DOI: 10.2478/s11535-006-0042-2 Research article CEJB 1(4) 2006 609–635

Visceral regeneration in the crinoid Antedon mediterranea: basic mechanisms, tissues and cells involved in gut regrowth Daniela Mozzi1, Igor Yu Dolmatov2, Francesco Bonasoro1, Maria Daniela Candia Carnevali1∗ 1

2

Department of Biology, University of Milan, 20133 Milan, Italy

Insitute of Marine Biology, 690041 Vladivostok, Russia

Received 21 September 2006; accepted 20 November 2006 Abstract: Crinoids are able to regenerate completely many body parts, namely arms, pinnules, cirri, and also viscera, including the whole gut, lost after self-induced or traumatic mutilations. In contrast to the regenerative processes related to external appendages, those related to internal organs have been poorly investigated. In order to provide a comprehensive view of these processes, and of their main events, timing and mechanisms, the present work is exploring visceral regeneration in the feather star Antedon meditteranea. The histological and cellular aspects of visceral regeneration were monitored at predetermined times (from 24 hours to 3 weeks post evisceration) using microscopy and immunocytochemistry. The overall regeneration process can be divided into three main phases, leading in 3 weeks to the reconstruction of a complete functional gut. After a brief wound healing phase, new tissues and organs develop as a result of extensive cell migration and transdifferentiation. The cells involved in these processes are mainly coelothelial cells, which after trans-differentiating into progenitor cells form clusters of enterocytic precursors. The advanced phase is then characterized by the growth and differentiation of the gut rudiment. In general, our results confirm the striking potential for repair (wound healing) and regeneration displayed by crinoids at the organ, tissue and cellular levels. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.  Keywords: Crinoids, visceral regeneration, cell differentiation/trans-differentiation, cell migration, cell proliferation

∗

E-mail: [email protected]

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Introduction

Regeneration of lost body parts following autotomy or traumatic mutilation is a widespread phenomenon in the animal kingdom. The ability to repair or replace damaged or lost organs and appendages increases the fitness of the individual organism and of the species. This fitness increase is the main advantage conferred by regenerative capabilities [1, 2]. The distribution of regenerative capabilities appears to be less related to phylogeny or the level of complexity of taxa [3] than to individual histogenetic and morphogenetic plasticity [1, 2]. According to some authorities [3–5], regenerative potential is an intrinsic characteristic of life, although it can be lost when their costs are higher than their benefits. Echinoderms offer a wide range of models, which are suitable for investigating different problems related to regeneration. They possess a striking regenerative potential, which is evident in both extant and extinct species [1, 6–9]. In spite of the large amount of available data on regenerative events in echinoderms, regeneration in this phylum was studied extensively mostly in the 19th and early 20th centuries [2, 10]. Because regenerative phenomena have been largely disregarded in more recent times, many well-known regenerative properties have not been investigated using modern approaches and techniques. In view of the remarkable regenerative potential of echinoderms and their close phylogenetic relationship to vertebrates, these animals offer unique and novel opportunities to study regenerative developmental processes and to explore in detail aspects related to the evolution of specific mechanisms of organ and tissue repair. Crinoids are the most ancient class of living echinoderms and they are well known for their regenerative potential. They are able to completely regenerate arms [2, 11–21], pinnules, cirri, and also viscera, including the whole gut, after self-induced or traumatic mutilation [1, 2]. While regenerative processes shown by external appendages have been extensively studied, the regeneration phenomena of internal organs of crinoids have been poorly investigated. The only available studies are very old and incomplete [22–24]. In spite of this, the common presence in nature of partially or completely eviscerated specimens and individuals with visceral regeneration in progress [7, 25–28], indicates that the internal organs of crinoids display striking regenerative capabilities. Thus, we re-explored visceral regeneration using a modern approach in preliminary pilot studies, wich provided encouraging results [29, 30]. In order to investigate comprehensively the main events of the overall process and mechanisms at tissue and cellular levels, the present work examines the histological and ultrustructural aspects of visceral regeneration in the common feather star Antedon meditteranea, the experimental model used by Dendy [24] and Przibram [22] in their historical experiments. The phenomenon of visceral regeneration was studied more exstensively in holothuroids, since the first half of the 20th century [31–35], and in asteroids by Anderson [36–38]. With regard to visceral regeneration in echinoderms, the most controversial topic has been the origin of the luminal epithelium. Different hypotheses for regeneration of the digestive

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epithelium in holothurians were discussed in a recent review by Garc´ıa-Arrar´as and Greenberg [35]. According to previous data [32, 33, 35] obtained in Holothuria glaberrima, after evisceration, the new digestive tube develops from the mesentery which anchored the original gut to the body wall. This phenomenon is apparently a typical epimorphic process, where a blastema-like structure is formed as a thickening of the edge of the mesentery. In the following stages, the regrowth of the intestinal tract can imply two alternative mechanisms of cell recruitment: a) from the remnants of the oesophagus and cloaca, through morphallactic mechanisms of tissue rearrangement and migration/proliferation of endodermally derived cells [31, 33, 39–44]; b) from coelomic epithelium, through proliferation/migration and trans-differentiation phenomena of new mesodermally derived progenitor cells. This second mechanism occurs typically when endodermally derived tissues are completely lost by evisceration [45–47]. Although more specific mechanisms at the cellular level have yet to be explored, including experiments utilizing cell culture methods [48], a plausible interpretation about elements involved and their possible roles has been proposed, with particular reference to the fundamental role of the mesothelium and the organogenetic potential of the coelomic epithelium, which also implies a presumptive mesodermal derivation of the luminal epithelium of the new gut. Taking into account the anatomical complexity and the central role of the digestive apparatus for the animal functional biology, these fundamental questions have an overall biological relevance. The specific mechanisms, through which A. mediterranea regenerates the whole visceral mass would seem to be a worthwhile target.

