RESEARCH ARTICLECellular and Muscular GrowthPatterns During SipunculanDevelopmentALEN KRISTOF 1 , TIM WOLLESEN 1 , ANASTASSYA S. MAIOROVA 2 ,AND ANDREAS WANNINGER 11 Department of Biology, Research Group for Comparative Zoology, University of Copenhagen,Copenhagen, Denmark2 A. V. Zhirmunsky <strong>Institut</strong>e of Marine Biology, Vladivostok, RussiaABSTRACTJ. Exp. Zool.(Mol. Dev. Evol.)314B, 2011Sipuncula is a lophotrochozoan taxon with annelid affinities, albeit lacking segmentation of theadult body. Here, we present data on cell proliferation and myogenesis during development ofthree sipunculan species, Phascolosoma agassizii, Thysanocardia nigra, andThemiste pyroides. Thefirst anlagen of the circular body wall muscles appear simultaneously and not subsequently as inthe annelids. At the same time, the rudiments of four longitudinal retractor muscles appear. Thissupports the notion that four introvert retractors were part of the ancestral sipunculan bodyplan.The longitudinal muscle fibers form a pattern of densely arranged fibers around the retractormuscles, indicating that the latter evolved from modified longitudinal body wall muscles. For ashort time interval, the distribution of S-phase mitotic cells shows a metameric pattern in thedeveloping ventral nerve cord during the pelagosphera stage. This pattern disappears close tometamorphic competence. Our findings are congruent with data on sipunculan neurogenesis, aswell as with recent molecular analyses that place Sipuncula within Annelida, and thus stronglysupport a segmental ancestry of Sipuncula. J. Exp. Zool. (Mol. Dev. Evol.) 314B, 2011. & 2011Wiley-Liss, Inc.How to cite this article: Kristof A, Wollesen T, Maiorova AS, Wanninger A. 2011. Cellular andmuscular growth patterns during sipunculan development. J. Exp. Zool. (Mol. Dev. Evol.)314B:[page range].The phylogenetic position and evolutionary origin of thesipunculans, a small and exclusively marine group of coelomate,vermiform animals that show no obvious segmental organizationin the adult stage, has been controversial for decades. They havebeen related to taxa as diverse as holothurians, echiurids,priapulids, phoronids, mollusks, or annelids (e.g., Åkesson, ’58;Hyman, ’59; Rice ’85; Scheltema, ’93; Cutler, ’94). Recently, anumber of independent molecular phylogenetic analyses havesuggested a close relationship to Annelida (including Echiura) oreven a nested position within this phylum (Boore and Staton,2002; Staton, 2003; Jennings and Halanych, 2005; Bleidornet al., 2006; Struck et al., 2007; Dunn et al., 2008; Hejnol et al.,2009; Mwinyi et al., 2009; Shen et al., 2009; Sperling et al., 2009;Zrzavy et al., 2009). The latter scenario has received significantsupport by a recent study, whereby topology tests significantlyreject the sistergroup relationship of Sipuncula and Annelida(Dordel et al., 2010). Moreover, ultrastructural similarities havebeen found in the foregut of certain sipunculans and polychaetesas well as in their collagenous cuticle (Tzetlin and Purschke,2006). This notion is further supported by recent developmentalstudies on sipunculans and echiurans that have revealedsegmental traits during neurogenesis (Hessling, 2002, 2003;Hessling and Westheide, 2002; Kristof et al., 2008; Wanningeret al., 2009). Given their proposed inclusion within Annelida iscorrect, it seems plausible to assume secondary loss of a onceGrant Sponsor: European Research Council; Grant number: MEST-CT-2005-020542; Grant Sponsors: Faculty of Science, University of Copenhagen;FEBRAS; Grant numbers: 10-III-B-06-089; 09-III-A-06-190; Grant Sponsor:RFFI; Grant numbers: 08-04-01001-a; 09-04-98584_r_vostok_a; 10-04-10062_k. Correspondence to: Andreas Wanninger, Department of Biology, ResearchGroup for Comparative Zoology, University of Copenhagen, <strong>Universitet</strong>sparken15, DK-2100 Copenhagen, Denmark. E-mail: awanninger@bio.ku.dkReceived 7 July 2010; Revised 4 October 2010; Accepted 1 December 2010Published online in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/jez.b.21394& 2011 WILEY-LISS, INC.
