Zoomorphology

DOI 10.1007/s00435-009-0095-7

ORIGINAL PAPER

Wound healing and arm regeneration in Ophioderma longicaudum and Amphiura Wliformis (Ophiuroidea, Echinodermata): comparative morphogenesis and histogenesis

Anna Chiara Maria Biressi · Ting Zou · Samuel Dupont · Carl Dahlberg · Cristiano Di Benedetto · Francesco Bonasoro · Michael Thorndyke · Maria Daniela Candia Carnevali

Received: 12 December 2007 / Revised: 3 November 2008 / Accepted: 25 March 2009© Springer-Verlag 2009

Abstract All species of the Ophiuroidea have exceptionalregenerative capabilities; in particular, they can replacearms lost following traumatic or self-induced amputation.In order to reconstruct this complex phenomenon, westudied arm regeneration in two diVerent ophiuroids,Ophioderma longicaudum (Retzius, 1805) and AmphiuraWliformis O. F. Müller, 1776, which are quite distantlyrelated. These species present contrasting regeneration anddiVerentiation rates and diVer in several ecological traits.The aim of this paper is to interpret the primary sequence ofmorphogenetic and histogenetic events leading to the com-plete reconstruction of a new arm, comparing the armregenerative processes of these two ophiuroid species withthose described in crinoids. Arm regeneration in ophiuroidsis considered an epimorphic process in which new struc-tures develop from a typical blastema formed from an accu-

mulation of presumptive undiVerentiated cells. Our resultsshowed that although very diVerent in some respects suchas, for instance, the regeneration rate (0.17 mm/week forO. longicaudum and 0.99 mm/week for A. Wliformis), mor-phogenetic and histogenetic aspects are surprisingly similarin both species. The regenerative process presents similarcharacteristics and follows a developmental scheme whichcan be subdivided into four phases: a repair phase, an earlyregenerative phase, an intermediate regenerative phase andan advanced regenerative phase. In terms of histogenesis,the regenerative events involve the development of newstructures from migratory pluripotent cells, which prolifer-ate actively, in addition in both cases there is a signiWcantcontribution from dediVerentiated cells, in particular ded-iVerentiating myocytes, although to varying extents. Thisevidence conWrms the plasticity of the regenerative phe-nomenon in echinoderms, which can apparently followdiVerent pathways in terms of growth and morphogenesis,but nevertheless involve both epimorphic and morphallac-tic contributions at the cellular level.

Keywords Brittle stars · Echinoderms · Morphology · Ophiuroids · Regeneration

Introduction

Echinoderma show a remarkable regenerative potentialexpressed at diVerent anatomical levels in both adults andlarvae (Candia Carnevali 2006; Eaves and Palmer 2003;Candia Carnevali and Bonasoro 2001a; Vickery et al. 2001;Thouveny and Tassava 1997). Regenerative phenomena,well known in all living echinoderms, display speciWcpeculiarities in terms of both phenomenology and strategicapplication in each individual group, and also are well

Electronic supplementary material The online version of this article (doi:10.1007/s00435-009-0095-7) contains supplementary material, which is available to authorized users.

A. C. M. Biressi (&) · C. Di Benedetto · F. Bonasoro · M. D. Candia CarnevaliDipartimento di Biologia “Luigi Gorini”, Università degli Studi di Milano, via Celoria 26, 20133 Milan, Italye-mail: anna.biressi@unimi.it

T. ZouDepartment of Physiology, Medical College of Nanchang University, 330006 Nanchang, Jiangxi, People’s Republic of China

S. Dupont · C. Dahlberg · M. ThorndykeThe Swedish Royal Academy of Sciences, Institution of Marine Ecology, Göteborg University, Kristineberg Marine Station, 566 Kristineberg, 45034 Fiskebackskil, Sweden

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documented in the fossil record (Oji 2001). Regeneration islargely employed for rebuilding appendages and organs lostafter traumatic or self-induced amputations. In addition, inmany cases, the isolated fragments (arms, body portions)can undergo a partial or total regeneration, even whendetached from the donor animal, and give rise, in extremecases, to the asexual development of a new individual(Ducati et al. 2004; Mladenov and Burke 1994; Wilkieet al. 1984; Emson and Wilkie 1980; Hyman 1955).

In ophiuroids, Wssion and regenerative phenomena arewell known and also remarkably well documented in fossilsof extinct species (Aronson 1987, 1992). In this group, armregeneration is the most explored phenomenon. However,there are only few older morphological/histological studiesdescribing arm regeneration in ophiuroid species (Morgulis1909; Zeleny 1903; DawydoV 1901), whilst recent litera-ture tends to focus speciWcally on physiological, ecologicalor molecular aspects of regeneration (Clark et al. 2007;Dupont and Thorndyke 2006, 2007; Bannister et al. 2005;Thorndyke et al. 2001, 2003; Makra and Keegan 1999;Sköld and Rosenberg 1996; Stancyk et al. 1994; Aronson1991; Walsh et al. 1986; Salzwedel 1974), leavingunsolved many fundamental morphological and histologi-cal questions, vital so as to provide basic informationneeded to better understand the evolutionary and adaptivesigniWcance of the regenerative phenomena throughoutEchinodermata. Preliminary studies on arm regeneration inophiuroid species suggested that the phenomenon can becomparable in its main aspects to arm regeneration incrinoid species (Thorndyke et al. 2001; Thorndyke andCandia Carnevali 2001). BrdU experiments also showedthat, in ophiuroid species, cell proliferation is a crucial pro-cess in the regenerative phenomenon, suggesting, by anal-ogy to crinoid species, a fundamental neurotrophic role forthe nervous system (Thorndyke et al. 2001). Indeed, exper-iments early in the twentieth century showed that the lackof a functional nerve could inhibit regeneration (Morgulis1909). More recent studies also underlined the potentialrole of the radial water canal as a major source of cells andregulatory factors during regeneration (Bannister et al.2005; Thorndyke et al. 2003). In spite of the renewed inter-est in ophiuroid regeneration, many general aspects of theregenerative process remain undeWned and are in urgentneed of clariWcation. The unfortunate absence of signiWcantcomprehensive studies focussing on arm regeneration inOphiuroidea was underlined by Wilkie (2001) in his recentreview of autotomy and regeneration in echinoderms.

This paper aims to describe in detail arm regeneration inophiuroids and provide an overview of general and essen-tial fundamental aspects of the phenomenon focussing onmorphogenesis and histogenesis.

Ophiuroidea are characterized by long and movablearticulated arms. The arms comprise a conspicuous double

endoskeletal component: a longitudinal series of vertebralossicles joined by movable articulations that form the inter-nal core of the arm. Besides this internal component, thearm is also provided with superWcial endoskeletal armour.Each vertebral ossicle is externally covered by four superW-cial skeletal plates, one aboral, one oral and two lateral armshields, these latter provided with spines. Adjacent verte-bral ossicles are connected by muscles and ligaments: twopairs of intervertebral muscles, namely oral and aboralmuscles, and one large intervertebral ligament. The nerveruns along the oral vertebral groove; it shows a typical gan-glionic structure and runs side by side with an epineuraloral canal (lined by an ectodermic-derived epithelium) anda hyponeural (coelomic derived) aboral canal. Aborally tothis hyponeural canal, the radial water canal runs in a decal-ciWed vertebral area; a pair of podia originates from theradial water canal at the level of each vertebral ossicle. Theradial haemal canal is located in the vertical septum of thehyponeural radial canal and branches into the podia(Hyman 1955).

Materials and methods

Experimental animals

Specimens (n = 140) of Ophioderma longicaudum(Retzius, 1805) (Ophiodermatidae, Ophiodermatina) werecollected in spring 2006, autumn 2006 and autumn 2007 byscuba divers in the Thyrrenian coast of Italy (Giglio Island,Tuscan Archipelago and Monte Argentario, Grosseto) at adepth of 30–50 m and a temperature of approximately16°C. They were left to acclimatize for several days priorthe experiment and maintained at 16°C in aerated 50 laquaria with artiWcial seawater (Instant Ocean, AquariumSystems) and fed once a week with a mixed diet based onchopped cuttleWsh and aquaculture Wsh food (Formula Oneand Prime Reef, Ocean Nutrition). Specimens (n = 60) ofAmphiura Wliformis O. F. Müller, 1776 (Amphiuridae,Gnathophiurina) were collected in spring 2005 and latewinter 2008 at 25–40 m depth in the vicinity of Kristine-berg Marine Station (KMF) Sweden, using a Petersen mudgrab. Individuals were immediately separated from the sed-iment cores by gentle rinsing to avoid breaking arms. Theywere then maintained in natural Xowing seawater at 14°C,with 1 cm of sediment from the collection site. Also in thiscase, the temperature stress was minimized by acclimationto 14°C for 2 weeks before the experiment.