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Experimental procedures

Specimens of A. mediterranea (Fig. 1A) were collected by scuba divers from the Tirrenian coast of Italy (Elba island) and maintained at 15-16◦C in a closed artificial sea-water system with a diet of invertebrate food for fine filter feeders (Coral Fliud – Marin). The visceral mass of each individual was removed from the calyx by gently pulling it after a superficial incision of the tegmen at the point of the arm branchings. This procedure is straightforward due to the loose connection of mesenterial layers anchoring the whole visceral mass to its calyx, and because of the weak defensive reaction shown by the animals, which involves only bending of the oral pinnules over the tegmen. Visceral regeneration was monitored at fixed times, 24 h, 2 days, 3 days, 5 days, 1 week, 2 and 3 weeks post evisceration (p.e.). Regenerating samples were observed in vivo under a Wild M3C Planapo stereomicroscope (SM). Regenerating and non regenerating control samples, consisting of whole calices and associated viscera or formed by isolated visceral masses, were collected and prepared for light microscopy (LM) and transmission electron microscopy (TEM), or for immunocytochemistry (ICC). Samples for LM and TEM were prefixed in 2% glutaraldehyde in 0.1 M and 0.1 Osm cacodylate buffer (pH 7.2) for 2 hours at 4-6◦ C and then, after an overnight washing in the same buffer, postfixed with 1% osmic acid in 0.1 M cacodylate buffer. After standard dehydration in an ethanol series, the samples were embedded in Epon-Araldite

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812. Semithin (1 µm) and thin (50 nm) sections were cut with a LKB Ultratome V and Reichert Ultracut E using a diamond knife. The semithin sections were transfered onto slides and stained with crystal violet-basic fuchsin for light microscopy. Ultrathin sections were mounted on copper grids and stained with uranyl acetate and lead citrate for electron microscopy. LM stained sections were observed and photographed with a Jenaval light microscope. TEM sections were observed and photographed in a Jeol 100SX electron microscope (for details see [13]). Other samples for LM were fixed for approximately 4 weeks in Bouin’s solution (satured aqueous solution of picric acid, 37% formaldehyde and 100% acetic acid – 15:5:1 v/v). After standard dehydration in an ethanol series and two 30 minute immersion in xylene, the samples were pre-embedded overnight in a mixture of xylene and paraffin. Then the specimens were embedded in paraffin with melting point of 56-58◦C. Paraffin sections (7 µm) were cut with a Reichert OmE microtome and stained with Milligan’s Trichrome stain [49]. Stained sections were observed and photographed as described above. Cell proliferation in regenerating tissues was monitored by employing a well-established ICC method to detect the incorporation of the thymidine analogue bromodeoxyuridine (BrdU) into DNA-synthesizing cells. For use with semithin Epon-Araldite sections, the standard BrdU-immunocytochemistry protocol for paraffin was modified as described in our previous papers [15, 17]. The incubation in BrdU was performed during the final 2 hours p.e. of the regeneration period in artificial sea-water solution at a final concentration of 0.05%. Immediately after BrdU incubation, the specimens were fixed in a solution of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PBS (pH 7.6) for 2 hours, and then were washed overnight in the same buffer. After standard dehydration in an ethanol series, the samples were embedded in Epon-Araldite 812 and the semithin sections (1 µm) were cut with a LKB Ultratome V. The resin was removed from the sections using a mixture of methanol (10 ml), propylene oxide (5 ml) and KOH (2 g). After washing in methanol and in distilled water, the sections were pretreated with H2 O2 in PBS in order to exclude the potential activity of endogenous peroxidases. After washing in PBS the specimens were incubated for 1 hour with 1% normal goat serum in PBS and Tween at room temperature. After a further series of washings in PBS, the samples were incubated overnight with a monoclonal anti-BrdU antibody (Cell Proliferation Kit, Amersham GE Healthcare) at 4◦ C. After washing in PBS, the samples were incubated for 3 hours with peroxidase anti-mouse IgG (Cell Proliferation Kit, Amersham GE Healthcare) at room temperature and, after a further series of washings in PBS, incubated for 5 minutes with 0.05% 3,3’-diaminobenzidine (DAB) and 0.03% H2 O2 in PBS. Finally they were washed in distilled water. Control tests were performed by omitting the primary antibody. The sections were observed and photographed with a Jenaval light microscope. Micrographs were processed by Adobe Photoshop 6.0 and Microsoft PowerPoint.

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Results

3.1 General anatomy The body of Antedon mediterranea consists of three main parts [7]: central calyx, ten arms and many aboral cirri (Figs. 1A,B). The calyx provides support and protection for all the internal organs, particularly for the visceral mass. This consists mainly of the digestive tube and the associated perivisceral coelomic cavities and haemal lacunae (Figs. 1B,C). The innervation is provided by nervous components of the ectoneural and hyponeural systems. A soft and loose membrane, called the tegmen, covers the oral surface of the calyx. The mouth, located in the center of the tegmen, is connected to five ciliated grooves (ambulacral grooves) arranged on the tegmen in a pentamerous pattern and directed towards the five pairs of arms (Figs. 1B,C). The gut of Antedon mediterranea is typically “U”-shaped and consists of three main parts: oesophagus, intestine and rectum. The oesophagus is a straight tract which descends from the mouth into the aboral part of the body and leads to the intestine, which bends back up to the oral side of the body, coiling in a wide clockwise spiral (Fig. 1B). The rectum is the region of the digestive tube, which ends in a prominent anal papilla, eccentrically projecting from the tegmen in an interradial position (Fig. 1B). The digestive tube has some outpockets at the level of both the oesophagus and the intestine [7, 50]. The ultrastructure of the gut epithelium is very similar to that of other species of crinoids [50]. In particular, the intestinal epithelium appears to be composed of three cell types: vesicular enterocytes and two different types of granular enterocytes.

