 5dpf in a few internal cells with a subterminal location and showing proliferative abilities, i.e., most probably in the mesodermal component of the prospective SAZ (see below for further discussion on the organization of the SAZ and the presence of ectodermal/mesodermal components). At 15dpf, most of the genes show a broad expression in the developing 4th segment both in internal and superficial cells, i.e., in both the developing mesoderm and ectoderm (Fig. 9). These cells are proliferating, as shown by EdU incorporation (Fig. 10E–I″), cyclin B and PCNA expression (Fig. S3I–J″), and likely correspond to progenitors/undifferentiated cells that will produce the differentiated tissues of the 4th segment. In addition to this large expression domain in the developing 4th segment, a strong expression in a few cells in front of the pygidium is also often observed (Figs. 4C–D″ and S2), suggesting that most of the studied genes are in fact expressed in both the SAZ and the developing 4th segment. In 15dpf worms that have not yet produced a 4th segment, only the expression in the SAZ is observed (Fig. 4B–B″ and not shown). A similar situation is observed in worms 11 days after caudal amputation as many of the studied genes are expressed in ectodermal and mesodermal domains posterior to the lastly formed segment and anterior to the pygidium (Fig. 9). Proliferative cells are observed in these zones (Figs. 11 and S3K–N′) that constitute the SAZ during post-caudal regeneration posterior elongation. In addition to being expressed in the SAZ, many of the genes are also expressed in the mesoderm of developing segments, like what has been observed in 15dpf worms (Fig. 9 and not shown). The expression of a large number of genes whose orthologs are predominantly or exclusively expressed in stem cells in other organisms, in the cells of the Platynereis SAZ suggests that the SAZ contains bona fide stem cells. The observation that most of these genes are also expressed in the PGCs in Platynereis reinforces the view that their combined expression constitutes a “stem cell signature”, thereby supporting the stem cell nature of the cells expressing these genes in the SAZ. The argument of the “stem cell

signature” must however be considered with caution as most of these genes are also expressed in mesodermal cells of the developing segments and for some in ectodermal progenitors just in front of the cytologically characterized ectodermal teloblasts, which correspond to (or at least include) non-stem cell populations. The expression in the SAZ may be at least in part in some types of progenitor cells that do not show “stemness” properties such as self-renewal and the ability to undergo asymmetric cell divisions. An often used indirect evidence for the stem cell nature of undifferentiated proliferative cells is based on the capacity of some well characterized stem cells to retain proliferation label within their nucleus over long chase periods (see for example Alié et al., 2011). We performed EdU pulse and chase experiments, incorporating EdU before posterior elongation actually starts (from 4dpf to 7dpf) and chasing until 21dpf when most worms have a well differentiated 4th segment (Fig. 10J–L). These experiments showed that the 4th segment has been produced through many rounds of divisions from a small population of initial cells, which is consistent with the presence of stem cells. Definitive demonstration of the presence and behavior of stem cells and progenitors in the SAZ would require individual cell lineage tracing experiments that are currently very difficult to perform in Platynereis due to a lack of convenient technical tools (Ferrier, 2012). Organization of the Platynereis SAZ and the formation of ectodermal and mesodermal derivatives during posterior elongation In clitellate annelids such as leeches, mesodermal and ectodermal derivatives are produced by independent stem cells, mesoteloblasts and ectoteloblasts, respectively (Weisblat and Shankland, 1985; Zhang and Weisblat, 2005). We wondered whether a similar situation could be observed in Platynereis. During post-caudal regeneration posterior elongation, our data strongly suggest that the SAZ comprises two clearly separated mesodermal and ectodermal domains (Figs. 8 and 9) as it is observed in the leeches. We indeed found that a ring of ectodermal cells located at the boundary of the lastly formed segment and the pygidium express some “stem cell genes” as well as Pdu-cdx, Pdu-evx, and Pdu-hox3. EdU incorporations and cyclin B expression indicate proliferative abilities – in these experiments, incomplete rings of ectodermal cells are often observed, indicating that the mitotic behavior of these cells is coordinated to a certain degree. Previously published BrdU pulse and chase experiments suggested that this ectodermal ring includes behave like stem cells (De Rosa et al., 2005) i.e. they divide asymmetrically. This ectodermal ring likely constitutes the ectodermal part of the SAZ and contains ectodermal stem cells (ectoteloblasts). We also observed that several “stem cell genes” (Figs. 8 and 9) are expressed in a subterminal ring-like mesodermal domain whose part of the cells are proliferating – this likely constitutes the mesodermal part of the SAZ and therefore includes the mesodermal stem cells (mesoteloblasts). Although we did not observe at earlier stages of posterior elongation (3 to 15dpf), clear ectodermal and mesodermal rings that would be comparable to the ones seen during post-caudal regeneration posterior elongation, most of the studied genes are expressed at 15dpf in large mesodermal and ectodermal domains that probably include both the teloblasts and the undifferentiated cells of the developing segments. These gene expression patterns in 15dpf worms, as well as the EdU labellings and gene expression analyses conducted during post-caudal regeneration posterior elongation, tend therefore to indicate that, during juvenile posterior elongation, two sets of independently renewed ectoteloblasts and mesoteloblasts are indeed at play. Although we do not have the sort of direct evidence provided by cell tracking, we do not see a posterior group of cell with a distinct molecular signature which would provide both ectodermal and mesodermal precursors in