Experimental arm regenerations were obtained by (1)stimulating the arm mechanically in its distal region inorder to induce autotomy (supplementary material); (2)leaving the autotomised animal to regenerate for a preWxedperiod; (3) collecting and processing the regenerating

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samples according to various protocols outlined below.Only a single regeneration was induced in each individual.Progress of regeneration was monitored at 3 days, 1 week,2, 3, 4, 5, 6, 10 and 12 weeks post-amputation (p.a.) forO. longicaudum and at 1, 2, 3, 4, 6, 8, 9, 12, 14, 16, 19, 24and 26 days p.a. for A. Wliformis. The regeneration anddiVerentiation rates within a species are dependent on therelative amount of tissue lost (‘length lost’: a key parame-ter, which represents the quantity of tissue needed to com-pletely regenerate an intact arm that has no obviousprevious history of regeneration; Dupont and Thorndyke2006; Clark et al. 2007). To reduce intraspeciWc variability,the position of autotomy in O. longicaudum was about one-third from the arm tip for individuals with a disc diameterof about 2 cm. In A. Wliformis, the position of autotomy wascalculated according to Dupont and Thorndyke (2006) toinduce regeneration with a regeneration rate of 0.99 mm/week and a diVerentiation rate of 21% per week.

Microscopy

Standard methods for morphological analysis by stereomi-croscopy (Leica MZ75, with Leica CLS 150XE lights, pro-vided with a camera Leica Digilux 18.102), opticalmicroscopy and transmission electron microscopy (TEM)were employed.

For histology, protocols for both standard paraYn waxand resin embedding/sectioning were employed. The use ofparaYn wax allowed the subsequent employment of stain-ing methods such as the trichrome stain or the Domagk’smethod, whilst resin provided high-quality histologicalsamples, in terms of tissue preservation, that are not achiev-able using paraYn wax.

For resin sections, regenerating arms were preWxedwith 2% glutaraldehyde in 0.1 M and 0.1 Osm cacodylatebuVer (pH 7.2) for 2 h for O. longicaudum and withSPAFG Wxative (for details see Ermak and Eakin 1976)for approximately 1 month for A. Wliformis, washed over-night in the same buVer, and post-Wxed with 1% osmicacid in 0.1 M cacodylate buVer (pH 7.2) for 2 h. No decal-ciWcation method was employed. After standard dehydra-tion in an ethanol series, the samples were embedded inEpon 812-Araldite. Semi-thin (0.96 �m) sections (Reic-hert Ultracut E with glass knives) were stained with crys-tal violet and basic fuchsin and observed under a Jenavallight microscope provided with a camera Panasonic GP-KR222; thin (0.07 �m) sections (Reichert Ultracut E withglass knives) were mounted on copper grids and stainedwith uranyl acetate and lead citrate for electron micros-copy. Thin sections were observed and photographed in aJeol 100SX electron microscope (for details see CandiaCarnevali et al. 1993).

For paraYn wax sections, regenerating samples ofO. longicaudum were Wxed in Bouin’s Wxative for 1 month.After washing several times with tap water, the sampleswere dehydrated in an ethanol series and then embedded inparaYn wax according to standard methods. Sections(8 �m), cut with a Reichert OmE, were stained according tothe Milligan trichromic staining technique and observedusing the Jenaval light microscope. For A. Wliformis, armswere Wxed with 4% paraformaldehyde in Wltered seawater(FSW) for 2 h at room temperature. They were then rinsedin FSW and decalciWed in 0.5 M EDTA (pH 8) for 24 h.The samples were subsequently dehydrated and embeddedin paraYn wax. Sections (5 �m) were stained withDomagk’s method after dewaxing and rehydration, andthen permanently mounted using PolyMount. Slides wereanalysed using a Leica microscope adapted for digitalimaging with a Canon S40 colour camera and Canon imag-ing software, all provided by Leica Microsystems. Imageswere processed by using Adobe Photoshop.

Measurements and statistics

When the regenerate started to diVerentiate, it was possibleto distinguish two distinct parts: the proximal diVerentiatedpart, comprising fully formed segments with clearly devel-oped ossicles, podia and spines, and the distal part thatremained undiVerentiated with no, or only poorly deWned,spines or ossicles (for more details on the method, seeDupont and Thorndyke 2006).

The total regenerated length (RL, mm) and the diVeren-tiated length (DL, mm) of each regenerate were measuredat each collection period. Regeneration rate (RR, mm/week) was calculated as the slope of the signiWcant(P < 0.0001, F = 175.85) simple linear regression betweenthe regenerated length (RL, mm) and time (weeks). AdiVerentiation index, DI as a percentage, was calculated asthe proportion in length of the regenerate that is com-pletely diVerentiated (0 if the regenerate is completelyundiVerentiated and 100% if the arm is completely diVer-entiated): DI = (DL/RL) £ 100. According to morphologi-cal and physiological studies, DI is a good indicator offunctional recovery of the tissue, this index being corre-lated to the timing of neuropeptide expression and physio-logical recovery of the nervous system (Dupont, personalobservation).

Simple linear regression models were used to test therelationship type between the variables. The Shapiro–Wilktest (Shapiro and Wilk 1965) was used to check whether thedata were normally distributed and the Levene test wasused to check whether variances were homogenous. Allstatistical analyses were performed using SAS/STAT®

software (SAS Institute Inc. 1990).

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Results

Whole-mount observations

Ophioderma longicaudum

Arm regeneration in O. longicaudum was monitored for anoverall period of 12 weeks from the autotomic amputationto the regrowth of a complete small-scale arm (Fig. 1).

Shortly after the traumatic event, the autotomy surfaceof the stump appeared to be strongly concave and partlyprotected/covered by the prominent edges of the externalskeletal shields (Fig. 1a). At 1 week p.a., the autotomysurface tended to acquire a Xattened shape, and only at2 weeks p.a. did a swelling become recognizable. The2-week presumptive blastema appeared as a small button,slightly emerging in the central-oral area of the autotomysurface. At 3 weeks p.a., the regenerating bud was

suYciently developed to clearly emerge from the superW-cial arm shields (Fig. 1c) appearing as a pale smooth bud.At 5–6 weeks p.a., the regenerate acquired a prominent andelongated shape, displaying a series of new arm shieldsclearly distinguishable in the transparent context of theblastemal tissue. At 12 weeks p.a. (Fig. 1d), the regeneratelooked like a pine cone, displaying larger and more deWnedarm shields with few developing spines particularly evidentin its proximal region.

Amphiura Wliformis

In A. Wliformis, arm regeneration was monitored for anoverall period of 24 days, from autotomy to the develop-ment of a complete small-scale arm (Figs. 2, 3, 4).

Immediately after autotomy, the autotomy surface waspartly covered and protected by the arm shields (1–3 daysp.a.; Fig. 3a, b). The vertebral ossicle was exposed, coveredby the remains of intervertebral muscles (Fig. 2a). After3 days, a new epithelium appeared on the aboral side tocover the entire wound (Fig. 2b, c). The Wrst appearance ofa small regenerative bud occurred at 3–4 days p.a. in thecentral-oral area at the level of the neural cord and theradial water canal (Fig. 2d). This was followed by thegrowth in length of a well-deWned smooth regenerating bud(4–6 days p.a.; Fig. 3c), and Wnally by the regrowth of aregenerating arm with new arm shields and podia (8–11 days p.a.). At 12 days p.a. (Fig. 3d), the arm shieldsbecame larger and deWned, and the proximal part of theregenerate progressively acquired the appearance of asmall-scale arm. At 19 days p.a. (Fig. 4a), the regeneratingarm could conventionally be subdivided into two parts, theproximal diVerentiated part (45% of total length) formed bycomplete functional segments with spines and podia, andthe distal undiVerentiated part. At 24 and 26 days p.a.(Fig. 4b), more than 90% (in length) of the regeneratingarm was fully diVerentiated.