3.2 0 hours post-evisceration (0 h p.e.) Immediately after evisceration, in the stereomicroscope the oral (upward facing) wound surface of the calyx appeared to be covered by coelomic and haemal fluids, which quickly coagulated to form a clot presumably protecting internal tissues from bacterial invasion. This clot was completely transparent and through it two concentric series of muscle pairs arranged in a pentamerous pattern were visible. In the centre of the inner muscle series, the hole of the “rosette”, with the remains of the axial organ tubules passing through into the chambered organ, and the shreds of the mesenterial laminae could be recognized (Fig. 2A). Despite significant loss of tissues and organs, the only sign of stress shown by the experimental specimens was the obvious close folding of oral pinnules and arms over the calyx, which lasted for some hours after evisceration. Subsequently the animals recovered completely and appeared to be healthy. They displayed usual feeding behaviour, responding to the presence of food by outstretching their arms and pinnules to form the typical filter fan. The animals did not show any other sign of stress throughout the rest of the regenerative period.

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A ambulacral groove

mouth

anal papilla rectum arms

haemal lacunae rosette

oesophagus intestine calyx hyponeural nerve anal papilla system mouth cirri

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Fig. 1 Antedon mediterranea. (A) Intact specimen. (B) General anatomy; the main external and internal organs are shown. (C) Comprehensive external view of the visceral mass (marked by the circle in Fig. B).

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ag m ac

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Fig. 2 SM comprehensive oral views of regenerating specimens of A. mediterranea at different regenerative stages. (A) 0 hours p.e.: two concentric series of pairs of muscle bundles (asterisks) are visible; the hole of the “rosette” is recognizable in the centre (arrow); bar = 400 µm. (B) 24 hours p.e.: the regenerating tissue consists of a thin, translucent-granulose layer (arrowhead) recognizable all along the borders of the injured area; bar = 500 µm. (C) 72 hours p.e.: a continuous translucent layer (arrowheads) covers the oral surface of the calyx; five regenerating ambulacral grooves can be recognized (arrow); bar = 800 µm. (D) 5 days p.e.: a first outline of mouth (m) and five new heterogeneous ambulacral grooves (arrows) are visible; bar = 500 µm. (E-G) 2 weeks p.e. (E) A real mouth (m) and well developed ambulacral grooves (ag) become evident; bar = 400 µm. (F) The pentagonal ridge around the mouth (m) is visible; bar = 400 µm. (G) A well developed anal cone (ac) is developed; bar = 130 µm.

3.3 24 hours post-evisceration (24 h p.e.) At 24 hours p.e. the earliest active repair events took place at the oral surface of the body, starting from the periphery of the calyx, all along the borders of the injured area.

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The regenerating tissues could be distinguished also in the stereomicroscope due to their more translucent-granulose appearance (Fig. 2B), possibly resulting from the extensive cell migration processes occurring in the area. In vertical LM and TEM sections of the whole regenerating calyx, the histological pattern showed that the cells involved in the early healing processes were the migratory elements described previously in regenerating crinoids [2, 29], namely progenitor cells (amoebocytes-coelomocytes) and granulocytes. The progenitor cells are morphologically undifferentiated cells migrating through the tissues (amoebocytes) or through the coelomic fluid (coelelomocytes – Fig. 3A), and are presumed to be stem cells [2]. They appeared to play a main role in this extensive migration process characterizing the early repair phase. These progenitor cells penetrated the wound region and formed a thick cellular layer, pluristratified and heterogeneous, irregularly covering the large injured area and forming an early cicatricial layer (Fig. 3B). As shown in the TEM, the cytoplasm of these undifferentiated elements contained small clear vesicles and well developed Golgi apparatus (Fig. 3C). Some of these progenitor cells appeared to be fused together to form obvious syncytia with roundish nuclei characterized by finely granular karyoplasm (Fig. 3D). Although less abundant than the progenitor cells, a number of typical granulocytes could also be detected in the cicatricial clot, their distinctive feature being the prominent electron-dense cytoplasmic granules (Fig. 3C). The cicatricial clot was already covered by a thin layer of epithelial cells possibly derived from the peripheral area of the calyx (Figs. 3B,E). In the TEM these cells looked flat and elongated, with long processes. Their cytoplasm contained mitochondria, cisternae of RER (rough endoplasmic reticulum) and clear vesicles (Fig. 3E). Typical septate junctions were common between adjacent cells (Fig. 3F), but a proper basal lamina was absent under this new epithelium (Fig. 3E). In histological sections, regenerating coelomic components (early outlines of coelomic cavities and septa) could also be detected under the covering cicatricial clot (Fig. 3B). In terms of cell proliferation, although the BrdU labelling showed scattered marked nuclei distributed in the different tissues, the reaction appeared to be sensibly stronger at the level of the cicatricial layer and the coelomic epithelium (Figs. 9A,B).

3.4 48 hours post-evisceration (48 h p.e.) At 48 hours p.e., the repair processes were at advanced stages. When examined in the stereomicroscope, the regenerative tissues were clearly seen to have formed a translucent tegmen covering the oral surface of the calyx, the only obvious exceptions being the central and the radial areas which looked different and not translucent. Under the regenerative layer, neither the inner series of muscles nor any of the rosette could be distinguished any more.

