EVOLUTION & DEVELOPMENT 13:1, 72–79 (2011) DOI: 10.1111/j.1525-142X.2010.00457.x

The segmental pattern of otx, gbx, and Hox genes in the annelid Platynereis dumerilii

Patrick R. H. Steinmetz,a,1 Roman P. Kostyuchenko,b Antje Fischer,a and Detlev Arendta,Ã aDevelopmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69012 Heidelberg, Germany bDepartment of Embryology, State University of St. Petersburg, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia ÃAuthor for correspondence (email: [email protected]) 1Present address: Department for Molecular Evolution and Development, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.

SUMMARY Annelids and arthropods, despite their distinct engrailed-expressingcells.Thefirstsegmentisa‘‘cryptic’’ classification as Lophotrochozoa and Ecdysozoa, present a segment that lacks chaetae and parapodia. This and the morphologically similar, segmented body plan. To elucidate the subsequent three chaetigerous larval segments harbor the evolution of segmentation and, ultimately, to align segments anterior expression boundary of gbx, hox1, hox4,andlox5 across remote phyla, we undertook a refined expression genes, respectively. This molecular segmental topography analysis to precisely register the expression of conserved matches the segmental pattern of otx, gbx,andHoxgene regionalization genes with morphological boundaries and expression in arthropods. Our data thus support the view that segmental units in the marine annelid Platynereis dumerilii. an ancestral ground pattern of segmental identities existed in We find that Pdu-otx defines a brain region anterior to the first the trunk of the last common protostome ancestor that was lost discernable segmental entity that is delineated by a stripe of or modified in protostomes lacking overt segmentation.

INTRODUCTION cockroaches; yet is not involved in the formation of anterior- most segments in the same species, which are presumably Arthropod and annelid brains are morphologically similar defined by a gap gene-driven mechanism (Stollewerk et al. with anterior supra-esophageal ganglia connected to a seg- 2003; Tautz 2004; Deque´ant and Pourquie´2008; Pueyo et al. mental chain of ventral ganglia via circum-esophageal con- 2008). In insects, myriapods, crustaceans, pycnogonids (sea nectives (Bullock and Horridge 1965). This shared spiders), chelicerates, and onychophorans, the three anterior morphology had traditionally been interpreted as obvious most segments are homologized based on conserved anterior homology and was used as strong argument in favor of com- Hox expression boundaries (hox1/labial in the second and mon descent of annelids and arthropods from segmented hox4/deformed in the third segment) (Hughes and Kaufman common ancestors (Articulata) (Cuvier 1817; Siewing 1985). 2002b; Jager et al. 2006; Eriksson et al. 2010) (Fig. 1A). Also, However, the homology of annelid and arthropod segments it is reasonable to assume that the anterior expression bound- has been called into question by the ‘‘New Animal Phylog- ary of fushi-tarazu/lox5 in the fourth segment, as found at eny’’ that places arthropods and annelids into distinct pro- least during some stages of development in the chelicerates tostome super-phyla (Ecdysozoa and Lophotrochozoa), Archegozetes and Cupiennius, the centipede Lithobius,the together with a majority of nonsegmented groups (Adoutte millipede Glomeris and the crustacean Daphnia, represents the et al. 2000). Convergent evolution of a segmented nervous ancestral arthropod state (Telford 2000; Hughes and Kauf- system in annelids and arthropods has accordingly been con- man 2002a). However, the complete change of function to a sidered a more parsimonious scenario than the multiple sec- pair-rule gene in insects and the dynamic anterior boundary in ondary loss of head segmentation (Davis and Patel 1999; some of the investigated arthropods (CNS expression in the Seaver 2003). third segment of Cupiennius; retraction to the fifth segment Recent molecular studies have elucidated different modes during later development in Daphnia) indicate that ftz is less of segment formation in anterior and posterior segments in conserved as a regional marker gene and thus less useful for various animal groups. The Delta/Notch/Hairy oscillating interphyletic comparison than the highly conserved hox1 and system plays a role in vertebrate somite formation and in hox4. Beyond that, the anterior expression boundary of segmentation from the posterior growth zone in spiders and gbx/unplugged appears to posteriorly abut otx in chordates,

