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

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1 EVOLUTION & DEVELOPMENT 13:1, (2011) DOI: /j X 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 Arendt a, a Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany b Department of Embryology, State University of St. Petersburg, Universitetskaya nab. 7/9, St. Petersburg, Russia Author for correspondence ( detlev.arendt@embl.de) 1 Present address: Department for Molecular Evolution and Development, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria. SUMMARY Annelids and arthropods, despite their distinct classification as Lophotrochozoa and Ecdysozoa, present a morphologically similar, segmented body plan. To elucidate the evolution of segmentation and, ultimately, to align segments across remote phyla, we undertook a refined expression analysis to precisely register the expression of conserved regionalization genes with morphological boundaries and segmental units in the marine annelid Platynereis dumerilii. We find that Pdu-otx defines a brain region anterior to the first discernable segmental entity that is delineated by a stripe of engrailed-expressing cells. The first segment is a cryptic segment that lacks chaetae and parapodia. This and the subsequent three chaetigerous larval segments harbor the anterior expression boundary of gbx, hox1, hox4, and lox5 genes, respectively. This molecular segmental topography matches the segmental pattern of otx, gbx, and Hox gene expression in arthropods. Our data thus support the view that an ancestral ground pattern of segmental identities existed in the trunk of the last common protostome ancestor that was lost or modified in protostomes lacking overt segmentation. INTRODUCTION Arthropod and annelid brains are morphologically similar with anterior supra-esophageal ganglia connected to a segmental chain of ventral ganglia via circum-esophageal connectives (Bullock and Horridge 1965). This shared morphology had traditionally been interpreted as obvious homology and was used as strong argument in favor of common descent of annelids and arthropods from segmented common ancestors (Articulata) (Cuvier 1817; Siewing 1985). However, the homology of annelid and arthropod segments has been called into question by the New Animal Phylogeny that places arthropods and annelids into distinct protostome super-phyla (Ecdysozoa and Lophotrochozoa), together with a majority of nonsegmented groups (Adoutte et al. 2000). Convergent evolution of a segmented nervous system in annelids and arthropods has accordingly been considered a more parsimonious scenario than the multiple secondary loss of head segmentation (Davis and Patel 1999; Seaver 2003). Recent molecular studies have elucidated different modes of segment formation in anterior and posterior segments in various animal groups. The Delta/Notch/Hairy oscillating system plays a role in vertebrate somite formation and in segmentation from the posterior growth zone in spiders and 72 cockroaches; yet is not involved in the formation of anteriormost segments in the same species, which are presumably defined by a gap gene-driven mechanism (Stollewerk et al. 2003; Tautz 2004; Deque ant and Pourquie 2008; Pueyo et al. 2008). In insects, myriapods, crustaceans, pycnogonids (sea spiders), chelicerates, and onychophorans, the three anterior most segments are homologized based on conserved anterior Hox expression boundaries (hox1/labial in the second and hox4/deformed in the third segment) (Hughes and Kaufman 2002b; Jager et al. 2006; Eriksson et al. 2010) (Fig. 1A). Also, it is reasonable to assume that the anterior expression boundary of fushi-tarazu/lox5 in the fourth segment, as found at least during some stages of development in the chelicerates Archegozetes and Cupiennius, the centipede Lithobius, the millipede Glomeris and the crustacean Daphnia, represents the ancestral arthropod state (Telford 2000; Hughes and Kaufman 2002a). However, the complete change of function to a pair-rule gene in insects and the dynamic anterior boundary in some of the investigated arthropods (CNS expression in the third segment of Cupiennius; retraction to the fifth segment during later development in Daphnia) indicate that ftz is less conserved as a regional marker gene and thus less useful for interphyletic comparison than the highly conserved hox1 and hox4. Beyond that, the anterior expression boundary of gbx/unplugged appears to posteriorly abut otx in chordates, & 2011 Wiley Periodicals, Inc.