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ec

mc

cc c

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n uc uc

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Fig. 3 Antedon mediterranea at 24 hours p.e. (A) Progenitor cell of coelomic derivation (coelomocyte; c) at TEM; bar = 1.5 µm. (B) LM vertical section: the first provisional cicatricial layer formed by migratory cells (mc) and an upper incomplete layer of epidermal cells (ec); under this cicatricial clot, sparse coelomic visceral components (arrows) could be detected; cc = coelomic cavities; bar = 200 µm. (C) TEM section: the migratory cells forming the first cicatricial layer are undifferentiated progenitor cells (uc) and granulocytes (gr); G = Golgi apparatus; g = granules; bar = 2 µm. (D) Syncytium of progenitor cells: the roundish nuclei (n) are characterized by finely granular karyoplasms; bar = 1.5 µm. (E) TEM section through the new thin epidermis; ec = epidermal cells; bar = 2 µm. (F) Detail of a septate junction (arrow) between two adjacent epidermal cells; bar = 0.5 µm. Vertical LM sections through the calyx showed that at this regenerative stage, the oral surface of the calyx was covered by a true epidermis forming a continuous epithelial layer over the cicatricial tissues. Under this new tegmen, the central region appeared empty and consisted mainly of a wide coelomic space incompletely subdivided by mesenterial

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laminae and septa (Fig. 4A). In sections close to the base of the arms, in particular, well defined cell clusters were commonly associated with the connective tissue of some coelomic septa (Fig. 4B). Such cell clusters (Fig. 4C), which were absent from sections close to central calyx (Fig. 4A), were presumptive primordia of a new gut. The TEM sections suggested that these cell clusters were the result of extensive morphogenetic processes related to the septa, namely cell migration involving the mesothelial lining which became deeply infolded and irregular in thickness. Here the coelothelium was frequently interrupted or absent, so that the related connective tissue, which consisted of an amorphous matrix and rare collagen bundles, was often exposed to the coelomic cavity. The mesothelial lining of the septa was irregularly formed by coelothelial cells (peritoneocytes – Fig. 4D) joined to each other only by small desmosomes. In terms of cytological features, these cells possesed a cilium, a roundish/oval nucleus, with a decondensed chromatin and a prominent nucleolus, as well as a cytoplasm rich in mitochondria, well developed Golgi apparatuses, small RER cisternae and ribosomal complexes (polysomes) (Fig. 4D). Although a basal lamina was present, it did not appear to be attached to the overlying epithelial cells, and hemidesmosomes were not found (Fig. 4D). In addition, groups of cells including presumptive dedifferentiating coelothelial cells (Fig. 4E) and derived progenitor cells (coelomocytes) (Fig. 4F) were found scattered within the connective tissue of the septum. The coelothelial origin of these cells was strongly suggested by the single cilium still present on their surface even when they are sunken in the connective tissue (Fig. 4E). On the basis of the unusual displacements of coelothelially derived cells throughout the underlying connective tissue and the obvious presence of long epithelial processes apparently crossing the basal membrane gaps, these intramesothelial cell clusters (Fig. 4G) can be interpreted as the result of an active migration/penetration process involving coelothelial and coelothelially derived cells. No basal membrane was present around these cell clusters, which were morphologically undifferentiated cells, quite similar to standard coelomocytes (Figs. 4G,H). In contrast, the coelothelially derived progenitor cells frequently showed the unusual presence of centrioles and rootlets of cilia close to the nucleus (Fig. 4I) and a number of large phagosomes including granulocytic granules (Fig. 4H). Junctional complexes were not present. Only spot desmosomes could be observed between adjacent cells (Fig. 4J). Scattered mitotic cells were also detectable (Fig. 4K). Once incorporated into the connective tissue layer, these coelothelium derived groups of progenitor cells could be considered as the presumptive enterocytic precursors. At the cluster periphery, many granulocytes (Fig. 4G) and scattered amoebocytes (see above) were found irregularly distributed among the enterocyte precursors. Amoebocytes were often polynucleated (Fig. 4L). Neural cells and processes were also detected in the connective tissue of the septa. The clusters of enterocytic precursors did not form a compact structure. Inner empty spaces were often observed among the cells, where extracellular matrix was lacking and a number of cell processes and cilia were detected.

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Fig. 4 Antedon mediterranea at 48 hours p.e. (A – C) LM sections. (A) Paraffin vertical section close to central calyx emphasizing the presence of many and large empty spaces identifiable as outlines of coelomic cavities (cc), incompletely subdivided by mesentherial septa (arrows); bar = 200 µm. (B) Paraffin vertical section close to the base of the arms; clu = cell cluster; cc = coelomic cavities; arrows = mesentherial septa; bar = 200 µm. (C) Detail of the cell cluster shown in B: it is formed by deep infoldings of the mesothelial lining; bar = 500 µm. (D - L) TEM sections. (D) The mesothelial lining of the septum consists of coelothelial cells (peritoneocytes - p); cl = cilia; bl = basal lamina; cc = coelomic cavity; bar = 2 µm. (E) Presumptive dedifferentiating coelothelial cell (dc) in the intramesothelial connective tissue; cn = connective tissue; cl = cilium; bar = 1.5 µm. (F) Progenitor cells (pc) in the connective tissue; bar = 1 µm. (G) Detail of intramesothelial cell cluster; c = coelomocytes; gr = granulocytes; bar = 7 µm. (H) Some coelomocytes of the cell cluster can be regarded as presumptive enterocytic precursors (ep); ph = phagosomes; bar = 2.5 µm. (I) Detail of an enterocytic precursor showing evident Golgi apparatus (marked G) and centrioli (cen) close to the nucleus (n); bar = 0.5 µm. (J) Detail of desmosomes (arrows) between adjacent enterocytic precursors; bar = 0.5 µm. (K) Mitotic cell of the intramesothelial cell cluster; bar = 1 µm. (L) Polynucleated amoebocytes; bar = 2.5 µm.