Microscopic anatomy

Ophioderma longicaudum

3 days p.a. At 3 days p.a., sagittal sections (Fig. 5a–c)showed that the concave autotomy surface was already cov-ered by a thin cellular layer (Fig. 5b); this layer, due to theepithelial aspect of its peripheral regions and the heteroge-neous and discontinuous structure of its central area,seemed to be partly derived from the expansion of the outerarm epidermis and partly by cell migration. Wound healingwas still incomplete in terms of the underlying cicatriciallayer which consisted of a few scattered cells distributed ina very loose stroma of connective tissue (Fig. 5c). The dis-tal ends of the main coelomic cavity and the radial water

Fig. 1 Ophioderma longicaudum. SM. Oral view of the regeneratingarm and of the stump. Autotomy surface on the right. a 3 days p.a. andb 1 week p.a. The autotomy surface is protected/covered by the oralarm shield. c 3 weeks p.a. The regenerative blastema (arrowhead) isvisible. d 12 weeks p.a. The regenerate displays deWned arm shields(arrowhead) and a few developing spines (arrow). Os oral arm shield,Ls lateral arm shield, S spines

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canal appeared to be hypertrophic and completely sealed oVpartly by clots of migratory cells and partly by contractionof the muscular components of the coelomic wall (Fig. 6a,b). Both the coelomic components and the radial nerve cordappeared retracted with respect to the autotomy surface.

Massive groups of cells, including phagocytes and smallcells (presumably free undiVerentiated coelomocytes),apparently involved in proliferation, could be detected inclose association with the radial nerve, the radial watercanal and the other coelomic structures. Wandering coe-lomocytes and phagocytes were also frequently observedinside both somatocoelic and hydrocoelic (radial watercanal) cavities (Fig. 6c–e). In addition, a number of distinctcellular elements were recognized amongst the usual popu-lations of free cells; these cells could be identiWed as myo-cytes at diVerent stages of dediVerentiation on the basis oftheir cytological features and staining aYnity (Fig. 6i).DediVerentiating myocytes were also detectable in the his-tological areas close to the intervertebral muscles, whosestructure was undergoing an extensive rearrangement par-ticularly evident in the last stump segment (Fig. 6g–i). Onthe other hand, due to the speciWc autotomy modalities, noremains of myocytes were present on the retained side ofthe autotomy wound; in fact, during autotomy, the interver-tebral muscles related to the autotomic articulation totallydetach from their proximal insertions into the vertebralossicle.

1 week p.a. At 1 week p.a. (Fig. 5d–f), the still concaveautotomy surface appeared to be healed by a conspicuousand thick cicatricial layer comprising a mass of small cells(presumptive undiVerentiated coelomocytes) and phago-cytes, distributed in a dense meshwork of Wbrous connec-tive tissue (Fig. 5e, f). Inside this thick cicatricial layer, theregrowing distal ends of the coelomic cavities and theradial water canal, still hypertrophic, as well as the radialnerve cord appeared to begin their regrowth forward,always surrounded by groups of small cells frequentlyinvolved in mitosis (Fig. 6f). At the same time, a largenumber of free cells, coelomocytes, phagocytes as well asdediVerentiating myocytes could be seen inside the coelo-mic and the radial water canals. DediVerentiating myocyteswere still frequently detectable in the intervertebral musclesof the stump, particularly in the peripheral disorganizedareas.

2 weeks p.a. At 2 weeks p.a., the microscopic anatomyanticipated what was more deWned in the following stages

Fig. 2 Amphiura Wliformis. SM. Frontal view of the autotomy surfaceof the stump. a Immediately after autotomy. The vertebral ossicle is ex-posed and partly covered by remains of intervertebral muscles. b2 days p.a. The Wrst appearance of a new epithelial layer is expandingfrom the aboral (arrows) towards the oral side. c 3 days p.a. A contin-uous epithelial layer covers all the wound surface (arrow). d 4 daysp.a. The early appearance of a small blastema (arrow) is visible in thecentral-oral area (at the level of the neural cord and the radial water ca-nal). As aboral arm shield, Ls lateral arm shield, Os oral arm shield, Ppodia, S spine, V vertebral ossicle

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(3–4 weeks). Sagittal sections (not shown) displayed theWrst outline of blastemal growth, indicated by a smallswelling of the cicatricial area; this consisted of a non-homogeneous mesenchymal stroma covered by a continu-ous epithelial layer. This early blastemal rudiment devel-oped in association with the ends of the radial water canaland radial nerve; these structures and the other canals (peri-visceral coelom and epineural and hyponeural sinuses)began to regrow in close association with groups of mitoticcells scattered in a dense mesenchymal layer. Within thecoelomic canals, the same types of free cells described inthe previous stages could be found, including dediVerentiat-ing myocytes.

3 weeks p.a. At 3 weeks p.a., the growth of the regenerat-ing blastema (Fig. 5g–i) was more pronounced than in theprevious stage and its histological structure became moredeWned showing evident signs of pattern formation and tis-sue diVerentiation. The reformed tracts of both the neural

and coelomic components were clearly recognizable withinthe regenerative bud and showed their characteristic ana-tomical and histological features (Fig. 5h). In particular, theregenerating radial nerve cord displayed a typical ganglion-ated pattern, whereas the main coelomic components (peri-visceral coelomic cavity, radial water canal, hyponeuralsinus) and the epineural sinus were clearly diVerentiatedfrom each other, each one presenting a hollow/hypertrophicor still “solid” structure. The coelomic epithelium appearedto be thickened in some areas due to the extensive prolifer-ation of the coelothelial cells, indicated by the frequentoccurrence of mitotic Wgures. Large aggregations of migra-tory cells (particularly coelomocytes), phagocytes and ded-iVerentiating myocytes were frequently detectable insidethe coelomic cavities, mainly in their distal regeneratingends. The blastemal structure looked rather heterogeneous(Fig. 5h, i). Also at this stage, all the outgrowths of the mainstructures were almost completely enveloped by a closesheath of small satellite cells (presumptive undiVerentiated

Fig. 3 Amphiura Wliformis. SM view of the regenerating arm.a Immediately after autotomy. b 3 days p.a. The autotomy surface iscovered by the prominent edges of the external skeleton shields.c 6 days p.a. An undiVerentiated regenerative blastema (arrow) isclearly recognizable. d 12 days p.a. Segmentation is in progress in the

proximal part of the regenerating tip (arrow) but no complete segmentswith spines and podia are formed. The distal part looks rather undiVer-entiated (arrowhead). ap autotomy plane, As aboral arm shield, Ls lat-eral arm shield, P podia, S spine

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blastemal cells) included in a dense mesenchymal matrix,particularly evident in the trichrome-stained paraYn waxsections. By contrast, around this inner sheath of densemesenchyme, an outer layer of very loose and almost acel-lular mesenchyme was present, then interrupted by addi-tional dense layers of presumptive blastemal cells whichtended to stratify in the apical blastemal cap close to thebasal processes of the epithelial cells. So the outer epithe-lium acquired a pluristratiWed appearance (Fig. 5i) andbecame strongly evident; it looked already covered by anormal thin cuticle.

4 and 5 weeks p.a. At 4 and 5 weeks p.a., sagittal sectionsshowed a well-developed regenerating arm with anadvanced level of tissue diVerentiation. In particular, thetypical serial ganglionated structure of the regeneratingnerve cord became very evident inside the regenerate. Themain coelomic cavity, the other canals of coelomic deriva-tion (radial water canal, hyponeural sinus) and the epineuralsinus appeared to be well developed and deWned; the out-

lines of new podia also began to appear. PresumptiveundiVerentiated coelomocytes, phagocytes and dediVerenti-ating myocytes were still found inside these new coelomiccanals, as described in previous stages. The regenerativeblastema maintained the multilayered heterogeneous struc-ture; in its inner core, blastemal mitotic cells included in arather Wbrous matrix were still grouped around thereformed portions of the coelomic canals and radial nervecord; in the apical cap of the regenerative blastema, thesesame cells tended to stratify in dense layers, the interspacesbetween these two “cellularized” part being Wlled by aloose acellular mesenchyme. In the old stump, the musclesshowed the characteristic disarranged pattern alreadydescribed in the previous stages; dediVerentiating myocyteswere still present, although they did not form the majorcomponent of the bundle.