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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post-caudal regeneration posterior elongation and expression patterns in 15dpf worms suggest that this situation is already in place when posterior elongation actually starts. We see no evidence of ingressing cells at the level of the SAZ that would suggest that a process akin to gastrulation would take place at this level. When do these two sets of teloblasts segregate? The embryonic origin of the ectoderm and mesoderm of the three first segments of the larva are already well known in Platynereis. As in a number of other spiralian species, they derive from two blastomeres of the D quadrant that appear early during the characteristic spiral cleavage of the egg, 2d for the ectoderm and 4d for the mesoderm (Ackermann et al., 2005). Lineage tracing experiments indicate that the 4d micromere produce the whole segmental mesoderm of the larva, as well as a small number of cells at the posterior tip of the 3dpf worms, that are also positive for the Vasa protein and proliferative (Rebscher et al., 2007). These cells probably correspond to the internal cells expressing stem cell markers that we detect and, as far as the Vasa protein labeling (Rebscher et al., 2007) and our WMISH profiles allow following their fate, they are quite convincingly at the origin of the mesodermal component of the SAZ. The origin of the ectodermal component of the SAZ in contrast remains an open question and three hypotheses can be considered: (1) these cells derive from the internal cells displaying stem cell signatures observed at 3 and 5dpf and which would thus be ectomesodermal in nature; (2) the ectoteloblasts originate from a small group of cells set aside early in development, derived from the 2d micromere and distinct from those giving rise to the mesoteloblasts; (3) ectoteloblasts are induced later, again from 2d micromere derived ectoderm, at the boundary between the third segment and the pygidium. Our data does not allow to unambiguously choose between these three possibilities. We do not know either how these two groups of cells are regenerated after caudal amputation. The first hypothesis fits well with the gene expression patterns of “stem cell genes” observed at 3 and 5dpf. Indeed, at 3dpf, at the putative location of the future SAZ, i.e., immediately anterior to the forming pygidium, we only found internal cells expressing the “stem cell genes” (as well as Pdu-cdx, Pdu-evx and Pdu-hox3) and that incorporate EdU. At 5dpf, the “stem cells genes” are still only expressed in internal cells and no EdU incorporation can be observed in superficial cells in front of the forming pygidium. This would suggest that the only stem cells present at these stages would be internal ectomesodermal stem cells that would later give rise to both the meso- and ectoteloblats. The hypothesis of a common origin for ectoteloblasts and mesoteloblasts would be reminiscent of the situation in vertebrates, in which tail bud stem cells have been shown to continuously produce both ectodermal and mesodermal progenitors during posterior elongation (e.g., Kanki and Ho, 1997; Davis and Kirschner, 2000; Mathis et al., 2001; Roszko et al., 2007; Wilson et al., 2009; Tzouanacou et al., 2009). We have however to mention that only “internal” ectodermal cells, i.e., the neuroectodermal cells of the neural tube (but not epidermal cells) are produced by these bi-potential tail bud stem cells in vertebrates (Tzouanacou et al., 2009) while in Platynereis we would have to assume that the epidermis would also be produced by ectomesodermal stem cells. In the second hypothesis, ectoteloblasts would be present in 3 and 5dpf worms but we would have failed to detect them through the expression of the studied “stem cell genes”. Interestingly, at 5dpf, Pdu-hox3 is expressed in an incomplete ring of ectodermal cells at the border between the 3rd segment and the pygidium, an expression that is clearly reminiscent to that of this gene during post-caudal regeneration posterior elongation. These Pdu-hox3-expressing cells might be the ectoteloblasts cells in 5dpf worms. However, none of the other studied genes seem to be expressed in these cells (Fig. 9). We however noticed that for most

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WMISH of the “stem cell genes” at 15dpf the labeling in the ectoderm is much weaker than in the mesoderm (and not observed in all the worms in contrast to the mesodermal labeling) – we do not know whether this is due to technical reasons or to a lower expression level in the ectoderm. It is therefore conceivable that there is some ectodermal expression of these genes at the earlier stages, in particular in the ectodermal cells that express Pdu-hox3 at 5dpf, but that we failed to detect it. A relatively late origin of the ectoteloblasts from pygidial cells (3rd hypothesis) would be supported by the expression of several genes, such as Pdu-gcm, Pdu-evx, Pdu-cdx, and Pdu-hox3 in pygidial cells at 3dpf and 5dpf stages (Fig. 9). Interestingly, Pdu-evx and Pdu-cdx are also expressed in the regenerating pygidium before post-caudal regeneration posterior elongation starts (De Rosa et al., 2005) and still expressed in cells in the pygidium of worms 11 days after caudal amputation. The pygidium per se is however unlikely to constitute a continuous source of ectoteloblasts because (i) most of the “stem cell genes” are never expressed in pygidial cells; (ii) the expression of the genes that are expressed in the pygidium in early stages tends to decrease or disappear at later stages when posterior elongation is in progress; (iii) a similar decrease is observed for EdU incorporation indicating that most cell divisions in the pygidium precede the start of posterior elongation. Posterior stem cells as a new category of animal stem cells In this work, we characterize a new type of stem cells in an organism in which their existence had only been so far postulated. In clitellate annelids and in most malacostracan crustaceans, teloblast cells had already been described cytologically. For the first time however, we are able to establish elaborate molecular signatures for two distinct populations of posterior stem cells, ectoteloblasts at the origin of the epidermis and neural tissues of worm's trunk and mesoteloblasts, which produce