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3.5 72 hours post-evisceration (72 h p.e.) At 72 hours p.e., the stereomicroscope revealed a continuous translucent layer covering the surface of the calyx (Fig. 2C), interrupted only at the level of the five radii by the thin, coloured outlines of the regrowing ambulacral grooves converging towards the oral centre (Fig. 2C). In vertical LM sections, the ambulacral epithelium of the grooves looked thickened and partly differentiated, whereas in adjacent areas, both the outer epithelium and the associated dermis were thinner and quite similar in appearance to the tegmen of noneviscerated animals (Fig. 5A). At this stage the first recognizable gut rudiment appeared. It consisted of a series of separate primordia formed by groups of presumptive enterocytic precursors (see above) and distributed, from the arm base to the central calyx, in the connective tissue of the coelomic septa (Fig. 5A). The timing of gut rudiment development varied appreciably among individuals. Two substages of development, early and late, could be recognized. At the early stage, the TEM sections revealed that the different gut primordia were still separated from the surrounding connective tissue by a thin defined basal lamina. Some primordia showed a clear internal ciliated cavity (Fig. 5B). The enterocytic precursors were characterized by numerous ovoid RER cisternae, well developed Golgi apparatuses and nuclei with large nucleoli and were joined to each other by spot desmosomes located in the apical cell portions (Fig. 5B). Neural processes containing small dense core vesicles, possibly representing the first components of the intestinal basiepithelial plexus, were detectable among the basal portions of the enterocyte precursors (Fig. 5C). At the end of this regenerative stage an early gut rudiment was clearly outlined in the centre of the calyx. At the level of the region where the mouth would develop, this rudiment was connected to the superficial thick layer (Fig. 5A). The enterocyte precursors appeared to have quickly reached a more advanced differentiation state, with the appearance of small mucous vacuoles in their apical cytoplasm (Fig. 5D), which looked particularly rich in polysomes. The ovoid RER vesicles were replaced by elongated cisternae which were laterally aligned along the plasmalemma (Fig. 5D). In addition, the enterocytes contained many large phagosomes including granulocytic granules (Fig. 5E). Specimens processed by the BrdU method still showed a widespread faint labelling in the different regenerating visceral tissues. Strongly labelled nuclei were only scattered at the level of the gut rudiment (Figs. 9C,D).

3.6 5 days post-evisceration (5 d p.e.) At 5 days p.e., the tegmen surface, when observed in the stereomicroscope, appeared to be furrowed by five heterogeneous bands which were slightly pigmented in some samples (Fig. 2D). Moreover, in most samples, a first outline of the mouth could be recognized in the tegmen centre (Fig. 2D).

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Fig. 5 Antedon mediterranea at 72 hours p.e. (A) LM vertical section of the calyx showing gut rudiments (grd) developing from the perypherical coelomic septa (arrow): two arrowheads indicate the region of the new tegmen where the mouth will develop; note the superficial layer connected to the gut rudiment; at the level of adjacent areas, both the outer epithelium and the associated dermis are thinner (single arrowhead); cc = coelomic cavities; bar = 700 µm. (B-E) TEM sections. (B) The gut primordia are formed by morphologically identifiable enterocytic precursors (ep); il = intestinal lumen; cl = cilia; arrow = apical desmosomes between adjacent enterocytic precursors; bl = basal lamina; bar = 5 µm. (C) Detail of a neural process of the newly developing basiepithelial plexus; bl = basal lamina; bar = 0.5 µm. (D,E) Sections of enterocytic precursors (ep) at advanced differentiation stage: the cell phenotype becomes increasingly evident. (D) il = intestinal lumen; cl = cilia; mv = mucous vacuoles; bar = 2.5 µm. (E) Enterocytes containing phagosomes (ph) including granulocytic granules; bar = 100 µm.

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In vertical LM sections, the gut rudiment appeared already to form a spiral tube, opening at one end at the centre of the disk (future mouth) and at the other end in an eccentric interradial position (future anus) (Fig. 6A). The inner lining of this gut primordium consisted of a luminal epithelium at an early stage of morphological differentiation. In the TEM, the earliest mucous cells could be distinguished in this epithelium, particularly in the future oesophagous (Fig. 6B). Throughout the gut rudiment, the enterocytes were characterized by cilia and microvilli on their luminal surface (Fig. 6C). The apical cytoplasm of these cells was partly filled with small mucous granules (Fig. 6C), whereas their central portion was occupied mainly with oval/roundish euchromatic nuclei including prominent nucleoli. Other ultrastrucural features included a number of free ribosomes, many mitochondria, a well developed Golgi apparatus, apparently involved in active synthesis of mucous material, some elongated cisternae of RER, aligned along the lateral plasmalemma, and also many large phagosomes which contained granulocytic granules at different stages of digestion, eventually discharged into the gut lumen (Figs. 6C,D). A loose network of neural processes running between the basal parts of the enterocytes formed the basiepithelial plexus. The gut rudiment was covered on its outer side by a coelomic epithelium composed of typical coelothelial cells (Fig. 6E). Their morphological features were quite similar to those of the peritoneocytes described above. Moreover, in some of these coelothelial cells, differentiation into typical myocytes was in progress, as suggested by the appearance of small bundles of myofilaments in their basal cytoplasm (Fig. 6F). The layer of loose connective tissue interposed between the two basal laminae, belonging respectively to the inner digestive epithelium and the outer coelothelium, contained a loose meshwork of collagen fibrils and numerous scattered granulocytes (Fig. 6E). These granulocytes occupied most of the gut wall space, their granules being at different stages of maturation and involved in typical degranulation phenomena. Fibroblasts-like cells were occasionally recognized between granulocytes. At this regenerative stage, the enterocytes of the new gut showed active cell division, as could be inferred from TEM sections, in which different mitotic figures were detected frequently (Fig. 6G).

3.7 1 week post-evisceration (1 w p.e.) At 1 week p.e. a small anal papilla developed. In the stereomicroscope it looked like a translucent protuberance located eccentrically from the tegmen at the level of one interradius. LM sections showed a well developed digestive tract, complete in all its main portions, i.e. oesophagus, intestine and rectum. It consisted mainly of a wide spiral tube with a rather large lumen, but did not present the typical infoldings and diverticula which could be seen in non-eviscerated animals. The oesophageal epithelium was characterized by the presence of many mucous cells

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which appeared yellow when stained with Milligan trichrome stain. The cytoplasm of these mucous cells contained numerous large heterogenous granules.