6 weeks p.a. Besides an obvious increase in size, at6 weeks p.a., the sagittal sections did not display signiWcantnovelties, in terms of histological pattern, with the marked

Fig. 4 Amphiura Wliformis. SM view of the regenerating arm. a19 days p.a. The regenerating arm can be divided into two parts, acompletely diVerentiated proximal part (45% in length) includingfunctional segments complete with spines and podia, and a partly

undiVerentiated distal part. b 26 days p.a. More than 90% in length ofthe regenerating tip is totally diVerentiated. ap autotomy plane, difdiVerentiated portion of the regenerate, undif undiVerentiated part ofthe regenerate

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exception of the Wrst appearance of skeletal spicules scat-tered in both the proximal and distal region of the regener-ate, where they tended to occupy the empty mesenchymalspaces noted in the previous stage. In transverse sections,these spicules, which were always included in a Wbrousconnective matrix, appeared to be developed and distrib-uted in both the peripheral and central areas of the arm,particularly between the main coelomic cavity and thehyponeural sinus.

10 and 12 weeks p.a. At 10 weeks p.a., the regenerateincreased in size and already showed anatomical featureswhich became more deWned at 12 weeks p.a.; at this stage,the regenerating arm looked like a well-diVerentiated mini-ature arm (Fig. 5j–l), at least as far as its proximal regionwas concerned. In terms of microscopic anatomy, this por-tion showed all the typical structural components (Fig. 5k):radial nerve with its ganglionated organization, epineural andhyponeural sinuses, radial water canal with podia, vertebral

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ossicles and the associated intervertebral ligaments andmuscles, a well-diVerentiated coelomic cavity and Wnallyexternal skeletal shields provided with typical spines couldbe easily recognized. In contrast, the tip of the regeneratingarm still consisted of a regenerative blastema showing thecharacteristic histological features already described in theprevious stages (Fig. 5l; see above, 3–4 weeks p.a.).

Amphiura Wliformis

1 day p.a. Both sagittal and frontal sections already showeda thin epithelial layer covering the wound area which was alsoprovided by a well-developed thick cicatricial layer (Fig. 7a)rich in Wbres and migratory cells. The distal injured ends ofthe main coelomic cavities and canals were already sealed oV;coelomocytes and phagocytes were frequently detectableinside them, mainly in the distal regenerating regions. In con-trast to what was seen in O. longicaudum, the stump autotomysurface was characterized by the presence of the muscularremains (aboral intervertebral muscles) belonging to the seg-ment involved in the autotomic process and detached by theirdistal skeletal insertions.

3 days p.a. At 3 days p.a. (Fig. 7b, c), the overall patternof the regenerating arm was more or less that describedin O. longicaudum at 1 week p.a., and was characterizedby the Wrst appearance of a small blastema, consisting of

a mesenchyme covered by an outer thick apparentlypluristratiWed epithelium. In both sagittal and frontalhistological sections, the rareWed structure of the blastemalmesenchyme comprising alternating cellular and acellularlayers was emphasized by the trichrome staining. The out-growth of the main coelomic cavity appeared to project intothe regenerative blastema and was associated with a sheathof Wbrous mesenchymal matrix. Groups of free cells couldfrequently be detected inside the coelomic cavity (Fig. 7c).The muscles of the stump, in particular the aboral musclebundles involved in autotomy (see above), acquired aremarkable disorganized pattern, showing a number ofdediVerentiating myocytes (Fig. 7b).

4–6 days p.a. At 4–6 days p.a. (Fig. 7d–f), the advancedprogress of the regenerative pattern was comparable to thatseen at 3 weeks in O. longicaudum. The blastema was welldeveloped and its structure became more complex andinhomogeneous (Fig. 7d). The blastemal mesenchyme wasWlled by cells (presumptive undiVerentiated coelomocytes;Fig. 7f) and the Wbrous matrix was poorly developed,except in the proximal region. A peculiar and distinctivefeature of the regenerating arm at this stage was theregrowth of the radial nerve cord, always associated withthe outgrowth of the perivisceral coelom and the radialwater canal, which showed often a solid structure. At thelevel of the stump, aggregations of cells (particularly coe-lomocytes; Fig. 7e), phagocytes and dediVerentiating myo-cytes were frequently detectable inside the coelomiccavities, mainly in the distal regenerating regions; the mus-cles showed the characteristic disarranged pattern alreadydescribed in the previous stages.

8–12 days p.a. At 8–12 days p.a. (Fig. 7g–i), both sagittaland frontal sections showed an evident progressive develop-ment. In particular, the proximal portion of the regeneratingarm was dominated by the outgrowths of (1) the radialnerve with its ganglionated structure, (2) the perivisceralcoelomic cavities, and (3) the radial water canal with newpodia. The distal portion of the regenerate still compriseda typical undiVerentiated blastema comparable to thatdescribed in the previous stages.

16–24 days p.a. At 24 days p.a., the microscopic anatomyof the regenerate (Fig. 7j–l) was comparable to thatdescribed in O. longicaudum at 12 weeks and the regeneratingarm was actually looking as a miniature arm (Fig. 7j, k). Inparticular, longitudinal sections, both sagittal and frontal, ofits proximal portion showed a typical histological patterncharacterized by (1) the prominent presence of the radialnerve cord, (2) diVerentiated coelomic components anddiVerentiated epineural sinus, (3) vertebral ossicles withassociated intervertebral ligaments and muscles, and (4)

Fig. 5 Ophioderma longicaudum. Light microscopy (LM). Semi-thinsagittal sections stained with crystal violet and basic fuchsin (a–e, g)and trichrome-stained paraYn wax section (f). a–c 3 days p.a. a Com-prehensive view of arm stump: the autotomy surface is markedly con-cave. b Detail of the thin epithelium (arrowhead) lining the wound: thehealing process is still incomplete in terms of underlying cicatriciallayer. c Detail of the loose connective layer underlying the thin healingepidermis; note some scattered phagocytes (arrowhead). d–f 1 weekp.a. d Comprehensive view of the arm stump: note the reduced concaveshape of the autotomy surface. e Detail of the thick cicatricial layerformed by layers of densely packed cells; note the regrowing distalends of the radial water canal and of the radial nerve. f Detail of the cic-atricial layer: the trichrome staining emphasizes the dense Wbrous com-ponent (cf). g–i 3 weeks p.a. g Comprehensive view of the arm stumpand its blastema. h Detail of g. The outgrowths of the radial nerve cord,radial water canal and other coelomic cavities are recognizable insidethe blastema; note a large mass of cells inside the main coelomic cav-ity. i Detail of the regenerative blastema. The blastema heterogeneousstructure consists of an inner sheath of blastemal cells (surrounding theradial nerve cord and the radial water canal), an outer layer of veryloose mesenchyme, a stratiWed dense layer of blastemal cells adjacentto the epidermis. j–l 12 weeks p.a. j Comprehensive view of the regen-erating arm showing its overall internal anatomy. k Detail of the prox-imal region of the regenerate: the arm tissues and structures are welldiVerentiated. Particularly relevant is the ganglionated structure of thenerve cord. l Detail of the apical portion of the regenerating arm: apartfrom the skeletal spicules, the tissue diVerentiation is still at an earlystage comparable to a blastemal one. n radial nerve, v vertebra, c maincoelomic cavity, m intervertebral muscles, ct connective tissue, l liga-ments, p podia, rwc radial water canal, e epineural sinus, Os oral armshield, As aboral arm shield, b blastema, ra regenerating arm

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superWcial skeletal arm shields. As previously described,the distal tip of the regenerate retained the features of anundiVerentiated blastema (Fig. 7l).

Sub-microscopic anatomy

Although preliminarily, the ultrastructural analysis carriedout so far allowed in both species to enter in a more detail

with respect to what seen at optical microscopy level and toidentify better the cellular phenotypes involved in armregeneration (Fig. 8).