all the mesodermal derivatives of the segments. These signatures are for a large part shared by the direct daughters of these two populations, i.e., the proliferative intermediate segmental precursors, which is a property of other stem cell systems. One important issue raised by these data is about the validity of a molecular fingerprint for stem cells. Many of the genes encode RNA-binding proteins that also play a role in germinal stem cells in bilaterians including Platynereis itself (Table 1 and Fig. 2). This fingerprint is also found in other types of stem cells across the animal tree. Studies in cnidarians, ctenophores, flatworms, and annelids have shown that in these aforementioned species a small set of genes (mainly piwi, vasa, nanos and pl10) are expressed in both germinal and somatic stem cells (e.g., Rebscher et al., 2007; Dill and Seaver, 2008; Gustafson and Wessel, 2010; Juliano et al., 2010; 2011; Alié et al., 2011; Millane et al., 2011; Giani et al., 2011). Our study extends this fingerprint by showing that additional genes considered as germ cell genes, such as pumilio and its related genes pufA and pufB, as well as three genes from the tudor family, are expressed in both somatic and germinal stem cells in Platynereis. This reinforces a previously underappreciated relationship between germinal stem cells and multipotent somatic stem cells and that has raised the hypothesis of a common genetic core for “stemness” in germinal and somatic stem cells (e.g., Juliano et al., 2010; Alié et al., 2011; Juliano et al., 2011; Leclère et al., 2012). There are two ways of considering these similarities: convergence or ancestry. In the former conception, the proteins expressed confer properties that are crucial for the maintenance and renewal capacity of a stem cell. For instance, Piwi, other proteins of the same family and the PiRNAs that are associated to them, have been shown to play important roles in epigenetic regulation, transposon silencing, and genome integrity (Juliano et al., 2010). It is conceivable that such

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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molecules, having first evolved their functions in germinal stem cells might have been co-opted multiple times in distinct types of somatic stem cells as they appeared independently in various animal groups. A weakness of this interpretation however appears when one considers that the vertebrate somatic stem cells usually do not express these genes, letting one to ask why molecules so useful generally for “stemness” have been left aside in the vertebrate lineage. In the ancestry interpretation, molecular fingerprints, irrespective of the actual functions of the genes involved, are inherited from ancestral cell types (Arendt, 2008). The RBP fingerprint would then indicate an ancient common origin for germinal and at least some types of somatic stem cells. While this sounds at first a far-fetched idea considering the diverse biologies of the various stem cell types displaying the fingerprint, it leads to a reasonable and further testable scenario. In cnidarians, interstitial cells (I-cells) are multipotent stem cells that can differentiate in both germinal and somatic cells (Gold and Jacobs, 2013). These I-cells express the RBP fingerprint (for example, Leclère et al., 2012, in the jellyfish Clytia). I-cells are thought to play important roles in the regenerative capacity of cnidarians, as well as in their asexual reproduction capabilities. I-cells may be representative of an ancestral pluripotent stem cell type present in the ancestor of eumetazoans, playing a role in high regenerative capabilities and capable of forming germ cells by epigenesis (Solana, 2013). These cells would have been inherited by bilaterians, but evolved in different ways in the various lineages. The neoblasts of planarians are themselves capable of differentiating into germ cells, capabilities that are unknown for the neoblast-like cells of clitellate annelids and the posterior stem cells of annelids. It is noteworthy that the formation of germ cells by epigenesis is widespread in bilaterians and may be ancestral (Extavour, 2007). Some gene expressions are not shared between the germinal and somatic posterior stem cells of Platynereis. Interestingly, the genes in question are mostly transcription factors (id, ap2, gcm, evx, cdx, hox3, hunchback). These discrepancies illustrate the difficulty of defining anything close to a “universal” stemness signature. Possibly, while the RBPs would be useful for generic stem properties and their expressions therefore broadly conserved in various bilaterian stem cell types, transcription factors would intervene in much more specific properties, such as restricting the pluripotency of these cells, and would therefore be much less conserved in stem cell types across bilaterians. One example to illustrate this idea is the set of genes, discovered in recent years, capable of maintaining and inducing mammalian embryonic stem cells. They comprise transcription factors such as Oct4, Klf4 and Nanog, which do not have clear orthologs in invertebrates and must have therefore evolved in the direct ancestral lineage of vertebrates. One remarkable example illustrated in this study is Platynereis hox3. hox3 is a homeobox gene part of the Hox cluster of Platynereis (Hui et al., 2012). In a majority of bilaterian models, Hox clusters display the remarkable property of colinear expression: the genes are involved in anterior-posterior body axis patterning and are expressed along the embryonic body axis in the same order as they are found on the chromosome. While Hox gene expression in Platynereis mostly follow this rule (Kulakova et al., 2007; Pfeifer et al., 2012), hox3 is a clear exception showing, instead of a “Hox-like” expression pattern, a strong expression in the ectodermal teloblast-like stem cells. We propose that the ancestral function of hox3 in axis patterning was lost in the annelid lineage and that hox3 was recruited for a new role in the posterior stem cell system. To support this hypothesis, the hox3 ortholog in C. teleta, a distantly related marine annelid, also displays an expression in the ectodermal teloblasts (Fröbius et al., 2008), suggesting that this new function evolved early in the annelid lineage.