2 Steinmetz et al. The segmental pattern of otx, gbx,and Hox genes 73 Fig. 1. Schematic summary correlating the posterior (orthodenticle/otx) and anterior expression borders of gbx/unplugged, hox1/labial, hox4/deformed and lox5/ fushi-tarazu to the position of parasegment and segment boundaries marked by engrailed expression in the neuroectodermal 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 Brand 2001; Hirth et al. 2003; Lowe et al. 2003; Steinmetz et al. 2007; Urbach 2007). However, the limited data set available for other arthropods (especially for gbx) does not yet allow firm conclusions to what extent this pattern can be generalized across arthropods. Thus, taken with caution in the cases of gbx/unplugged and ftz, the set of conserved anterior and posterior expression boundaries of orthologous homeobox genes constitutes a conserved molecular topography, an arthropod ground plan of segment identities (Fig. 1A) that can be used as a basis for comparing segment identities between arthropods and annelids. Previous studies revealed only few molecular similarities between annelid and arthropod segment formation. In the phyllodocid annelid Platynereis dumerilii (Prud homme et al. 2003), but not in other annelids investigated (Patel et al. 1989; Wedeen and Weisblat 1991; Seaver et al. 2001; Seaver and Kaneshige 2006), orthologs of the arthropod segment polarity genes wingless (wg) and engrailed (en) demarcate early forming segment boundaries. In addition, recent observations show that in Platynereis and insects, but so far not observed in other annelids (Kang et al. 2003; Seaver and Kaneshige 2006), the Hedgehog signaling system is required for the establishment and maintenance of segmental morphology supporting homology of segmentation at least within protostomes (Dray et al. 2010). Remarkably, however, the annelid pattern of en and wg isoutofregisterbyhalfasegmentwith the arthropod pattern, in that adjacent wg and en stripes demarcate segment boundaries in the annelid as opposed to parasegmental boundaries in the arthropods (located between the later-appearing segment boundaries; Fig. 1A) (Damen 2002b; Prud homme et al. 2003). As in arthropods, an anterior-to-posterior sequence of otx 1 (Bruce and Shankland 1998; Arendt et al. 2001), gbx 1 (Steinmetz et al. 2007) and Hox 1 regions (Kourakis et al. 1997; Irvine and Martindale 2000; Kulakova et al. 2007; Fro bius et al. 2008) appears to convey regional identities to the series of anterior segments in the annelid embryo and larva, as inferred from various expression studies. However, while the expression of Pdu-otx appears to be presegmental and that of the Hox genes staggered in the segmental region, a precise alignment of these molecular regions with early developing segmental boundaries has not been undertaken. The matter is further complicated by the unclear affiliation of the tentacular cirri which are head appendages present directly anterior of the first chaetigerous segment in Platynereis (Hempelmann 1911) and many other phyllodocids (Pleijel and Dahlgren 1998; Rouse and Pleijel 2001). It is unclear if they represent a first, incomplete or cryptic segment (Hempelmann 1911) similar to the reduced head segments in other polychaetes (Rouse and Pleijel 2001), or a nonsegmental part of the peristomium (Schroeder and Hermans 1975). Does this cryptic segment indeed exist and how do the anterior and posterior expression boundaries of otx, gbx, and Hox-genes relate to the first molecular and morphological manifestations of segmentation in an annelid? How does this pattern compare with the arthropod ground pattern? Here, by reassessing engrailed expression as marker for anterior segment boundaries (Prud homme et al. 2003) and by analysis of early segmental blocks of musculature, we corroborate the existence of a first, most anterior, cryptic larval segment in the Platynereis larva. By double whole-mount in situ hybridization (Tessmar-Raible et al. 2005) we spatially correlate the stripes of en expression (and thus, the prospective boundaries of all larval segments) with the anterior expression boundaries of Hox and gbx genes. We show that the first larval segment expresses gbx, directly abutting the otx 1 peristomium, and spatially correlate the anterior expression