Coelomocytes and phagocytes At all the diVerent stages,it was possible to conWrm that the main cytotypes detectedas free elements inside the coelomic cavities or migratorycells in the blastemal tissues are phagocytes (Fig. 8a) and

Fig. 6 Ophioderma longicaudum. LM. Semi-thin sagittal sections ofthe stump and the regenerating arm, stained with crystal violet andbasic fuchsin. a Detail of the wound healing at 3 days p.a. showing thecontribution to sealing by contraction of the muscular wall of the coe-lomic canals (arrowheads). b Detail of the wound healing at 3 days p.a.showing the contribution to sealing by clots of free coelomic cells(arrowhead); note a large migratory phagocyte (arrow) in the maincoelomic cavity. c Detail of phagocytes (arrow) and coelomocytes(arrowhead) migrating in the main coelomic cavity at 3 days p.a.d Delamination process of coelomocytes (arrowhead) from the coelo-thelium at 3 days p.a. e Migratory coelomocytes inside the radial watercanal at 3 weeks p.a. f Detail of the coelomic epithelium in the regen-

erative blastema at 3 weeks p.a.: evident mitotic Wgures can be seen inthe coelothelial cells (arrowhead). g Intervertebral muscle (m) of thestump at 3 days p.a. showing a rather disorganized pattern: the detailrefers to the second intervertebral muscle from the autotomy surface.Note a massive group of presumptive dediVerentiating myocytes (rec-ognizable for their fusiform shape and staining properties) close to theaboral bundle (arrowhead). h Detail of the aboral intervertebral mus-cles (m) at 3 days p.a. (Wrst intervertebral muscle from the autotomysurface). Masses of dediVerentiating myocytes (arrowhead) appear tobe displaced from the periphery of the bundle towards the coelomiccavity (c). i Presumptive dediVerentiating myocytes (arrowhead)migrating inside the coelomic cavity at 3 weeks p.a

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coelomocytes (Fig. 8b, c). In both species, phagocytes arerecognizable for their eccentric nucleus and for the pres-ence of a large number of cytoplasmic inclusions and resid-ual bodies of diVerent size and density. On the other hand,coelomocytes displayed the typical undiVerentiated mor-phology characterized by a central nucleus surrounded bysmall cytoplasm. Also in this case, there was no signiWcantultrastructural diVerence between the coelomocytes of thetwo species.

DediVerentiating myocytes The third cytotype alwaysinvolved in regeneration processes was represented bydediVerentiating myocytes. In both O. longicaudum andA. Wliformis, in fact, myocytes at diVerent stages ofdediVerentiation (Fig. 8d) were constantly detectable inthe peripheral area of the stump muscles; these cells werecharacterized by obvious processes of progressive disor-ganization of their contractile apparatus resulting in avery irregular and peculiar distribution of acto-myosinWlament bundles. DediVerentiating myocytes are dramati-cally diVerent in ultrastructural features from the normalmuscle Wbres constituting the central portion of the bundle(Fig. 8e).

DiVerentiating cells In the two samples, at the level of theblastemal portion, we could detect early signs of diVerentia-tion processes related to some speciWc cell types, evenwhen the light microscopy is revealing only an apparentlyundiVerentiated blastema. In some cellular phenotypes, thediVerentiation in progress could be easily tested in the lightof speciWc distinctive features; for instance, coelothelialcells can be recognized on the basis of their cilia and typicaljunctional complexes (Fig. 8f), myocytes (belonging to thecoelothelium itself) on the basis of the clear primordium ofcontractile apparatus (Fig. 8h), neural cells on the basis oftheir growing varicose processes (Fig. 8g). In addition, theepidermic component of the neural cord became to bediVerentiated and evident (Fig. 8i). These encouragingpreliminary results strongly suggest that an appropriatedetailed investigation at TEM should be dedicated to acomprehensive ultrastructural analysis of the regenerationprocesses in these and other ophiuroid species (work inprogress).

Comparative growth and diVerentiation rates

For O. longicaudum, the regeneration process is muchslower than for A. Wliformis. In terms of morphogenesis, the1 day p.a. stage of A. Wliformis appeared approximately tocorrespond to the 3 days p.a. stage in O. longicaudum andthe 24 days p.a. stage in A. Wliformis to 12 weeks in O. lon-gicaudum, suggesting a morphogenesis three times faster inA. Wliformis. Growth and diVerentiation rates are alreadywell characterized for A. Wliformis (Dupont and Thorndyke

2006) and the observed RR and DI conWrmed the predictionof the model. In O. longicaudum, the regeneration rate(mm/week) was calculated as the slope of the signiWcantsimple linear regression between the regenerated length(mm) and time (week). Analysis of the slope of the lineproduced an average rate of arm regeneration of0.167 § 0.013 mm/week (Fig. 9). After 12 weeks p.a., 70%in length of the regenerate was fully diVerentiated.

Discussion

In the present study, we selected two common ophiuroidspecies, O. longicaudum and A. Wliformis, which are dis-tantly related and diVer signiWcantly in morphology, physi-ology, size, behaviour and habitat (Table 1).

O. longicaudum belongs to Ophiodermatidae (Ophioder-matina) and is a common and representative epibenthic spe-cies of the Mediterranean Sea and of the eastern AtlanticOcean. It lives in shallow waters (0–70 m depth) and is car-nivorous (Tortonese 1965). The disc diameter variesbetween 1 and 3 cm, with arms of medium length. A. Wlifor-mis belongs to the Amphiuridae (Gnathophiurina) and pre-dominate on many sublittoral soft bottoms down to 200 mdepth in the North Sea and the Mediterranean Sea (Rosen-berg 1995). It is a typical infaunal suspension feeder (Sköldand Rosenberg 1996; Salzwedel 1974), with a disc diameterup to 10 mm and with very long and thin arms. Naturalautotomy may occur at the level of any arm articulation(Wilkie 2001; Emson and Wilkie 1980; Hyman 1955).

It is relevant that in these two species the arm autotomicprocesses diVer signiWcantly; in O. longicaudum, in fact,both the oral and the aboral intervertebral muscles involvedin the autotomic articulation separate symmetrically fromtheir proximal insertions to the vertebral ossicle (Wilkie2001; this study); in contrast, in A. Wliformis, the oral inter-vertebral muscles separate from their proximal insertions,whereas the aboral intervertebral muscles separate fromtheir distal insertions (Wilkie 2001; this study). Since autot-omy is obviously the prelude to the regenerative processes,in terms of causes and eVects, these diVerent basic modali-ties of automutilation could inXuence the outcome of thefollowing processes of wound healing and regenerativedevelopment.

Regeneration rate

Our study revealed important diVerences in the regenera-tion rate (RR), with A. Wliformis regenerating three timesfaster than O. longicaudum. With a RR of 0.17 mm/week,O. longicaudum is the slowest regenerating speciesrecorded to date, even slower than the Antarctic speciesOphionotus victoriae Bell, 1902 (see Clark et al. 2007, for a

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review of the literature on RR). This rate is similar to thatobserved by Zeleny (1903) in O. longicaudum (previouslynamed Ophioglypha lacertosa). In a slightly diVerentexperiment (no feeding and the arm cut at disc level), RRbetween 0.15 and 0.48 mm/week was calculated. A relationbetween the disc diameter and the RR was observed, themaximal rate being exhibited by individuals with disc

diameter of 12–15 mm. Individuals with disc diameterclose to 2 cm (used in our experiment) gave a RR of0.15 mm/week. Several parameters can inXuence the RR inophiuroids at diVerent levels: (i) interspeciWc (e.g. tempera-ture, see Clark et al. 2007); (ii) intraspeciWc (food quality,number of regenerating arms, hypoxia, pollutants, etc.);(iii) intraindividual (e.g. length lost, see Dupont and

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Thorndyke 2006). Nevertheless, the slow RR observed inO. longicaudum cannot be explained by size or temperaturesince other species with similar size and temperature rangeregenerate more quickly. For example, Ophiura ophiura(Linnaeus, 1816) with a disc diameter up to 30 mm regen-erates more than 1 mm/week at 14°C (Dupont, personalcommunication). IntraspeciWc factors such as culture condi-tions (e.g. food quality, water parameters) can be excludedsince animals were fed with a high nutritive diet, once-a-week feeding being an optimized solution used also in otherstudies (only to quote some: McAlister and Stancyk 2003;Clark et al. 2007) and the water parameters during theexperiment were always optimal. Another factor which wasconsidered is the muscle detachment pattern (and thereforethe resulting availability of a source of myocytes at thewound site) during the autotomic process. This factoractually does not appear to be of major importance sinceO. ophiura which follows the same detachment pattern ofO. longicaudum (Wilkie 2001) does actually show aremarkable higher RR. The observed low RR can, there-fore, be considered a representative character of the Ophio-dermatidae.