The ancestry of posterior elongation Posterior elongation (posterior growth) is the process by which some animals elongate their antero-posterior axis in an anterior to posterior temporal progression through the progressive addition of new tissues in the posterior part of their body. Posterior elongation is found in both segmented and unsegmented species that belong to the three main branches of bilaterians and has been hypothesized to be a feature already present in Urbilateria, the last common ancestor of all bilaterians (reviewed in De Rosa et al. (2005) and Martin and Kimelman (2009)). This hypothesis is mainly based on expression data suggesting an involvement of the Wnt pathway and evx, cdx, and brachyury genes in posterior elongation in various species as well as on functional data in vertebrates and some arthropods indicating the requirement of the Wnt pathway and cdx in the process (see Introduction for references). Evidence for the homology of posterior elongation in the different bilaterian lineages is however limited as the process of posterior elongation itself remains poorly characterized outside vertebrates and clitellates (these latter however probably correspond to a very derived condition and are therefore of limited usefulness for bilaterian wide comparisons). Functional data have only been obtained in a limited set of species. Our discovery of posterior stem cells sustaining elongation in Platynereis do not provide direct arguments to the debate of the ancestry of posterior elongation in bilaterians, but this study provides nevertheless a new ground for comparing it with the situation in other bilaterian lineages. Neither the tail bud posterior stem cells postulated from clonal analysis in vertebrates nor the SAZ of short germ arthropods display anything similar to the RBP fingerprint. However in amphioxus, a chordate that present post-embryonic posterior growth, vasa and nanos are expressed in the SAZ (Wu et al., 2011), suggesting the presence of posterior stem cells similar to the annelids. The presence of a common type of posterior stem cells in protostome and deuterostome lineages may in fact be an indication of the ancestral nature of post-embryonic posterior elongation. To further test this hypothesis, it would be interesting to consider unsegmented groups that do present posterior elongation such as hemichordates or nemerteans. Another perspective is to explore the niche of these posterior stem cells. It will be important to establish whether signaling pathways involved in posterior elongation in vertebrates, such as Wnt, Notch, and FGF pathways may have similar roles in Platynereis. We identified several members of these pathways in Platynereis and started to use small molecule inhibitors to study their function during development (Demilly et al., 2013; Gazave and Balavoine, unpublished). Cell proliferation profiles and gene expression patterns characterized in this study will undoubtedly help to determine the possible roles of these pathways during Platynereis posterior elongation.

Acknowledgments This work was funded by the CNRS, the Agence Nationale de la Recherche (France) (ANR grant “ANNELIVERTEBR”), the Institut Universitaire de France, and the Who am I? laboratory of excellence (No. ANR-11-LABX-0071) funded by the French Gouvernement through its “Investments for the Future” program operated by ANR under Grant no. ANR-11-IDEX-0005-01. JB was supported by a fellowship of the Association pour la recherche sur le cancer (ARC jeunes chercheurs). The authors wish to thank Dr. Nicole Rebscher for the gift of the Platynereis anti-Vasa antibody. We also acknowledge the platform ImagoSeine staff for help in confocal imagery. We are grateful to the Genoscope and the EMBL for giving access to unpublished Platynereis ESTs and genomic sequences. We are also thankful to two anonymous referees for valuable suggestions on the manuscript.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2013.07.013.