3 74 EVOLUTION & DEVELOPMENT Vol. 13, No. 1, January--February 2011 boundaries of hox1, hox4, and lox5 with the second, third, and fourth larval segment. Overall, this arrangement matches the arthropod ground pattern. Furthermore, the first, gbx 1 Platynereis segment and its putative homologous arthropod parasegment share the absence of a commissure and a ganglion located on the circum-esophageal connectives while all subsequent (para-) segments have segmental ganglia with commissures. Our results indicate homology of the anterior most four (para-) segments in annelids and arthropods, which would imply the frequent loss of this pattern concomitant with segmentation in many protostome groups. MATERIAL AND METHODS Animal culture Platynereis embryos and larvae were obtained from an established breeding culture at the EMBL Heidelberg. Labeling of cellular outlines Trochophore larvae were incubated for 15 min in 5 mm BODI- PY564/570 coupled to propionic acid (Invitrogen, San Diego, CA, USA) in natural sea water, then rinsed twice in natural sea water and mounted in a 1:1 mixture of natural sea water and 7,5% MgCl 2 to prevent muscular contractions. Wholemount in situ hybridization and immunohistochemistry Larvae were fixed in 4% paraformaldehyde/1.75% PBS Tween-20 for 2 h. Single color and double fluorescent wholemount in situ hybridizations followed established protocols (Tessmar-Raible et al. 2005). For immunohistochemistry, a monoclonal antibody raised against acetylated a-tubulin (1:250; SigmaT6793, Sigma, St. Louis, MO, USA) was used. Phalloidin labeling Embryos were fixed in 4% PFA in PBS10.1% Tween-20 (PTW), for 50 min at room temperature, rinsed in PTW 2 20 min and stored in PTW at 41C up to maximum 7 days. The larvae were Proteinase K-digested and postfixed as described previously (Tessmar-Raible et al. 2005). The larvae were incubated 1 3 nights shaking at 41C in rhodamine phalloidin (Molecular Probes) 1:100. Afterwards the larvae were washed 3 10 min and 3 to 5 30 min in PTW and stored in 87% glycerol containing 2.5 mg/ml of antiphotobleaching reagent DABCO (Sigma) at 41C. Larvae were mounted between a slide and a cover slip, separated by three layers of adhesive tape. Confocal microscopy and image analysis Single confocal images were taken on a Leica TCS SPE (Leica Microsystems CMS, Mannheim, Germany) with a 40 oil immersion objective at mm intervals. Surface rendering and 3D modeling of muscle structures were performed using Imaris (Bitplane) and colorized in Photoshop (Adobe). All other 3D reconstructions were done using ImageJ. RESULTS AND DISCUSSION The molecular comparison of arthropods and annelid segments had so far been mainly hampered by the difficulty to unequivocally assign regional marker gene expression to early developing segments and regions in annelids. Here, we reassess the previously described engrailed gene (Prud homme et al. 2003) as an anterior segment boundary marker to precisely correlate homeobox gene expression borders before the occurrence of morphological segment boundaries in Platynereis. Anterior to the previously described three ectodermal rows of engrailed 1 cells that extend from the ventral midline to the dorso-lateral appendage-forming regions (Prud homme et al. 2003), we identify an additional, shorter row that persists through larval development, visible from 24 hpf onwards (Fig. 2, A F, and H). Two-color double in situ hybridization shows that this anteriormost engrailed 1 cell row posteriorly abuts the otx 1 peristomium at the anterior border of the trunk ventral nerve cord anlage (Fig. 3, A D). Between 24 and 32 hpf, a fifth row of en 1 cells appears in the prospective growth zone of the pygidial region (Fig.2,C F,arrow).Between36and48hpf(whenmediolateral patterning occurs and neural differentiation starts), the engrailed 1 rows disintegrate in the medial neuroectoderm (Fig. 2, C F), similar to the medial portion of cell rows positive for some NK homeobox genes that likewise show segmental expression at earlier stages (Saudemont et al. 2008). During the same period, segmental structures such as chaetal sacs (Fig. 2E) or ciliary bands (Fig. 2H, marked by a-tubulin in situ hybridization) become morphologically apparent on the lateral side (Steinmetz et al. 2007), critically depending on Hedgehog signaling (Dray et al. 2010). Consistent with previous reports, we show by double in situ hybridization that the persisting lateral parts of the engrailed 1 rows are positioned at the anterior segment boundary abutting the ciliated band-marker a-tubulin at posterior segment boundaries (Fig. 2H). These findings depict an apparent discrepancy between morphological and molecular segmental markers: we observe four rows of engrailed 1 cells as opposed to only three pairs of ciliated bands and parapodia. Likewise, four nk4/tinmanexpressing rows of cells were reported previously (Saudemont et al. 2008). To solve this mismatch, we have inspected larval morphology more closely and observe a distinct, chaetal-saclike 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 the corresponding region that is innervated by axons projecting onto the axonal bundles left and right of the mouth (circum-esophageal ring connectives) without forming a commissure (Fig. 4, A F) (Gilpin-Brown 1958). These cirri resemble the second, posterior pair of tentacular cirri that form from the first chaetigerous segment during metamor-

4 Steinmetz et al. The segmental pattern of otx, gbx,and Hox genes 75 Fig. 2. Single (A F) and two-color (H) in situ hybridization 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 reconstruction of confocal sections. I IV, larval segments; Arrowheads, 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 boundaries; 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 process of cephalization (Hempelmann 1911). Also, muscle stainings with rhodamine-phalloidin reveal that after 5 days of development, the anterior tentacular cirri region exhibits a muscle organization similar to subsequent chaetigerous segments (Fig. 4, G J). Although fewer in number, the anterior

5 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 segmentsfinto pairs of ventral oblique muscles (Fig. 4, H J, yellow and blue) and more medial appendage muscles (Fig. 4, H J, orange). While the ventral oblique muscles are morphological similar and thus directly comparable between first and subsequent larval segments (Fig. 4, H J, yellow and blue), the v-shaped appendage muscles of the first larval segment differ from more complex parapodial appendage muscles of the subsequent segments, hampering the direct correlation of the v-shaped muscles reaching toward the cirri to a particular set of parapodial muscles (Fig. 4, H J, orange). These morphological observations together with the existence of a first engrailed stripe anterior to the first chaetigerous segment reveal the existence of a first, cryptic segment in the anterior tentacular cirri region, as proposed already a century ago (Hempelmann 1911). This notion is further supported by recent fate-mapping studies that show that the anterior pair of cirri does not originate from the otx 1 peristomium (developing from the 2a, 2b, and 2c micromeres), but like all following metameric segments from the 2d micromere (Ackermann et al. 2005). To establish the molecular identities of the now four Platynereis larval segments (one cryptic plus three chaetigerous segments), we spatially correlated the anterior boundaries of otx, gbx, hox1, hox4, and lox5 expression with the engrailed 1 anterior segment boundaries and the otx 1 peristomium by two-color double WMISH (Fig. 3). We found that the previously identified boundary between peristomial otx and gbx expression (Steinmetz et al. 2007) coincides with the anterior border of the cryptic first segment demarcated