Regenerative process

In spite of the diVerences outlined above, O. longicaudumand A. Wliformis show remarkable similarities in terms ofbasic mechanisms of arm regeneration. The complete

regenerative process, in terms of representative stages fromthe early post-autotomic stage to the development of aminiature functional arm complete with all its anatomicalfeatures, can be schematically subdivided into four mainphases: a repair phase, an early regenerative phase, an inter-mediate regenerative phase and an advanced regenerativephase (summarized in Fig. 10).

The repair phase (compare Figs. 5a–c, 7a, 10a), whichcovers a p.a. period of 3 days in O. longicaudum and 1 dayin A. Wliformis, is essentially characterized by wound-healing processes. These include: (1) sealing of the coelo-mic canals and cavities, by clots of free coelomic cellsand contraction of the coelomic wall; (2) the completere-epithelialization of the autotomy surface, through plasticreorganization of the epidermis and contributions frommigratory cells; (3) early indications of rearrangement ofthe tissues damaged by the autotomic process. The presenceof a loose connective tissue stroma, often discontinuous,beneath the outer epithelium clearly indicates an incom-plete healing process at this stage. Although true regenerativephenomena are not yet apparent, signiWcant preparatoryprocesses, crucial for subsequent regeneration, such as cellmigration, proliferation and dediVerentiation begin to takeplace.

The early regenerative phase (compare Figs. 5d–f, 7b, c,10b), which covers the 3 days to 1 week p.a. period inO. longicaudum and the 1–3 days p.a. period in A. Wlifor-mis, is characterized by the start of true developmentalregenerative phenomena. During this phase, the completehealing of the epithelial layer is accompanied by extensivemigration and proliferation of cells leading to the formationof a thick subepithelial pseudo-cicatricial layer consisting ofa dense mesenchyme Wlled with undiVerentiated cells andscattered phagocytes. The presence of these migratory cells,which remain abundant inside the coelomic cavities, is particu-larly evident immediately outside the cavities, at the ampu-tated ends of the coelomic components and the radial nervecord. These terminal structures are externally surroundedby a few continuous layers of actively dividing undiVerenti-ated cells (presumptive blastemal cells) and no longerappear retracted but project forward to deWne a localizedgrowth centre that can be considered a pre-blastemal area.

The intermediate regenerative phase (compare Figs. 5g–i, 7d–i, 10c), which covers the 1–3 weeks p.a. period inO. longicaudum and the 3–12 days p.a. period in A. Wliformis,is characterized by the formation and development of arecognizable regenerative blastema. This grows distallyfrom the oral part of the autotomy surface, in correspon-dence with the regrowing ends of the radial nerve cord andthe radial water canal. It is covered by a well-deWned outerepithelium, which acquires a multilayered appearance dueto an underlying stratiWcation of presumptive blastemalcells. Key phenomena of morphogenesis and diVerentiation

Fig. 7 Amphiura Wliformis. LM. Semi-thin semi-sagittal sections,stained with crystal violet and basic fuchsin (a–e, g–j, l) and paraYnwax section (k) stained with Domagk’s method. a Comprehensiveview of the autotomized arm at 1 day p.a. The wound is covered by athin epithelium layer (arrowhead). b 3 days p.a. Detail of the autotomysurface. An early blastema is present. Due to the slightly oblique sec-tion plane, the presence of the remains of the oral intervertebral muscleWbres is also revealed (*). c 3 days p.a. Detail of the stump. Largemigratory phagocytes (arrowhead) can be found inside the radial watercanal. d–f 4 days p.a. d Section of the regenerating arm at the level ofthe radial water canal. The blastema is well developed. Early diVeren-tiation processes, mainly related to the skeletal spicules, are in progressclose to the radial water canal and the radial nerve. e Detail of d. Freecoelomocyte in the radial water canal (arrowhead). f Detail of the blas-tema. A well deWned outer epithelium covers a dense accumulation ofpresumptive blastemal cells (*). g–i Regenerating arm at 8 days p.a. gComprehensive view of stump and regenerate. The regenerating arm iswell developed. The section plane involves the muscles of the stump.h Detail of dediVerentiating myocytes (arrow) derived from the muscleremains (aboral intervertebral muscles) on the autotomy plane. i Detailof the radial water canal showing a migratory phagocyte (arrowhead).j–l Regenerating arms at 16 days p.a. j Detail showing the serial newpodia (Wrst and last one indicated by arrowhead) along the miniaturearm. k Section at the level of the radial water canal showing the ana-tomical features of the new arm. The speciWc staining emphasizes theconnective tissue stroma. l Detail of the apical region of the regeneratewhich retains blastemal characteristics. ap autotomy plane, b blastema,c coelomic cavity, m muscle; n radial nerve, p podia, ra regeneratingarm, rwc radial water canal, v vertebrae

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Zoomorphology

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take place at this stage in the regenerative blastema; this isparticularly evident in association with the radial nerve,which progressively develops its typical ganglionated orga-nization, and in the perivisceral coelomic cavity and theradial water canal which begin to acquire their distinctivefeatures.

Despite its resemblance in some cases to a typical blas-tema, the regenerative bud of an ophiuroid arm shows aheterogeneous structure with an advanced level of tissue

diVerentiation, rather diVerent from the condition of a typi-cal blastema (Sanchez Alvarado 2000). Its structure is, infact, deWned by a central core-of-growth formed by thediVerentiating regrowing ends of the diverse coelomicstructures and the radial nerve cord. This is surrounded by aperipheral area formed by distinct cellular and acellularmesenchymal layers and Wnally covered by an outer epithe-lium. On the basis of the extensive presence of free cellsinside the coelom-derived cavities, particularly at the levelof both the regenerate and the distal stump, and also takinginto account the evidence of proliferation in the coelothelialwall of these tracts, it is plausible to suggest that the coelot-helia continue to act as the primary source of new cells tobe employed in the regenerative regrowth of all the coelo-mic components. The extent to which this coelomic contri-bution of new cells is employed more widely in theblastema for the regrowth of other non-coelomic tissuesand structures is still an open question, even though thehigh density of migratory cells externally associated to thedeveloping coelomic structures seems to support thishypothesis. Certainly, in these regenerative phases, therecruitment of undiVerentiated cells of coelomic origin iscomplemented by a continuous recruitment of dediVerenti-ated cells derived from muscles.

During the advanced regenerative phase (compareFigs. 5j–l, 7j–l, 10d), which covers the 3–12 weeks p.a.period in O. longicaudum and the 11–24 days p.a. period inA. Wliformis, extensive growth, morphogenesis and diVer-entiation lead to the formation of a miniature arm. Duringthis phase, the histological pattern of the arm becomes pro-gressively more deWned: its proximal portion, in particular,acquires the distinctive internal and external features of acomplete functional arm, whereas its distal part looks stillundiVerentiated and retains typical blastemal features.

Fig. 8 Transmission electron microscopy (TEM). Resin sections ofregenerating arms at diVerent stages. a Ophioderma longicaudum:3 days p.a. Detail of the connective tissue of the stump close to theautotomy surface showing a large phagocyte surrounded by a stromaof collagen Wbrils (*). b O. longicaudum: 1 week p.a. Detail of a coe-lomic canal showing a coelomocyte (arrowhead) detaching from thecoelomic epithelium. c Amphiura Wliformis: 8 days p.a. Free coelomo-cyte in the hyponeural sinus close to the regenerate. d A. Wliformis:8 days p.a. Detail at the level of the autotomy plane showingdediVerentiating myocytes of the aboral muscles: an evident processof disorganization of the contractile apparatus is in progress (*). e A.Wliformis: 16 days p.a. Detail of a normal muscle bundle of the stump.The myocytes display the usual regular arrangement of contractile Wl-aments (*). f O. longicaudum: 12 weeks p.a. Detail at the regrowingcoelomic canal at the level of the blastemal region showing diVerenti-ation in progress in the coelomic epithelium. The coelothelial cellsalready display the typical cilia (arrowheads) and junctional com-plexes (arrows). g O. longicaudum: 12 weeks p.a. Detail of the apicalblastemal region. The subepithelial undiVerentiated layer shows anumber of varicose processes obviously belonging to neural cells(arrowhead) amongst presumptive coelomocytes. h O. longicaudum:12 weeks p.a. Detail of the apical blastema at the level of the regrow-ing coelom. A presumptive early myocytes (arrowhead) can be distin-guished appearing in the context of grouped coelomocytes, probablyboth destined to give raise to the coelomic wall. i A. Wliformis: 16 daysp.a. Detail of the apical blastemal region showing presumptive epithe-lial elements of the nerve (arrowhead) recognizable amongst the stillundiVerentiated cells of the subepidermal area

Fig. 9 Ophioderma longicaudum. Relationship between the time (in weeks) and the length of the regenerating arm (in mm). Open circle, raw data;black circle, mean § standard error of mean. No measurable regeneration prior to week 2 and growth rate of 0.17 mm/week

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Zoomorphology

During the following period, regenerate growth continuesin terms of length, as well as progressive diVerentiation ofnew structures. The regenerated arm maintains a diVerentsize and colour with respect to the old stump for some time,reacquiring its original size and becoming undistinguish-able from the remaining non-regenerating arm only aftermany months. This sequence of events and processesemployed in both repair and regenerative phenomena issurprisingly comparable between the two species, support-ing the idea of a common regenerative pathway in ophiu-roids, which although very versatile and plastic, remainsuniform in its basic features.