References Ackermann, C., Dorresteijn, A., Fischer, A., 2005. Clonal domains in postlarval Platynereis dumerilii (Annelida: Polychaeta). J. Morphol. 266, 258–280. Alié, A., Leclère, L., Jager, M., Dayraud, C., Chang, P., Le Guyader, H., Quéinnec, E., Manuel, M., 2011. Somatic stem cells express Piwi and Vasa genes in an adult ctenophore: ancient association of “germline genes” with stemness. Dev. Biol. 350, 183–197. Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552. Anne, J., 2010. Arginine methylation of SmB is required for Drosophila germ cell development. Development 137, 2819–2828. Appleford, P.J., Woollard, A., 2009. RUNX genes find a niche in stem cell biology. J. Cell. Biochem. 108, 14–21. Arendt, D., 2008. The evolution of cell types in animals: emerging principles from molecular studies. Nat. Rev. Genet. 9, 868–882. Barreau, C., Paillard, L., Méreau, A., Osborne, H.B., 2006. Mammalian CELF/Brunolike RNA-binding proteins: molecular characteristics and biological functions. Biochimie 88, 515–525. Beck, C.W., Slack, J.M.W., 1999. A developmental pathway controlling outgrowth of the Xenopus tail bud. Development 126, 1611–1620. Bolognesi, R., Farzana, L., Fischer, T.D., Brown, S.J., 2008. Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum. Curr. Biol. 18, 1624–1629. Boswell, R.E., Prout, M.E., Steichen, J.C., 1991. Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos. Development 113, 373–384. Bowman, S.K., Rolland, V., Betschinger, J., Kinsey, K.A., Emery, G., Knoblich, J.A., 2008. The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila. Dev. Cell 14, 535–546. Chawengsaksophak, K., de Graaff, W., Rossant, J., Deschamps, J., Beck, F., 2004. Cdx2 is essential for axial elongation in mouse development. Proc. Natl. Acad. Sci. USA 101, 7641–7645. Chekulaeva, M., Hentze, M.W., Ephrussi, A., 2006. Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles. Cell 124, 521–533. Chevenet, F., Brun, C., Banuls, A.L., Jacq, B., Christen, R., 2006. TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinfo. 7, 439. Copf, T., Rabet, N., Celniker, S.E., Averof, M., 2003. Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana. Development 130, 5915–5927. Davis, R.L., Kirschner, M.W., 2000. The fate of cells in the tailbud of Xenopus laevis. Development 127, 255–267. Demilly, A., Simionato, E., Ohayon, D., Kerner, P., Garcès, A., Vervoort, M., 2011. Coe genes are expressed in differentiating neurons in the central nervous system of protostomes. PLoS One 6, e21213. Demilly, A., Steinmetz, P., Gazave, E., Marchand, L., Vervoort, M., 2013. Involvement of the Wnt/β-catenin pathway in neurectoderm architecture in Platynereis dumerilii. Nat.Commun. 4, 1915, http://dx.doi.org/10.1038/ncomms2915. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469. De Rosa, R., Prud'homme, B., Balavoine, G., 2005. Caudal and even-skipped in the annelid Platynereis dumerilii and the ancestry of posterior growth. Evol. Dev. 7, 574–587. Dill, K.K., Seaver, E.C., 2008. Vasa and nanos are coexpressed in somatic and germ line tissue from early embryonic cleavage stages through adulthood in the polychaete Capitella sp. I. Dev. Genes Evol. 218, 453–463. Dohle, W., Scholtz, G., 1988. Clonal analysis of the crustacean segment: the discordance between genealogical and segmental borders. Development 104, 147–160. Dorresteijn, A.W.C., O'Grady, B., Fischer, A., Porchet-Henere, E., Boilly-Marer, Y., 1993. Molecular specification of cell lines in the embryo of Platynereis (Annelida). Roux's Arch. Dev. Biol. 202, 264–273. Eckert, D., Buhl, S., Weber, S., Jäger, R., Schorle, H., 2005. The AP-2 family of transcription factors. Genome Biol. 6, 246. Edgar, R.C., 2004. Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Extavour, C.G., Akam, M., 2003. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130, 5869–5884. Extavour, C.G., Pang, K., Matus, D.Q., Martindale, M.Q., 2005. vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms. Evol. Dev. 7, 201–215. Extavour, C.G., 2007. Evolution of the bilaterian germ line: lineage origin and modulation of specification mechanisms. Integr. Comp. Biol. 47, 770–785.