Fig. 6 Antedon mediterranea at 5 days p.e. (A) LM vertical section showing the gut rudiment which forms a spiral tube; il = intestinal lumen; m = future mouth; bar = 170 µm. (B-G) TEM sections. (B) Section of the oesophagous rudiment showing a large mucous cell (mc); the base of the adjacent ciliated cell (clc) is also recognizable; mg = mucous granules; mcv = microvilli; bar = 2 µm. (C) Gut rudiment: detail of the apical region of the enterocytes; mg = mucous granules; mcv = microvilli; cl = cilia; ph = phagosomes; bar = 2.5 µm. (D) Gut rudiment: detail of the enterocytes; G = Golgi apparatus; RER = rough endoplasmic reticulum; ph = phagosomes; bar = 1 µm. (E) Wall of the gut rudiment: detail of coelothelial cell (cl) of the outer colelomic epithelium; the underlying layer of loose connective tissue (con) is shown; gr = granulocytes; bar = 20 µm. (F) Detail of the regenerating coelothelium (cl); some subapical myocytes are developing; bundles of myofilaments (my) in the basal cytoplasm are recognizable; bar = 1 µm. (G) Detail of the mitotic nucleus of an enterocyte; bar = 1.5 µm.

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In paraffin sections the intestinal epithelium showed a more bright and homogeneous colour. With TEM it appeared to be composed mainly of columnar vesicular enterocytes (Fig. 7A), connected to each other by typical junctional complexes, including well recognizable septate junctions (Fig. 7C). They looked similar to that of enterocytes at 5 days p.e., containing large mucous granules which partly filled their apical cytoplasm (Fig. 7A), numerous free ribosomes and mitochondria, elongated lateral sub-plasmalemmal RER cisternae (Fig. 7A), and a well developed Golgi apparatus, actively involved in the synthesis of mucous material (Fig. 7B). Besides this main cell population, a second type of enterocytes, type I granular enterocytes, could be recognized at this stage. In these, cells large fusiform electron dense granules (Figs. 7A,D) were detectable in both apical and basal cytoplasm (Fig. 7A). In addition, these type I granular enterocytes were rich in well-developed roundish RER cisternae and Golgi bodies, and displayed large ovoid nuclei with finely granular karyoplasm and prominent central nucleoli (Fig. 7D). The rectal wall was lined a tall epithelium. The luminal cells had microvilli and cilia on their apical surface (Fig. 7E), and contained small RER cisternae, free ribosomes, mitochondria, and a well developed Golgi apparatus with small clear vesicles grouped in the supranuclear cytoplasm (Fig. 7E). At this stage, the outer coelomic epithelium of the gut wall still appeared to be composed of poorly differentiated, cuboidal cells (Fig. 7F); nevertheless, it was already associated with a basiepithelial neural plexus (Fig. 7G). The connective layer, interposed between the inner luminal epithelium and the outer coelothelium, was rather thick but loose and consisted mainly of an amorphous extracellular matrix and wandering coelomocytes. Haemal lacunae were common in the connective tissue layer of the intestinal wall, even in paraffin sections, lacking only at the level of the rectum. The oral and aboral coelomic spaces were subdivided into numerous partly or wholly separate cavities by abundant septa. At the level of each septum, the mesothelium was very thin and flat, and its coelothelial cells showed the same morphology and distinctive features as standard perytoneocytes. As far as cell proliferation is concerned, at this stage, the employment of the BrdU method allowed us to detect a relevant proliferation activity in the tissues. A number of BrdU labelled nuclei were found in both the luminal epithelium and coelothelium (Figs. 9E,F). In the TEM, vesicular enterocytes in the gut luminal epithelium appeared to be frequently involved in mitosis. During mitotic cycle these cells maintained their junctions and mucous granules (Fig. 7H).

3.8 2 weeks post-evisceration (2 w p.e.) At 2 weeks p.e. (Fig. 2E), a clear pentagonal ridge around the mouth, formed by the prominent borders of the ambulacral grooves, became evident in the stereomicroscope (Fig. 2F). The anal cone also appeared to be well developed (Fig. 2G). In vertical LM sections, the digestive tube was larger and more developed than at the previous stage. In general, the whole intestinal lumen appeared to be much wider than in

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non-eviscerated samples, with many internal infoldings without diverticula. Some fecal residues were found in the lumen of the rectum.

A

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Fig. 7 Antedon mediterranea at 1 week p.e. (A-F) TEM sections. (A) The new intestinal epithelium is formed by vesicular (ve) and granular (gr) enterocytes I; il = intestinal lumen; cl = cilia; mcv = microvilli; mg = mucous granules; bar = 7 µm. (B) Detail of the basal region of a vesicular enterocyte showing well developed Golgi bodies; bar = 1 µm. (C) Detail of the apical region of two vesicular enterocytes showing a typical junctional complex; note the septate junction (arrow); bar = 0.4 µm. (D) Granular enterocyte I; g = granules; bar = 2 µm. (E) Epithelium of the rectum: the cell mucous content is reduced; mcv = microvilli; G = Golgi apparatus; bar = 1 µm. (F) Outer coelomic epithelium (cl) of the gut wall showing cuboidal cells; cc = coelomic cavity; bl = basal lamina; my = myocytes; bar = 3 µm. (G) Detail of the basiepithelial neural plexus of the coelomic epithelium; np = neural process; bar = 1 µm. (H) TEM section of vesicular enterocyte involved in mitosis; mg = mucous granules; bar = 2.5 µm.