2KRISTOF ET AL.segmented body plan rather than an initial evolutionary steptoward segmentation in these animals. Interestingly, the lack ofcertain annelid key features, such as segmentation, coelomiccavities, nuchal organs, and chaetae, is also known for a varietyof interstitial, parasitic, and sessile annelid representatives, but toa much lesser extent for large burrowing forms, such asearthworms (reviewed in Bleidorn, 2007).Segmentation is usually considered a concerted repetition oforgans or organ systems that form subsequently from a posteriorgrowth zone along the anterior–posterior axis of an animal(Willmer, ’90). Studies on the cell proliferation patterns and theontogeny of organ systems typically associated with annelidsegments (e.g., subsequently emerging sets of paired perikaryaassociated with the ventral nerve cords, body wall ring muscles,nephridia) have proven to be ideal markers to assess whether ornot a taxon has derived from a segmented ancestor (Müller andWestheide, 2000; Hessling, 2002, 2003; Hessling and Westheide,2002; de Rosa et al., 2005; Seaver et al., 2005; Bergter et al.,2007; Brinkmann and Wanninger, 2008; Kristof et al., 2008;Wanninger, 2009). Furthermore, the growing number ofimmunocytochemical studies on neuro- and myogenesis of anumber of lophotrochozoan taxa allows for a comparison ofnervous and muscle system patterning pathways in putativelysegmented and nonsegmented clades (e.g., Hay-Schmidt, 2000;Croll and Dickinson, 2004; McDougall et al., 2006; Bergter et al.,2007; Wanninger, 2008; Wanninger et al., 2008, 2009). Althoughsome variation in annelid segment formation has been reported(Seaver et al., 2005; Brinkmann and Wanninger, 2010), thesegmentation process driven from a posterior growth zone isconsidered to be the ancestral condition for Annelida (Anderson,’66; de Rosa et al., 2005; Seaver et al., 2005; Wanninger et al.,2009). Herein, we compare the tempo–spatial distribution ofproliferating cells in Thysanocardia nigra and Themiste pyroideswith growth patterns reported for annelids. We supplement thiswork with data on myogenesis in Phascolosoma agassizii,T. nigra, and T. pyroides, and thus provide insights into theevolution of the myogenic bodyplans within the Lophotrochozoa.MATERIAL AND METHODSAnimalsAdult P. agassizii were collected in the vicinity of the FridayHarbor Laboratories (Washington) and were kept in thelaboratory until gametes were released. After fertilization, thedeveloping larvae were maintained in natural seawater atambient temperature (101C). Development was followed until15 days post-fertilization (dpf) when animals had reached the latepelagosphera stage. Adult specimens of T. nigra and T. pyroideswere obtained from Crenomytilus grayanus mussel beds. Musselaggregations were collected by scuba divers at depths of 4–8 mfrom the Vostok Bay, Sea of Japan (Russia). Adults were placed insmall tanks (15–30 specimens each) with ambient seawater(20–221C) until spawning occurred. In addition, fertilizationexperiments were performed. Adult specimens were cut open andgametes were transferred into glass jars. The eggs were fertilizedwith a few drops of a diluted sperm suspension. Embryos, larvae,and juveniles were reared in Petri dishes and glass vessels at17–191C. Elongation of the anterior–posterior axis started at3 dpf in both species. Metamorphosis occurred at 10 dpf inT. nigra and at 15 dpf in T. pyroides. Development was followedin both species until the first juvenile stages, which alreadyshowed the anlagen of the primary tentacles (i.e., at 18 dpf inT. pyroides and 15 dpf in T. nigra).EdU Labeling, F-Actin Staining, Confocal Laserscanning Microscopy,and 3D ReconstructionProliferating cells were visualized by in vivo labeling with thenucleotide analogue 5-ethynyl-2 0 -deoxyuridine (EdU) that isincorporated during the syn<strong>thesis</strong> phase (S-phase) of the cell cycle.Larvae were incubated in EdU (Invitrogen, Taastrup, Denmark),diluted in filtered seawater in the following concentrations andtime intervals at 17–191C: 250 mMfor1hr,8mMfor6hr,and5mMfor 24 hr. After EdU treatment and before F-actin staining, larvaewere anesthetized by adding drops of a 3.5 or 7% MgCl 2 solution tothe seawater and were subsequently fixed in 4% paraformaldehydein 0.1 M phosphate-buffered saline (PBS) (pH 7.3) for 1.5 hr at roomtemperature or overnight at 41C. This procedure was followed bythree washes (15 min each) in 0.1 M PBS (pH 7.3) with 0.1% sodiumazide (NaN 3 ). Until further processing, samples were stored in 0.1 MPBSwith0.1%NaN 3 at 41C.After storage, larvae were rinsed in 0.1 M PBS (pH 7.3) formore than 6 hr, followed by incubation in a blocking andpermeabilization solution (saponin-based permeabilization andwash reagent with 1% BSA) overnight at 41C. Incorporated EdUwas detected with a Click-iT EdU Kit (Cat] C3005, Invitrogen,Taastrup, Denmark). The larvae were incubated with the reactioncocktail provided by the supplier (7,5 mL Alexa Flour 488 azide,30 mLCuSO 4 ,1313mL EdU reaction buffer, and 150 mL EdU bufferadditive) for 24 hr at 41C. Some specimens were additionallyincubated for 24 hr at 41C in a polyclonal rabbit anti-serotoninprimary antibody (Calbiochem, Cambridge; dilution 1:200). Thespecimens were rinsed three times for 15 min in PBS. This wasfollowed by incubation with a goat anti-rabbit Alexa 594secondary antibody (dilution 1:300; Invitrogen, Taastrup, Denmark)in 0.1 M PBS for 24 hr at 41C. Finally, the specimens werewashed three times for 15 min each in the saponin-based washreagent with 1% BSA, incubated with the nucleic acid stain4 0 , 6-diamidino-2-phenylindole (DAPI; 1:10 dilution; Invitrogen,Taastrup, Denmark), and mounted in Fluoromount G (Southern-Biotech, Birmingham, Alabama) on glass slides.For F-actin labeling, the stored larvae were washed threetimes for 15 min in 0.1 M PBS, permeabilized for 6 hr in 0.1 MPBS containing 4% Triton X-100 at 41C, and incubated in a 1:40dilution of Alexa Flour 488 phalloidin (Invitrogen, Taastrup,J. Exp. Zool. (Mol. Dev. Evol.)