21

Ferrier, D.E., 2012. Evolutionary crossroads in developmental biology: annelids. Development 139, 2643–2653. Fernandéz-Taboada, E., Moritz, S., Zeuschner, D., Stehling, M., Schöler, H.R., Saló, E., Gentile, L., 2010. Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid body organization and planarian stem cell proliferation. Development 137, 1055–1065. Fischer, A.H., Henrich, T., Arendt, D., 2010. The normal development of Platynereis dumerilii (Nereididae, Annelida). Front. Zool. 30, 7–31. Fischer, A.H., Arendt, D., 2013. Mesoteloblast‐like mesodermal stem cells in the polychaete Annelid Platynereis dumerilii (Nereididae). J. Exp. Zool. B Mol. Dev. Evol. 320, 94–104. Fröbius, A.C., Matus, D.Q., Seaver, E.C., 2008. Genomic organization and expression demonstrate spatial and temporal Hox gene colinearity in the lophotrochozoan Capitella sp. I. PLoS One 3, e4004. Giani Jr., V.C., Yamaguchi, E., Boyle, M.J., Seaver, E.C., 2011. Somatic and germline expression of piwi during development and regeneration in the marine polychaete annelid Capitella teleta. Evodevo 5, 2–10. Gold, D.A., Jacobs, D.K., 2013. Stem cell dynamics in Cnidaria: are there unifying principles? Dev. Genes Evol. 223, 53–66. Gonsalvez, G.B., Rajendra, T.K., Wen, Y., Praveen, K., Matera, A.G., 2010. Sm proteins specify germ cell fate by facilitating oskar mRNA localization. Development 137, 2341–2351. Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27, 221–224. Guindon, S., Lethiec, F., Duroux, P., Gascuel, O., 2005. PHYML Online–a web server for fast maximum likelihood-based phylogenetic inference. Nucleic. Acids Res. 33 (Web Server issue), W557–W559. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. Guo, T., Peters, A.H., Newmark, P.A., 2006. A Bruno-like gene is required for stem cell maintenance in planarians. Dev. Cell 11, 159–169. Gustafson, E.A., Wessel, G.M., 2010. Vasa genes: emerging roles in the germ line and in multipotent cells. Bioessays 32, 626–637. Hartl, M., Mitterstiller, A.M., Valovka, T., Breuker, K., Hobmayer, B., Bister, K., 2010. Stem cell-specific activation of an ancestral myc protooncogene with conserved basic functions in the early metazoan Hydra. Proc. Natl. Acad. Sci. USA 107, 4051–4056. Harris, R.E., Pargett, M., Sutcliffe, C., Umulis, D., Ashe, H.L., 2011. Brat promotes stem cell differentiation via control of a bistable switch that restricts BMP signaling. Dev. Cell 20, 72–83. Hitoshi, S., Ishino, Y., Kumar, A., Jasmine, S., Tanaka, K.F., Kondo, T., Kato, S., Hosoya, T., Hotta, Y., Ikenaka, K., 2011. Mammalian Gcm genes induce Hes5 expression by active DNA demethylation and induce neural stem cells. Nat. Neurosci. 14, 957–964. Holley, S.A., 2007. The genetics and embryology of zebrafish metamerism. Dev. Dyn. 236, 1422–1449. Hui, J.H.L., McDougall, C., Monteiro, A.S., Holland, P.W.H., Arendt, D., Balavoine, G., Ferrier, D.E.K., 2012. Extensive chordate and annelid macrosynteny reveals ancestral homeobox gene organization. Mol. Biol. Evol. 29 (1), 157–165. Iavarone, A., Lasorella, A., 2006. ID proteins as targets in cancer and tools in neurobiology. Trends Mol. Med. 12, 588–594. Jacobs, D.K., Hughes, N.C., Fitz-Gibbon, S.T., Winchell, C.J., 2005. Terminal addition, the Cambrian radiation and the Phanerozoic evolution of bilaterian form. Evol. Dev. 7, 498–514. Janssen, R., Le Gouar, M., Pechmann, M., Poulin, F., Bolognesi, R., Schwager, E.E., Hopfen, C., Colbourne, J.K., Budd, G.E., Brown, S.J., Prpic, N.M., Kosiol, C., Vervoort, M., Damen, W.G., Balavoine, G., McGregor, A.P., 2010. Conservation, loss, and redeployment of Wnt ligands in protostomes: implications for understanding the evolution of segment formation. BMC Evol. Biol. 10, 374. Johnstone, O., Lasko, P., 2001. Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu. Rev. Genet. 35, 365–406. Juliano, C.E., Swartz, S.Z., Wessel, G.M., 2010. A conserved germline multipotency program. Development 137, 4113–4126. Juliano, C., Wang, J., Lin, H., 2011. Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms. Annu. Rev. Genet. 45, 447–469. Kataoka, N., Diem, M.D., Kim, V.N., Yong, J., Dreyfuss, G., 2001. Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicingdependent exon-exon junction complex. EMBO J. 20, 6424–6433. Kanki, J.P., Ho, R.K., 1997. The development of the posterior body in zebrafish. Development 124, 881–893. Kee, B.L., 2009. E and ID proteins branch out. Nat. Rev. Immunol. 9, 175–184. Kerner, P., Degnan, S.M., Marchand, L., Degnan, B.M., Vervoort, M., 2011. Evolution of RNA-binding proteins in animals: insights from genome-wide analysis in the sponge Amphimedon queenslandica. Mol. Biol. Evol. 28, 2289–2303. Kerner, P., Zelada González, F., Le Gouar, M., Ledent, V., Arendt, D., Vervoort, M., 2006. The expression of a hunchback ortholog in the polychaete annelid Platynereis dumerilii suggests an ancestral role in mesoderm development and neurogenesis. Dev. Genes Evol. 216, 821–828. Knoepfler, P.S., 2008. Why myc? An unexpected ingredient in the stem cell cocktail. Cell Stem Cell 2, 18–21. Kulakova, M., Bakalenko, N., Novikova, E., Cook, C.E., Eliseeva, E., Steinmetz, P.R., Kostyuchenko, R.P., Dondua, A., Arendt, D., Akam, M., Andreeva, T., 2007. Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa). Dev. Genes Evol. 217, 39–54.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i