72 & 2011 Wiley Periodicals, Inc. Steinmetz et al. The segmental pattern of otx, gbx,andHoxgenes 73

Fig. 1. Schematic summary correlating the posterior (orthodenticle/otx) and ante- rior expression borders of gbx/unplugged, hox1/labial, hox4/deformed and lox5/ fushi-tarazu to the position of paraseg- ment and segment boundaries marked by engrailed expression in the neuroectoder- mal regions of the hypothetical ancestral arthropod (A) and the annelid Platynereis (B). Ventral view. Segment abbreviations represent extant chelicerate, crustacean and insect segments having evolved from a common ancestral segment. An1, first antennal; An2, second antennal; Ch, cheliceral; Ic, intercalary; L1, first leg; L2, second leg; Md, mandibulary; Mx, maxillary; Pp, pedipalpal; perist, peristomium; prost., prostomium; ps, parasegments; pyg, pygidium; I–VI, Platynereis larval segments. Light gray in (B): developing parapodial appendages; Dark gray, mouth opening; Yellow, neuroectoderm. After (Li et al. 1996; Rogers and Kaufman 1996; Damen et al. 1998; Hirth et al. 1998; Telford and Thomas 1998; Telford 2000; Arendt et al. 2001; Damen 2002b; Hughes and Kaufman 2002b; Prud’homme et al. 2003; Simonnet et al. 2006; Kulakova et al. 2007; Steinmetz et al. 2007).