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The oesophageal epithelium contained an abundance of mucus cells which were responsible for its yellow colour in LM sections. This cell type was even more frequent in the epithelium of the rectum. At this regenerative stage all the intestinal epithelium was characterized by a hypertrophic apical mucous band. In the TEM, the differentiation of the gut epithelium had progressed. A third type of enterocyte, the type II granular enterocyte, was recognizable. These cells were distinguished by their numerous supranuclear roundish granules containing heterogenous electron-dense material and prominent Golgi apparatus (Fig. 8A). Their ovoid nuclei showed finely granular karyoplasm and large nucleoli associated with the inner perinuclear membrane (Fig. 8A).

g

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Fig. 8 Antedon mediterranea at 2 and 3 weeks p.e. (A-C) 2 weeks p.e.: TEM sections. (A) Detail of a granular enterocyte II; g = granules; G = Golgi apparatus; bar = 5 µm. (B) Regenerating gut wall: the outer coelothelium and the underlying connective tissue are shown; cl = coelothelium; my = myocytes; con = connective tissue; np = neural processes of the subcoelothelial nerve plexus; bar = 3 µm. (C) Myocyte (my) in the basal region of the coelothelium; bl = basal lamina; bar = 1 µm. (D) 3 weeks p.e.: LM vertical section through the new gut - the digestive tube is quite similar to that of control samples; il = intestinal lumen; cc = coelomic cavities; bar = 750 µm. The outer coelomic epithelium surrounding the gut appeared to be well differentiated and already consisted of the usual components: apical peritoneocytes, subapical myocytes and neural elements of the coelothelial plexus (Figs. 8B,C). Wide haemal lacunae, containing haemal fluid, were visible in the spaces between the basal membranes of luminal and coelomic epithelia (Fig. 8B).

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3.9 3 weeks post-evisceration (3 w p.e.) At 3 weeks p.e., the functional morphology of the new gut was quite similar to that of noneviscerated control samples. In LM paraffin sections, the digestive tube was characterized by a great number of infoldings and diverticula (Fig. 8D).

cc ce cl cc

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Fig. 9 ICC BrdU method. (A) 24 hours p.e.: semithin resin section showing sparse labelling (cell proliferation) particularly at the level of the cicatricial layer (ce) and the coelomic epithelium (cl); cc = coelomic cavities; bar = 100 µm. (B) Detail of A showing marked labelling of nuclei at the level of coelomic epithelium (cl); cc = coelomic cavities; bar = 25 µm. (C) 72 hours p.e.: semithin resin section showing a widespread labelling - a strong reaction is found at the level of the gut rudiment (arrows); bar = 125 µm. (D) Detail of C showing strongly labelled nuclei at the level of gut rudiment (arrows); bar = 50 µm. (E) 1 week p.e.: semithin resin section of the regenerating gut wall showing labelling (cell proliferation) in both the inner intestinal ephitelium (le) and outer coelothelium (cl); il = intestinal lumen; bar = 100 µm. (F) Detail of E showing markedly labelled nuclei in the inner intestinal ephitelium (arrows); bar = 50 µm.

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The gut wall appeared to be thickened and reinforced by a proper connective tissue component and its own well developed muscle layer. Food remains were easily detectable in the intestinal lumen. A general reduction of the wide empty spaces seen in the previous stages, including coelomic cavities and haemal lacunae, could be observed in the whole regenerated visceral mass (Fig. 8D).

4

Discussion

In order to discuss appropriately the specific mechanisms involved in the process of visceral regeneration, it is convenient to briefly analyse and reconstruct the complex sequence of events in their main aspects phase by phase. In Antedon mediterranea the regenerative process of the digestive tract can be subdivided into three main stages. The first stage, 0-48 hours p.e., is characterized by pronounced rearrangement of the damaged tissues, followed by wound healing processes and re-epithelization of the calyx oral surface. During this stage the oral surface of the exposed calyx is covered by a clot of migratory cells (progenitor cells, such as amoebocytes and coelomocytes, and granulocytes), by a film of coelomic and hemal fluid, and finally by a thin layer of migrating and expanding ectodermal cells. At the end of this early stage the coelomic cavity is sealed off and isolated again from the external environment. The second stage, 2-7 days p.e., is characterized by active morphogenetic processes involving the mesenterial septa located at the arm bases. In particular, coelothelial cells appear to dedifferentiate and migrate from the coelothelium, penetrating into the connective tissue of the mesenterial septa where they apparently undergo a subsequent transdifferentiation into enterocytes. During this process, the coelothellially derived cells tend to progressively lose their distinctive features and undergo activation of their nuclei. Following this extensive migration and presumptive transdifferentiation processes, these coelothelially derived progenitor cells form distinct clusters in the connective tissue layer of the septa. These cells can be considered as “enterocytic precursors” and appear to be involved in very active synthetic activities suggested by the presence of well developed GA and abundant polysomes. The granulocytes also migrate into the intramesenterial connective tissue, where they release their granules. The released granules appear to be taken up in the enterocytic precursors, which display granulocytic-granules containing phagosomes up to 7 day p.e., a stage when the lining of the new gut is completely reformed. One hypothesis is that the granulocytic granules contain regulatory factors which can contribute to or induce the differentiation/transdifferentiation process of the coelothelial cells. This role of granulocytes in regeneration is not new. Smith et al. [51] described a similar involvement of granular cells in nerve regeneration in cockroaches. In addition, this hypothesis seems to be consistent with the fact that, as shown by previous immunocytochemical results, the granulocytic granules of crinoids specifically contain at least some key regulatory factors such as Substance-P and TGF-ß [52]. As cell migration seems to be restricted spatially to the mesenterial septa at the base