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Kuo, M.W., Wang, S.H., Chang, J.C., Chang, C.H., Huang, L.J., Lin, H.H., Yu, A.L., Li, W. H., Yu, J., 2009. A novel puf-A gene predicted from evolutionary analysis is involved in the development of eyes and primordial germ cells. PLoS One 4, e4980. Laurenti, E., Wilson, A., Trumpp, A., 2009. Myc's other life: stem cells and beyond. Curr. Opin. Cell Biol. 21, 844–854. Le, S.Q., Gascuel, O., 2008. An improved general amino acid replacement matrix. Mol. Biol. Evol. 25, 1307–1320. Leclère, L., Jager, M., Barreau, C., Chang, P., Le Guyader, H., Manuel, M., Houliston, E., 2012. Maternally localized germ plasm mRNAs and germ cell/stem cell formation in the cnidarian Clytia. Dev. Biol. 364, 236–248. McGregor, A.P., Pechmann, M., Schwager, E.E., Feitosa, N.M., Kruck, S., Aranda, M., Damen, W.G., 2008. Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider. Curr. Biol. 18, 1619–1623. Martin, B.L., Kimelman, D., 2009. Wnt signaling and the evolution of embryonic posterior development. Curr. Biol. 19, R215–R219. Martin, B.L., Kimelman, D., 2008. Regulation of canonical Wnt signaling by Brachyury is essential for posterior mesoderm formation. Dev. Cell. 15, 121–133. Massari, M.E., Murre, C., 2000. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol. Cell. Biol. 20, 429–440. Mathis, L., Kulesa, P.M., Fraser, S.E., 2001. FGF receptor signalling is required to maintain neural progenitors during Hensen's node progression. Nat. Cell Biol. 3, 559–566. Mayer, G., Kato, C., Quast, B., Chisholm, R.H., Landman, K.A., Quinn, L.M., 2010. Growth patterns in Onychophora (velvet worms): lack of a localised posterior proliferation zone. BMC Evol. Biol. 10, 339. Millane, R.C., Kanska, J., Duffy, D.J., Seoighe, C., Cunningham, S., Plickert, G., Frank, U., 2011. Induced stem cell neoplasia in a cnidarian by ectopic expression of a POU domain transcription factor. Development 12, 2429–2439. Mito, T., Sarashina, I., Zhang, H., Iwahashi, A., Okamoto, H., Miyawaki, K., Shinmyo, Y., Ohuchi, H., Noji, S., 2005. Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus. Development 132, 2069–2079. Nimmo, R., Antebi, A., Woollard, A., 2005. mab-2 Encodes RNT-1, a C. elegans Runx homologue essential for controlling cell proliferation in a stem cell-like developmental lineage. Development 132, 5043–5054. Niwa N., Akimoto-Kato A., Sakuma M., Kuraku S. and Hayashi S., Homeogenetic inductive mechanism of segmentation in polychaete tail regeneration, Dev. Biol., http://dx.doi.org/10.1016/j.ydbio.2013.04.010, in press. Okano, H., Kawahara, H., Toriya, M., Nakao, K., Shibata, S., Imai, T., 2005. Function of RNA-binding protein Musashi-1 in stem cells. Exp. Cell Res. 306, 349–356. Parisi, M., Lin, H., 2000. Translational repression: a duet of Nanos and Pumilio. Curr. Biol. 10, R81–R83. Parma, D.H., Bennett Jr., P.E., Boswell, R.E., 2007. Mago Nashi and Tsunagi/Y14, respectively, regulate Drosophila germline stem cell differentiation and oocyte specification. Dev. Biol. 308, 507–519. Peel, A.D., Chipman, A.D., Akam, M., 2005. Arthropod segmentation: beyond the Drosophila paradigm. Nat. Rev. Genet. 6, 905–916. Pfeifer, K., Dorresteijn, A.W., Fröbius, A.C., 2012. Activation of Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Dev. Genes Evol.. 222, 165–179. Potten, C.S., Booth, C., Tudor, G.L., Booth, D., Brady, G., Hurley, P., Ashton, G., Clarke, G., Sakakibara, S., Okano, H., 2003. Identification of a putative intestinal stem cell marker and early lineage marker, Musashi1. Differentiation 71, 28–41. Prud'homme, B., de Rosa, R., Arendt, D., Julien, J.F., Pajaziti, R., Dorresteijn, A.W., Adoutte, A., Wittbrodt, J., Balavoine, G., 2003. Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation. Curr. Biol. 13, 1876–1881. Rebscher, N., Zelada-González, F., Banisch, T.U., Raible, F., Arendt, D., 2007. Vasa unveils a common origin of germ cells and of somatic stem cells from the posterior growth zone in the polychaete Platynereis dumerilii. Dev. Biol. 306, 599–611. Rebscher, N., Lidke, A.K., Ackermann, C.F., 2012. Hidden in the crowd: primordial germ cells and somatic stem cells in the mesodermal posterior growth zone of the polychaete Platynereis dumerillii are two distinct cell populations. Evodevo 3, 9.