hemichordates, polychaetes, and Drosophila (Rhinn and Hox 1 regions (Kourakis et al. 1997; Irvine and Martindale Brand 2001; Hirth et al. 2003; Lowe et al. 2003; Steinmetz 2000; Kulakova et al. 2007; Fro¨bius et al. 2008) appears to et al. 2007; Urbach 2007). However, the limited data set convey regional identities to the series of anterior segments in available for other arthropods (especially for gbx) does not the annelid embryo and larva, as inferred from various ex- yet allow firm conclusions to what extent this pattern can be pression studies. However, while the expression of Pdu-otx generalized across arthropods. Thus, taken with caution in appears to be presegmental and that of the Hox genes stag- the cases of gbx/unplugged and ftz, the set of conserved an- gered in the segmental region, a precise alignment of these terior and posterior expression boundaries of orthologous molecular regions with early developing segmental boundaries homeobox genes constitutes a conserved molecular topogra- has not been undertaken. The matter is further complicated phy, an ‘‘arthropod ground plan’’ of segment identities (Fig. by the unclear affiliation of the ‘‘tentacular’’ cirri which are 1A) that can be used as a basis for comparing segment iden- head appendages present directly anterior of the first tities between arthropods and annelids. chaetigerous segment in Platynereis (Hempelmann 1911) Previous studies revealed only few molecular similarities and many other phyllodocids (Pleijel and Dahlgren 1998; between annelid and arthropod segment formation. In the Rouse and Pleijel 2001). It is unclear if they represent a first, phyllodocid annelid Platynereis dumerilii (Prud’homme et al. incomplete or ‘‘cryptic’’ segment (Hempelmann 1911) similar 2003), but not in other annelids investigated (Patel et al. 1989; to the reduced head segments in other polychaetes (Rouse and Wedeen and Weisblat 1991; Seaver et al. 2001; Seaver and Pleijel 2001), or a nonsegmental part of the peristomium Kaneshige 2006), orthologs of the arthropod segment polarity (Schroeder and Hermans 1975). Does this ‘‘cryptic’’ segment genes wingless (wg)andengrailed (en) demarcate early form- indeed exist and how do the anterior and posterior expression ing segment boundaries. In addition, recent observations boundaries of otx, gbx, and Hox-genes relate to the first mo- show that in Platynereis and insects, but so far not observed lecular and morphological manifestations of segmentation in in other annelids (Kang et al. 2003; Seaver and Kaneshige an annelid? How does this pattern compare with the arthro- 2006), the Hedgehog signaling system is required for the pod ground pattern? establishment and maintenance of segmental morphology Here, by reassessing engrailed expression as marker for supporting homology of segmentation at least within protos- anterior segment boundaries (Prud’homme et al. 2003) and by tomes (Dray et al. 2010). Remarkably, however, the annelid analysis of early segmental blocks of musculature, we cor- pattern of en and wg isoutofregisterbyhalfasegmentwith roborate the existence of a first, most anterior, ‘‘cryptic’’ lar- the arthropod pattern, in that adjacent wg and en stripes de- val segment in the Platynereis larva. By double whole-mount marcate segment boundaries in the annelid as opposed to in situ hybridization (Tessmar-Raible et al. 2005) we spatially parasegmental boundaries in the arthropods (located between correlate the stripes of en expression (and thus, the prospec- the later-appearing segment boundaries; Fig. 1A) (Damen tive boundaries of all larval segments) with the anterior ex- 2002b; Prud’homme et al. 2003). As in arthropods, an ante- pression boundaries of Hox and gbx genes. We show that the rior-to-posterior sequence of otx 1 (Bruce and Shankland first larval segment expresses gbx, directly abutting the otx 1 1998; Arendt et al. 2001), gbx 1 (Steinmetz et al. 2007) and peristomium, and spatially correlate the anterior expression 74 EVOLUTION&DEVELOPMENT Vol. 13, No. 1, January--February 2011 boundaries of hox1, hox4,andlox5 with the second, third, RESULTS AND DISCUSSION and fourth larval segment. Overall, this arrangement matches the arthropod ground pattern. Furthermore, the first, The molecular comparison of arthropods and annelid seg- gbx 1 Platynereis segment and its putative homologous ar- ments had so far been mainly hampered by the difficulty to thropod parasegment share the absence of a commissure and unequivocally assign regional marker gene expression to early a ganglion located on the circum-esophageal connectives developing segments and regions in annelids. Here, we reas- while all subsequent (para-) segments have segmental ganglia sess the previously described engrailed gene (Prud’homme with commissures. Our results indicate homology of the an- et al. 2003) as an anterior segment boundary marker to pre- terior most four (para-) segments in annelids and arthropods, cisely correlate homeobox gene expression borders before the which would imply the frequent loss of this pattern concom- occurrence of morphological segment boundaries in Platy- itant with segmentation in many protostome groups. nereis. Anterior to the previously described three ectodermal rows of engrailed 1 cells that extend from the ventral midline to the dorso-lateral appendage-forming re- MATERIAL AND METHODS gions (Prud’homme et al. 2003), we identify an additional, shorter row that persists through larval development, visible Animal culture from 24 hpf onwards (Fig. 2, A–F, and H). Two-color double Platynereis embryos and larvae were obtained from an established in situ hybridization shows that this anteriormost eng- breeding culture at the EMBL Heidelberg. railed 1 cell row posteriorly abuts the otx 1 peristomium at the anterior border of the trunk ventral nerve cord anlage Labeling of cellular outlines (Fig. 3, A–D). Between 24 and 32 hpf, a fifth row of en 1 cells Trochophore larvae were incubated for 15 min in 5 mMBODI- appears in the prospective growth zone of the pygidial region PY564/570 coupled to propionic acid (Invitrogen, San Diego, CA, (Fig.2,C–F,arrow).Between36and48hpf(whenmedio- USA) in natural sea water, then rinsed twice in natural sea water lateral patterning occurs and neural differentiation starts), the and mounted in a 1:1 mixture of natural sea water and 7,5% engrailed 1 rows disintegrate in the medial neuroectoderm MgCl to prevent muscular contractions. 2 (Fig. 2, C–F), similar to the medial portion of cell rows pos- itive for some NK homeobox genes that likewise show seg- Wholemount in situ hybridization and mental expression at earlier stages (Saudemont et al. 2008). immunohistochemistry During the same period, segmental structures such as chaetal Larvae were fixed in 4% paraformaldehyde/1.75% PBS Tween-20 sacs (Fig. 2E) or ciliary bands (Fig. 2H, marked by a-tubulin for 2 h. Single color and double fluorescent wholemount in situ hybridizations followed established protocols (Tessmar-Raible in situ hybridization) become morphologically apparent on et al. 2005). For immunohistochemistry, a monoclonal antibody the lateral side (Steinmetz et al. 2007), critically depending raised against acetylated a-tubulin (1:250; SigmaT6793, Sigma, on Hedgehog signaling (Dray et al. 2010). Consistent with St. Louis, MO, USA) was used. previous reports, we show by double in situ hybridization that the persisting lateral parts of the engrailed 1 rows are posi- Phalloidin labeling tioned at the anterior segment boundary abutting the ciliated Embryos were fixed in 4% PFA in PBS10.1% Tween-20 (PTW), band-marker a-tubulin at posterior segment boundaries for 50 min at room temperature, rinsed in PTW 2  20 min and (Fig. 2H). stored in PTW at 41C up to maximum 7 days. The larvae were These findings depict an apparent discrepancy between Proteinase K-digested and postfixed as described previously (Tess- morphological and molecular segmental markers: we observe mar-Raible et al. 2005). The larvae were incubated 1–3 nights four rows of engrailed 1 cells as opposed to only three pairs shaking at 41C in rhodamine–phalloidin (Molecular Probes) 1:100. of ciliated bands and parapodia. Likewise, four nk4/tinman- Afterwards the larvae were washed 3  10 min and 3 to 5  30 min expressing rows of cells were reported previously (Saudemont in PTW and stored in 87% glycerol containing 2.5 mg/ml of anti- et al. 2008). To solve this mismatch, we have inspected larval photobleaching reagent DABCO (Sigma) at 41C. Larvae were morphology more closely and observe a distinct, chaetal-sac- mounted between a slide and a cover slip, separated by three layers of adhesive tape. like anlage anterior to that of the first chaetigerous, ‘‘real’’ larval segment at 52 hpf (Fig. 2G, ‘‘I’’). At about 5 days of development, an anterior pair of tentacular cirri develops in Confocal microscopy and image analysis the corresponding region that is innervated by axons project- Single confocal images were taken on a Leica TCS SPE (Leica ing onto the axonal bundles left and right of the mouth Microsystems CMS, Mannheim, Germany) with a  40 oil im- mersion objective at 0.5–1 mm intervals. Surface rendering and 3D (circum-esophageal ring connectives) without forming a modeling of muscle structures were performed using Imaris (Bit- commissure (Fig. 4, A–F) (Gilpin-Brown 1958). These cirri plane) and colorized in Photoshop (Adobe). All other 3D recon- resemble the second, posterior pair of tentacular cirri that structions were done using ImageJ. form from the first chaetigerous segment during metamor- Steinmetz et al. The segmental pattern of otx, gbx,andHoxgenes 75