Epimorphosis versus morphallaxis

In the light of our Wndings, arm regeneration in both theOphiodermatidae and Amphiuridae can be classiWed as anepimorphic process, involving the formation of a regenera-tive blastema as a main developmental “reference centre”in which proliferation, morphogenesis and diVerentiationphenomena take place under control of presumptive regula-tory factors. Paradoxically, an atypical aspect of this blaste-mal regeneration is the blastema itself, which is far frombeing a uniformly dense blastemal mesenchyme covered bya thin epithelium, as in a typical blastema (Sanchez Alva-rado 2000). This is true for the outer epithelial layer and iseven more evident for the internal components, whichappear rather diVerent in structure with respect to a stan-dard blastema, which is well exempliWed by the regenera-tive blastema described in crinoid arm regeneration (CandiaCarnevali and Bonasoro 2001b). This apparent paradoxicalsituation and these atypical characters can be explained inthe light of the following: (1) a recognizable early blastemais lacking in ophiuroids, in which, due to the speciWc ana-tomical complexity and post-autotomy tissue retraction atthe autotomy surface, early processes lead only to thedevelopment of a pre-blastemal area, corresponding to ablastema from a cellular point of view, but not comparable

from the morphology because of its Xattened shape; (2) theregenerative bud, apparently identiWable as a blastema,does not correspond to a true blastema in terms of develop-mental stage, but is at an advanced level of growth anddiVerentiation and should be considered a small regeneratingarm, its protruding region being only the distal end of aregenerate that is much longer, with the proximal portionwithin the old stump. New evidence implies that the term“blastemal” must be used for ophiuroid regeneration withcare.

Our present results give also an indication of the celltypes involved. As suggested by previous studies (Thorndykeet al. 2001; Bannister et al. 2005), the Wrst candidates forcells responsible for the growth of the regenerated struc-tures are the undiVerentiated cells, including original pro-genitor cells of coelomic derivation (coelomocytes). Alsoinvolved are dediVerentiated elements (myocytes) derivedfrom diVerentiated tissues of the autotomised arm, whichmigrate and concentrate in the blastemal area, where theyproliferate actively and rediVerentiate/transdiVerentiate intonew cell types.

Current evidence suggests two diVerent mechanisms ofcellular recruitment. The Wrst mechanism is the recruitmentof undiVerentiated progenitor cells from the coelotheliumand implies proliferation and migration inside the coelomiccavities (Figs. 6a–f, 10) and, possibly, throughout the tis-sues. A signiWcant pilot role for the coelomic structures hasbeen suggested since the classic regeneration studies byDawydoV (1901) on Amphiura sp., and was recently con-Wrmed in A. Wliformis by Bannister et al. (2005, 2008), whodescribed the radial water canal as the major source of cellsemployed during the overall regenerative process and thesite of expression of key developmental genes. Other cyto-types involved speciWcally in regeneration have yet to bedeWned. There appears to be no speciWc local reservoir ofundiVerentiated cells that can migrate through the tissues;this may represent a limitation for ophiuroids. In contrast incrinoids, such reserve elements present extensively around

Table 1 Schematic comparison between Ophioderma longicaudum and Amphiura Wliformis

Ophioderma longicaudum Amphiura Wliformis Reference

Taxon Ophiodermatidae Amphiuridae Smith et al. (1995)

Habitat Epibenthic Infaunal Tortonese (1965), Rosenberg (1995)

Depth Shallow (<70 m) Sublittoral (<200 m) Tortonese (1965), Rosenberg (1995)

Feeding Carnivorous Suspension feeder Tortonese (1965), Rosenberg (1995)

Disc diameter Up to 30 mm Up to 10 mm

Disc–arm ratio 1:5 1:12 Dupont and Thorndyke (2006), this study

Autotomy: oral muscles separate from Proximal insertions Proximal insertions Wilkie (1978, 2001)

Autotomy: aboral muscles separate from Proximal insertions Distal insertions Wilkie (1978, 2001)

Arm regenerating (%) 40 86 Dupont and Thorndyke (2006), this study

Regenerating rate (mm/week) 0.17 0.5–3.5 Dupont and Thorndyke (2006), this study

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Zoomorphology

the nerve cord as a sheath of satellite cells (amoebocytes)and actively involved in regeneration (Candia Carnevaliand Bonasoro 2001b) are currently considered possiblestem cells. At the present state of knowledge, it is diYcultto establish if true stem cells, i.e. completely pluripotentialneoblast-like cells, are involved in echinoderm regenera-tion.

On the other hand, the involvement of dediVerentiatedmyocytes in the regenerative processes indicates that there

is a second important recruitment mechanism involvingextensive local phenomena of rearrangement and recyclingof existing arm tissues. The constant presence of groups ofdediVerentiating myocytes from the early repair to theadvanced regenerative phases in the areas close to the inter-vertebral muscles of the stump, in the interspaces betweenmuscles and coelomic cavities, and inside the canals them-selves (Figs. 6g–i, 7h, 8d, 10), provides strong evidence forthe role of dediVerentiated cells.

Fig. 10 Generalized scheme of ophiuroid arm regeneration (based onA. Wliformis, emphasizing the common aspects of the regenerative pro-cess). a The repair phase: sealing of coelomic canals and cavities byclotting of free coelomic cells and contraction of muscles; re-epitheli-alization of the autotomy surface through reorganization of epidermisand contribution from migratory cells; rearrangement of tissues, nerve,connective tissues, muscles and epithelia; formation of a loose connec-tive stroma beneath the epithelium; cell migration, proliferation anddediVerentiation begin to take place. b The early regenerative phase:complete healing of the epithelial layer; migration and proliferation ofcells form a dense mesenchyme with undiVerentiated cells and phago-cytes; free cells inside the coelomic cavities; the coelomic componentsand the radial nerve are surrounded by a few continuous layers ofdividing undiVerentiated cells (blastemal cells) and projects forward

deWning a pre-blastemal area. c The intermediate regenerative phase:formation of a regenerative blastema growing orally together with theregrowing ends of the radial nerve cord and the radial water canal; amultilayered epithelium ensheathes the blastema; morphogenesis anddiVerentiation in the regenerative blastema, particularly the radialnerve forms its ganglionated structure along with the coelomic com-partments. DediVerentiated myocytes and free coelomic cells supportswith new cells. d The advanced regenerative phase: extensive growth,morphogenesis and diVerentiation form a miniature arm; the proximalpart acquires the distinctive internal and external features of a completefunctional arm whereas the distal portion is undiVerentiated and main-tains typical blastemal features. m muscles, v vertebrae, n radial nerve,g ganglion, b blastema, rwc radial water canal, c main coelomic cavity,ct connective tissue, p podia

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Conclusion

As well as providing a detailed description of the regenera-tive process of the arm in two species of the Ophiuroideaand showing the Wrst evidence of co-existence of epimor-phic and morphallactic processes in ophiuroid regeneration,the present results provide further conWrmation of the plas-ticity of the regeneration potential in echinoderms.