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of the arms, the first recognizable groups of enterocytic precursors develop in these same septa. For this reason, five separate gut rudiments are developed in parallel, which are located in the radii and consist of individual cell groups. At an advanced stage, the same rudiments grow to reach the central part of the calyx. On 2-3 days p.e., enterocyic precursors are already polarized cells, and typical cilia develop on their apical surface. In the central part of each cell group, an inner cavity starts to form and the morphological differentiation of the precursor cells takes place in parallel. These cells synthesize a proper basal lamina, and the transformation of synthetic apparatus occurs. In the early phase, the cytoplasm of the precursors contains roundish RER cisternae and subsequently transforms quickly into narrow cisternae laterally oriented along the plasmalemma. The GA is developed to a different extent and appears to participate in active mucus synthesis. At this stage, the junctions between precursors also change. Spot desmosomes are replaced by proper desmosomes, and typical septate junctions start to form. On 5 day p.e., separate groups of precursors appear to fuse together to form a continuous tubular gut rudiment extending from the mouth to the anal opening. The gut epithelium is composed of actively secreting differentiated enterocytes. The formation of the gut from discontinuous multiple rudiments occurs also in some species of holothurians [33, 53–57]. In these animals, the regenerating digestive tube develops from anterior and posterior rudiments, which grow towards each other along the gut mesenterial lamina. In some cases, these primordia can originate from different cell sources, with the posterior rudiment developing from endodermal cells of the cloaca, and the anterior rudiment developing from mesodermal cells of the coelomic epithelium [46, 47]. The important role played by the mesentherial septa is also demonstrated in asteroids [36–38]. The endodermal and mesodermal cells migrate through these structures to reach the regeneration sites and the cellular components of the new gut appear to be derived from the mesenteries. The third stage, 2-3 weeks p.e., is characterized by the complete reconstruction of the anatomical and histological features of the digestive tube. At this stage, the enterocyte differentiation is completed and the digestive lining contains all types of cells occurring in non-eviscerated samples. Following this stage, the gut grows in size mainly due to an increased deposition of extracellular matrix in the connective tissues. On the whole, our results show that in the crinoid A. mediterranea the regeneration of the digestive tract and associated organs is an efficient and rapid process, in spite of the complexity of the structures involved. In fact, the overall process takes approximately 3 weeks. Indeed, a small and at least partially functional gut is already developed at days 5-7 p.e. At this stage, the intestinal lining is well differentiated and the gut may be functional. It should be emphasised that crinoids also display their usual feeding behavior immediately following traumatic evisceration, outstretching their arms and pinnules to form a filter fan. During regeneration, crinoids may absorb organic nutrients dissolved in the water, employing an uptake process which takes advantage of the extensive surfaces of their ambulacral grooves [26]. Because of this unique strategy, it is possible that eviscerated animals can probably receive the necessary nutrients and obtain the energy

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for regenerating the whole visceral effectively and quickly. Many species of echinoderms can autotomize portions of their bodies [58], including visceral organs, and very often they retain parts of the digestive structures and tissues, which provide cellular sources for regeneration of a new gut. For instance, after disk autotomy in ophiuroids, a new stomach regenerates from small remnants of the old oesophagus and stomach [44, 59]. Aspidochirotid holothurians retain the oesophagus and cloaca after evisceration [7] and gut regeneration involves reorganization and growth of these remnants [33, 35, 43, 57]. Fission of apodid holothurians and surgical transection of juveniles of dendrochirotid holothurians result in a loss of the posterior parts of the body. Regeneration of these digestive systems occurs by a morphallactic reorganization of remaining regions of the intestine [41, 42]. In our evisceration experiments on A. mediterranea, all endodermal tissues were excised upon removal of the visceral mass. Our results show that in this case enterocytic precursors developed by transdifferentiation from coelothelially derived cells, i.e. from mesodermal cells. Similar results were also obtained in experiments on the holothurian Eupentacta fraudatrix [45, 47]. In this dendrochirotid holothurian the new gut develops from two rudiments, anterior and posterior. In the posterior rudiment, the digestive epithelium originates from the endodermally derived lining epithelium of the cloaca due to proliferation of vesicular enterocytes. In the anterior rudiment, the enterocytes develops de novo through direct transdifferentiation of the coelomic epithelial cells (both myoepithelial and peritoneal). In A. mediterranea, the development of the main components of the digestive tract occurs between the 2rd and 7th days p.e. This period appears to be the most significant time in the overall regeneration process. During this stage, both regrowth of all the anatomal structures and differentiation of the main cell types occur. These events indicate that gut regeneration in A. mediterranea is very similar to other regeneration processes involving the gut and other echinoderm organs [1, 12, 32, 33, 42, 43, 46, 47]. In all these cases, three phases of repair/regeneration can be distinguished. The first phase corresponds to a repair stage of wound healing and inflammatory reactions. The second phase is an early regenerative stage of active morphogenesis, and possibly dedifferentiation/transdifferentiation and early differentiation, during which a small but functional rudimentaly organ develops. The third phase of regeneration involves the growth and advanced differentiation of the reformed structures and tissues. Proliferation activity reaches its peak during the second stage [12, 15, 17, 60]. In Antedon mediterranea, during the whole process of visceral regeneration, no blastema-like structure can be recognized. This observation suggests that the regenerative processes are carried out by employing morphallactic phenomena of cell recruitment through migration. The extensive employment of cell migration in the regenerative development and reconstruction of new tissues from the remnants of the old tissues can be interpreted as clear signs of morphallaxis [2, 18, 32, 35]. On the other hand, although a certain cell proliferation activity is evident, its contribution is rather modest, even though it becomes more important after the first week of regeneration. Cell proliferation activity reaches its

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maximum at 7 days p.e., when the new digestive system is completely reformed but needs to undergo rapid and active growth. The presence of a relevant mitotic activity at the level of both the coelomic and digestive epithelium is also confirmed in holothurians [35]. Overall, our results further confirm the striking plasticity and the outstanding repair/regenerative potential displayed by these animals at level of their organs, tissues and cells, underlying once more the importance to explore neglected echinoderm regenerative models for a promising applied employment in regenerative medicine.

Acknowledgment This study has received financial support from MIUR COFIN 2003 Research Project. The Authors are particularly grateful to the Fondazione Cariplo and Landau Net-Work Centro Volta for the grant repeatedly given to Dr Igor Y. Dolmatov in support to his research activity in Milano.

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