6 Steinmetz et al. by the anterior most engrailed cell row (Fig. 3, A F). In the segmented trunk, the anterior expression borders of hox1 (Fig.3,GandH),hox4 (Fig. 3, I and J), and lox5 (Fig.3,K The segmental pattern of otx, gbx,and Hox genes 77 and L) register with the Pdu-en stripes of the second, third, and fourth larval segment, respectively. However, only the dorsal part of the hox4 and the ventral part of the lox5 anterior expression boundaries overlap engrailed and thus coincide with the annelid segment boundaries (Fig. 3, I L). The remaining hox4 and lox5, and the entire gbx and hox1 anterior boundaries lie posteriorly adjacent of engrailed in the middle of the annelid segment (corresponding to the arthropod segment boundary). A comparison between Platynereis segment and arthropod parasegment identities thus reveals striking molecular and morphological similarities (summarized in Fig. 1): in both cases, an anterior most gbx/unplugged 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 unknown) and is conserved in insects with the exception of fushitarazu 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 homologous 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 musculature (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, antennae; 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.

7 78 EVOLUTION & DEVELOPMENT Vol. 13, No. 1, January--February 2011 (Damen 2002a; Steinmetz et al. 2007). In Platynereis, it demarcates a fundamental morphological border between the head that originates from all four embryonic quadrants in a quadriradial-symmetrical manner, and the segmented trunk ectoderm (deriving entirely from the bilateral-symmetrically dividing 2d micromere) (Ackermann et al. 2005). So far, the ancestral position of the otx/gbx boundary in the frame of the developing arthropod segments is not clear due to the lack of comparative gbx expression data and missing otd/engrailed co-localization data for all arthropod groups except Drosophila (Hirth et al. 2003) and Tribolium (Li et al. 1996). However, the restricton of otd expression to the protocerebral regions of Tribolium (Li et al. 1996) and chelicerates (Telford and Thomas 1998; Simonnet et al. 2006) suggests an ancestral otx/gbx boundary at the anterior border of the first parasegmentfcomparable to the situation in Platynereis (Fig. 1). The position in Drosophila, where otd expression stretches into deutocerebral regions (Hirth et al. 2003; Urbach 2007), is so far not described for other arthropod species and appears secondarily modified. Further comparative co-expression studies will more clearly position the ancestral otx/gbx expression boundary in the developing arthropod brain. We thus found high similarities between the Platynereis and the arthropod ground pattern of Hox expression. However, the Platynereis Hox expression data is less similar to other previously investigated annelids such as the polychaetes Capitella (Fro bius et al. 2008) and Chaetopterus (Irvine and Martindale 2000) or the leech Helobdella (Kourakis et al. 1997), possibly due to considerable tagmatization of the trunk (Chaetopterus, Capitella) (Irvine et al. 1999; Fro bius et al. 2008), or due to the secondary loss of a larval stage (Helobdella) in those species. An annelid ground pattern of Hox expression in the anterior segments is thus difficult to discern (Fro bius et al. 2008). Thus, the similarities between Platynereis and the arthropod ground pattern can be interpreted either as an extreme case of homoplasy (i.e., ectodermal segmentation evolving twice independently in very similar spatial relationship to pre-existing regions), or as inheritance from a last common ancestor (and subsequent secondary modification in other groups). As the tagmatization of Chaetopterus, Capitella and of the leeches is derived within annelids, we tend to favor the latter scenario. Yet, evolutionary conservation of the otx/gbx/hox ground pattern in a specific segmental framework together with the conserved role in Hedgehog signaling in establishment of segmental morphology (Dray et al. 2010) would imply that the protostome ancestor already possessed segmented ectoderm that was subsequently lost in the lineages leading to today s nonsegmented protostomes (e.g., nematodes, flatworms, and mollusks). The recent recognition of echiurans and sipunculans, nonsegmented at adult stages, as derived from segmented annelid ancestors shows that loss of segmentation might have occurred more often than assumed previously (Buchberger et al. 1996; McHugh 1997; Struck et al. 2007; Kristof et al. 2008). We propose that secondary loss of segmentation might be directly connected with the loss of the Hox ground pattern, as identified here. 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