There are general similarities in arm regeneration inophiuroid and crinoid species (Candia Carnevali 2006;Candia Carnevali and Bonasoro 2001b; Thorndyke andCandia Carnevali 2001; Thorndyke et al. 2001). Bothinvolve the development of new structures from migratorypluripotent cells, which actively proliferate forming aregenerative blastema, and in both cases dediVerentiationphenomena are important, although to varying extents.Another common aspect seems to be the crucial role playedby the nervous system, which is well known as a promoter/inducer of regenerative processes in both vertebrates andinvertebrates. The problem of the neurotrophic role playedby the nervous system during regeneration has beenexplored in ophiuroids since the historical studies carriedout by Morgulis (1909) and there is also good recent evi-dence supporting the role of the radial nerve cord in regen-erative phenomena in ophiuroids (Candia Carnevali 2006;Thorndyke et al. 2001). This crucial aspect is currentlyunder study in our laboratories and would complete theoverall view of arm regeneration in ophiuroids.

Acknowledgments This work has been supported by grants of Min-istry of University and Research (MiUR) COFIN2003 and CO-FIN2006. T. Zou was supported by a grant of the Chinese–Italianprogram.

References

Aronson RB (1987) Predation on fossil and recent ophiuroids. Paleo-biology 13:187–192

Aronson RB (1991) Predation, physical disturbance and sublethal armdamage in ophiuroids: a Jurassic-recent comparison. Mar EcolProg Ser 74(1):91–97

Aronson RB (1992) Biology of a scale-independent predator–preyinteraction. Mar Ecol Prog Ser 89:1–13

Bannister R, McGonnell IM, Graham A, Thorndyke MC, Beesley PW(2005) Afuni, a novel transforming growth factor-beta gene isinvolved in arm regeneration by the brittle star Amphiura Wlifor-mis. Dev Genes Evol 215:393–401

Bannister R, McGonnell IM, Graham A, Thorndyke MC, Beesley PW(2008) Coelomic expression of a novel bone morphogenetic pro-tein in regenerating arms of the brittle star Amphiura Wliformis.Dev Genes Evol 218:33–38

Candia Carnevali MD (2006) Regeneration in Echinoderms: repair, re-growth, cloning. Invertebr Surv J 3:64–76

Candia Carnevali MD, Bonasoro F (2001a) Introduction to the biologyof regeneration in echinoderms. Microsc Res Tech 55:365–368

Candia Carnevali MD, Bonasoro F (2001b) Microscopic overview ofcrinoid regeneration. Microsc Res Tech 55:403–426

Candia Carnevali MD, Lucca E, Bonasoro F (1993) Mechanism ofarm regeneration in the feather star Antedon mediterranea: heal-ing of wound and early stages of development. J Exp Zool267:299–317

Clark MS, Dupont S, Rossetti H, Burn G, Thorndyke MC, Peck LS(2007) Delayed arm regeneration in the Antarctic brittle star Oph-ionotus victoriae. Aquat Biol 1:45–53

DawydoV C (1901) Beiträge zur Kenntnis der Regenerationserschei-nungen bei den Ophiuren. Z Wiss Zool 69:202–234

Ducati CC, Barker MF, Candia Carnevali MD (2004) Regenerativepotential and Wssiparity in the forcipulate starWsh Coscinasteriasmuricata. In: Heinzeller T, Nebelsick JH (eds) Echinoderms:München. Taylor & Francis, London, pp 113–118

Dupont S, Thorndyke MC (2006) Growth or diVerentiation? Adaptiveregeneration in the brittlestar Amphiura Wliformis. J Exp Biol209(Pt 19):3873–3881

Dupont S, Thorndyke MC (2007) Bridging the regeneration gap:insights from echinoderm models. Nat Rev Genet. doi:10.1038/nrg1923-c1

Eaves AA, Palmer AR (2003) Widespread cloning in echinoderm lar-vae. Nature 425:146

Emson RH, Wilkie IC (1980) Fission and autotomy in Echinoderms.Oceanogr Mar Biol Ann Rev 18:155–250

Ermak TH, Eakin RM (1976) Fine structure of the cerebral and pygid-ial ocelli in Chone ecaudata (Polychaeta: Sabellidae). J Ultra-struct Res 54:243–260

Hyman LH (1955) The invertebrates: Echinodermata, vol IV.McGraw-Hill, New York

Makra A, Keegan BF (1999) Arm regeneration in Acrocnida brachiata(Ophiuroidea) at Little Killary, west coast of Ireland. Proc R IrAcad B 99(2):95–102

McAlister JS, Stancyk SE (2003) EVects of variable water motion onregeneration of Hemipholis elongata (Echinodermata, Ophiuroi-dea). Invertebr Biol 122(2):166–176

Mladenov PV, Burke RD (1994) Echinodermata: asexual propagation.In: Adiyodi KG, Adiyodi RG (eds) Reproductive biology ofinvertebrates, vol VI, part B: asexual propagation and reproduc-tive strategies. Oxford and Hill, New Delhi, pp 339–383

Morgulis S (1909) Regeneration in the brittlestar Ophiocoma pumiliawith reference to the inXuence of the nervous system. Proc AmAcad Arts Sci 44:655–659

Oji T (2001) Fossil record of Echinoderm regeneration with specialregard to crinoids. Microsc Res Tech 55(6):397–402

Rosenberg R (1995) Benthic marine fauna structured by hydrodynamicprocesses and food availability. Neth J Sea Res 34:303–317

Salzwedel H (1974) Arm-Regeneration bei Amphiura Wliformis(Ophiuroidea). VeröV Inst Meeresforsch Bremerh 14:161–167

Sanchez Alvarado A (2000) Regeneration in the metazoans: why doesit happen? Bioessays 22:578–590

SAS Institute Inc. (1990) SAS/STAT user’s guide, version 6, 4th edn.SAS Institute, Cary

Shapiro SS, Wilk MB (1965) An analysis of variance test for normality(complete samples). Biometrika 52(3/4):591–611

Sköld M, Rosenberg R (1996) Arm regeneration frequency in eightspecies of Ophiuroidea (Echinodermata) from European seaareas. J Sea Res 35(4):353–362

Smith AB, Paterson GLJ, Lafay B (1995) Ophiuroid phylogeny andhigher taxonomy: morphological, molecular and palaeontologicalperspectives. Zool J Linn Soc 114:213–243

Stancyk SE, Golde HM, Pape Lindstrom PA, Dobson WE (1994)Borne to lose. I. Measures of tissue loss and regeneration by thebrittlestar Microphiopholis gracillima (Echinodermata: Ophiu-roidea). Mar Biol 118:451–462

Thorndyke MC, Candia Carnevali MD (2001) Growth Factors andregeneration in echinoderms (invited review). Can J Zool79:1171–1208

123

Zoomorphology

Thorndyke MC, Patruno M, Moss C, Beesley PW, Mallefet J (2001)Cellular and molecular bases of arm regeneration in brittlestars.In: Barker M (ed) Echinoderms 2000. Swets & Zeitlinger, Lisse,pp 323–326

Thorndyke MC, Patruno M, Dewael Y, Dupont S, Mallefet J (2003)Regeneration in the ophiuroid Amphiura Wliformis: cell biology,physiology and luminescence. In: Féral J-P, David B (eds) Echi-noderm research 2001. Swets & Zeitlinger, Lisse, pp 193–199

Thouveny Y, Tassava RA (1997) Regeneration through phylogenesis.In: Ferretti P, Gérardieu J (eds) Cellular and molecular basis ofregeneration. Wiley, Chichester, pp 9–43

Tortonese E (1965) Fauna d’Italia, vol 6: Echinodermata. Calderoni,Bologna

Vickery MC, Vickery MS, Amsler CD, Mc Clintock JB (2001) Regen-eration in echinoderm larvae. Microsc Res Tech 55(6):364–373

Walsh GE, McLaughlin LL, Louie MK, Deans CH, Lores EM (1986)Inhibition of arm regeneration by Ophioderma brevispina(Echinodermata, Ophiuroidea) by tributyltin oxide and triphenyl-tin oxide. Ecotoxcol Environ Saf 12(1):95–100

Wilkie IC (1978) Arm autotomy in brittlestars (Echinodermata: Ophiu-roidea). J Zool Lond 186:311–330

Wilkie IC (2001) Autotomy as prelude to regeneration in echinoderms.Microsc Res Tech 55(6):369–396

Wilkie IC, Emson RH, Mladenov PV (1984) Morphological andmechanical aspects of Wssion in Ophiocomella ophiactoides(Echinodermata: Ophiuroidea). Zoomorphology 10(5):310–322

Zeleny C (1903) A study of the rate of regeneration of the arms in thebrittle-star, Ophioglypha lacertosa. Biol Bull 6(1):12–17

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