Fig. 2. Single (A–F) and two-color (H) in situ hybrid- ization of the Platynereis anterior segment boundary marker Pdu-engrailed (A–F, H) and the ciliary marker Pdu-a-tubulin (H) indicates the presence of four larval segments, also visualized by the fluorescent, cellular outline marker propionic acid-BODIPY564/570 (G). Lateral view tilted anteriorly (A–E) or ventral view (F–H). (G) Single confocal section. (H) 3D reconstruc- tion of confocal sections. I–IV, larval segments; Arrow- heads, engrailed rows at anterior larval segment boundaries; Arrows, engrailed row in the prospective posterior growth zone; Continuous line, position of the prototroch ciliated band; Dotted lines, segment bound- aries; Dashed line in (G), outlines of the invaginated parapodial and tentacular cirri ‘‘sacs’’; Crosses, the position of the parapodial sacs; Dashed line in (F), approximate border of the ventral plate neuroectoderm. phosis (Fig. 4B), when this segment loses its chaetae in the of development, the anterior tentacular cirri region exhibits a process of cephalization (Hempelmann 1911). Also, muscle muscle organization similar to subsequent chaetigerous seg- stainings with rhodamine-phalloidin reveal that after 5 days ments (Fig. 4, G–J). Although fewer in number, the anterior 76 EVOLUTION & DEVELOPMENT Vol. 13, No. 1, January--February 2011