Romero-Lanman, E.E., Pavlovic, S., Amlani, B., Chin, Y., Benezra, R., 2011. Id1 Maintains embryonic stem cell self-renewal by up-regulation of Nanog and repression of Brachyury expression. Stem Cells Dev. 21, 384–393. Roszko, I., Faure, P., Mathis, L., 2007. Stem cell growth becomes predominant while neural plate progenitor pool decreases during spinal cord elongation. Dev. Biol. 304, 232–245. Scholtz, G., 1990. The formation, differentiation and segmentation of the postnaupliar germ band of the amphipod Gammarus pulex L. (Crustacea, Malacostraca, Peracarida). Proc. R. Soc. London B 239, 163–211. Scholtz, G., 1993. Teloblasts in decapod embryos: an embryonic character reveals the monophyletic origin of freshwater crayfishes (Crustacea, Decapoda). Zool. Anz. 230, 45–54. Schwamborn, J.C., Berezikov, E., Knoblich, J.A., 2009. The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. Cell 136, 913–925. Seaver, E.C., Thamm, K., Hill, S.D., 2005. Growth patterns during segmentation in the two polychaete annelids, Capitella sp. I and Hydroides elegans: comparisons at distinct life history stages. Evol. Dev. 7, 312–326. Shimizu, T., Bae, Y.K., Muraoka, O., Hibi, M., 2005. Interaction of Wnt and caudalrelated genes in zebrafish posterior body formation. Dev. Biol. 279, 125–141. Siomi, M.C., Mannen, T., Siomi, H., 2010. How does the royal family of Tudor rule the PIWI-interacting RNA pathway? Genes Dev. 24, 636–646. Solana, J., 2013. Closing the circle of germline and stem cells: the primordial stem cell hypothesis. Evodevo 4, 2. Song, M.H., Huang, F.Z., Chang, G.Y., Weisblat, D.A., 2002. Expression and function of an even-skipped homolog in the leech Helobdella robusta. Development 129, 3681–3692. Spassov, D.S., Jurecic, R., 2003. The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function? IUBMB Life 55, 359–366. Tessmar-Raible, K., Steinmetz, P.R., Snyman, H., Hassel, M., Arendt, D., 2005. Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. Biotechniques 39, 460–464. Tharun, S., 2009. Roles of eukaryotic Lsm proteins in the regulation of mRNA function. Int. Rev. Cell. Mol. Biol. 272, 149–189. Thomson, T., Lasko, P., 2005. Tudor and its domains: germ cell formation from a Tudor perspective. Cell Res. 15, 281–291. Tzouanacou, E., Wegener, A., Wymeersch, F.J., Wilson, V., Nicolas, J.F., 2009. Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis. Dev. Cell 17, 365–376. Van de Ven, C., Bialecka, M., Neijts, R., Young, T., Rowland, J.E., Stringer, E.J., Van Rooijen, C., Meijlink, F., Nóvoa, A., Freund, J.N., Mallo, M., Beck, F., Deschamps, J., 2011. Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone. Development 138, 3451–3462. Van Rooijen, C., Simmini, S., Bialecka, M., Neijts, R., van de Ven, C., Beck, F., Deschamps, J., 2012. Evolutionarily conserved requirement of Cdx for postoccipital tissue emergence. Development 139, 2576–2583. Wang, C.Q., Jacob, B., Nah, G.S., Osato, M., 2010. Runx family genes, niche, and stem cell quiescence. Blood Cells Mol. Dis. 44, 75–86. Wang, W.D., Melville, D.B., Montero-Balaguer, M., Hatzopoulos, A.K., Knapik, E.W., 2011. Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population. Dev. Biol. 360, 173–185. Weisblat, D.A., Shankland, M., 1985. Cell lineage and segmentation in the leech. Philos. Trans. R. Soc. London B. Biol. Sci. 312, 39–56. Wilson, V., Olivera-Martinez, I., Storey, K.G., 2009. Stem cells, signals and vertebrate body axis extension. Development 136, 1591–1604. Wolff, C., Sommer, R., Schroder, R., Glaser, G., Tautz, D., 1995. Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium. Development 121, 4227–4236. Wu, H.R, Chen, Y.T., Su, Y.H., Luo, Y.J., Holland, L.Z., Yu, J.K., 2011. Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Dev. Biol. 353, 147–159. Zhang, S.O., Weisblat, D.A., 2005. Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta. Development 132, 2103–2113.

Please cite this article as: Gazave, E., et al., Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells. Dev. Biol. (2013), http://dx.doi.org/10.1016/j.ydbio.2013.07.013i