Fig. 3. Schematic summary (A), two- (D, F, H, J, L) and single-color (B, C, E, G, I, K) whole-mount in situ hybridization correlating Platynereis otx (A,C,D),gbx (A, E, F), hox1 (A,G,H),hox4 (A, I, J), and lox5 (A, K, L) expression to the anterior segment boundary marker gene Pdu-engrailed (A, B, D, F, H, J, L). Blue stain in (D, F, H, J, L): nuclear DAPI stain. Continuous line: prototroch ciliated band position. Dashed lines in (A): segment boundaries. I–IV: position of larval segments. Color coding in (A) as in Fig. 1. All lateral views tilted anteriorly. tentacular cirri muscles are arrangedFas in chaetigerous seg- century ago (Hempelmann 1911). This notion is further sup- mentsFinto pairs of ventral oblique muscles (Fig. 4, H–J, ported by recent fate-mapping studies that show that the yellow and blue) and more medial appendage muscles (Fig. 4, anterior pair of cirri does not originate from the otx 1 H–J, orange). While the ventral oblique muscles are morpho- peristomium (developing from the 2a, 2b, and 2c micro- logical similar and thus directly comparable between first and meres), but like all following metameric segments from the 2d subsequent larval segments (Fig. 4, H–J, yellow and blue), the micromere (Ackermann et al. 2005). v-shaped appendage muscles of the first larval segment differ To establish the molecular identities of the now four from more complex parapodial appendage muscles of the Platynereis larval segments (one cryptic plus three chaetiger- subsequent segments, hampering the direct correlation of the ous segments), we spatially correlated the anterior boundaries v-shaped muscles reaching toward the cirri to a particular set of otx, gbx, hox1, hox4,andlox5 expression with the eng- of parapodial muscles (Fig. 4, H–J, orange). railed 1 anterior segment boundaries and the otx 1 per- These morphological observations together with the exis- istomium by two-color double WMISH (Fig. 3). We found tence of a first engrailed stripe anterior to the first chaetigerous that the previously identified boundary between peristomial segment reveal the existence of a first, ‘‘cryptic’’ segment in otx and gbx expression (Steinmetz et al. 2007) coincides with the anterior tentacular cirri region, as proposed already a the anterior border of the ‘‘cryptic’’ first segment demarcated Steinmetz et al. The segmental pattern of otx, gbx,andHoxgenes 77 by the anterior most engrailed cell row (Fig. 3, A–F). In the and L) register with the Pdu-en stripes of the second, third, segmented trunk, the anterior expression borders of hox1 and fourth larval segment, respectively. However, only the (Fig.3,GandH),hox4 (Fig. 3, I and J), and lox5 (Fig.3,K dorsal part of the hox4 and the ventral part of the lox5 an- terior expression boundaries overlap engrailed and thus co- incide with the annelid segment boundaries (Fig. 3, I–L). The remaining hox4 and lox5, and the entire gbx and hox1 an- terior boundaries lie posteriorly adjacent of engrailed in the middle of the annelid segment (corresponding to the arthro- pod segment boundary). A comparison between Platynereis segment and arthropod parasegment identities thus reveals striking molecular and morphological similarities (summa- rized in Fig. 1): in both cases, an anterior most gbx/un- plugged 1 and hox1/labial-negative (para-) segment lacking commissural axons is followed by a second hox1/labial 1,a third hox4/deformed 1 and a fourth lox5/fushi-tarazu 1 (para-) segment. This prospective ancestral pattern is found highly conserved in chelicerates (Rogers and Kaufman 1996; Damen et al. 1998; Telford and Thomas 1998; Telford 2000; Damen 2002b) (where, however, unplugged expression is so far un- known) and is conserved in insects with the exception of fushi- tarazu that adopted a novel function as a pair-rule gene (Hughes and Kaufman 2002b). This comparison indicates evolutionary correspondence of the first antennal/cheliceral segment in arthropods with the first larval (cryptic) segment in the annelid (Fig. 1) bearing the first pair of tentacular cirri. This would suggest that the first insect antennae are homol- ogous to the anterior pair of tentacular cirri in Platynereis,but not to annelid or onychophoran antennae, which both form from the most anterior six3 1 territory (Steinmetz et al. 2010). The Platynereis otx/gbx boundary lies, as in all bilaterians so far investigated, anterior of the Hox-expressing region

Fig. 4. Relative position (A, B), innervation (C–F) and muscula- ture (G–J) of head appendages and anterior segments before (A, C–J) and after metamorphosis (B). (A) Seven days old; (B) 5 weeks old; (C–J) 5 days old Platynereis worms. (C, E) Reconstructions of confocal stacks of acetylated a-tubulin-immunostained animals visualizing the axonal scaffold and ciliary bands. (D, F) Schematics of animals in (C, E) highlighting the main axonal fibers. Frame in (E) marks approximately the region magnified in (C). Z-projection (G), 3D-reconstruction of confocal sections (H, I) and schematics (J) of rhodamine–phalloidin stained animals. Color code in (D, F): Red, cerebral ganglion axons; Pink, Circum-esophageal connective axons innervating the anterior tentacular cirri and the esophageal nervous system; Brown, Axons of the first chaetigerous and following segments. Color code in (H–J): Yellow, anterior oblique muscles; Blue, posterior oblique muscles; orange, ‘‘cirral’’ and ‘‘parapodial’’ muscles; background colors depicting segmental identities as in Fig. 1. (A, B) Dorsal views. (C, D, H) Ventral– lateral views. (E–G, I, J) ventral views. Scale bar: 40 mm. a, anten- nae; plp, palpae; peris, peristomium; prost, prostomium; am, antennal muscle; 1.pp, first pair of parapodia; 2.pp, second pair of parapodia; atc, anterior pair of tentacular cirri; ptc, posterior pair of tentacular cirri; I–IV, segments 1–4. 78 EVOLUTION & DEVELOPMENT Vol. 13, No. 1, January--February 2011

(Damen 2002a; Steinmetz et al. 2007). In Platynereis,itde- et al. 1996; McHugh 1997; Struck et al. 2007; Kristof et al. marcates a fundamental morphological border between the 2008). We propose that secondary loss of segmentation might head that originates from all four embryonic quadrants in a be directly connected with the loss of the Hox ground pattern, quadriradial-symmetrical manner, and the segmented trunk as identified here. Notably, animals lacking all sign of seg- ectoderm (deriving entirely from the bilateral-symmetrically mentation, such as the nematode Capitella elegans (Aboo- dividing 2d micromere) (Ackermann et al. 2005). So far, the baker and Blaxter 2003), the abalone Haliotis asinina ancestral position of the otx/gbx boundary in the frame of the (Hinman et al. 2003), and the squid Euprymna scolopes (Lee developing arthropod segments is not clear due to the lack of et al. 2003), also lack all traces of these molecular regions comparative gbx expression data and missing otd/engrailed (Graham et al. 1989; Holland et al. 1992; Simeone et al. 1992; co-localization data for all arthropod groups except Droso- Tallafu and Bally-Cuif 2002; Hughes and Kaufman 2002b; phila (Hirth et al. 2003) and Tribolium (Li et al. 1996). How- Hirth et al. 2003). Further comparative analysis of other seg- ever, the restricton of otd expression to the protocerebral mented and nonsegmented ecdysozoans and lophotrochozoan regions of Tribolium (Li et al. 1996) and chelicerates (Telford groups, including the nonsegmented echiurans and sipuncul- and Thomas 1998; Simonnet et al. 2006) suggests an ancestral ans that likewise appear to be annelid-derived (Buchberger otx/gbx boundary at the anterior border of the first paraseg- et al. 1996; McHugh 1997; Struck et al. 2007; Kristof et al. mentFcomparable to the situation in Platynereis (Fig. 1). 2008), are needed to solve this issue. The position in Drosophila,whereotd expression stretches into deutocerebral regions (Hirth et al. 2003; Urbach 2007), Acknowledgment is so far not described for other arthropod species and appears This work was funded by a fellowship from the Luxembourg secondarily modified. 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