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Microscopic Anatomy of the digestive system in normal and regenerating specimens of the brittlestar Amphipholis kochii.


Echinoderms are renowned for their regenerative abilities (Hyman, 1955). They show wound healing and can replace lost external appendages (ambulacral tube-feet, spines, and tentacles) and internal organs (intestine, muscles, and nervous system); for reviews, see Dolmatov (1999); Candia Carnevali (2006). Despite the long history of echinoderm studies, some problems relating to regeneration phenomena are unsolved, particularly with regard to pathways and the sources of cells for the replacement of lost organs. One reason for this gap in knowledge is that morphogenetic processes in the echinoderms show great diversity and often differ from those described in other animals. For example, the intestine of holothurians can regenerate in five different ways, and within a single species the regeneration of the intestine varies according to the stage of the life cycle and the method of wounding (Dolmatov, 2009).

The regeneration of the digestive system in echinoderms cannot be described as either "epimorphosis" or "morphal-laxis." The major characteristic of epimorphic regeneration is the formation of a particular structure, a blastema, at the wounded area. The blastema is an aggregation of undifferentiated actively proliferating cells, which later gives rise to the lost organ. The anlage is formed as a superstructure over the undamaged part. Morphallaxis is considered to be restoration through reorganization of the remaining part of the damaged organ or animal, with proliferation making little or no contribution. On the one hand, in some holothurians the intestine regenerates after evisceration as an outgrowth from remaining structures (esophagus and cloaca); that is, it demonstrates one feature of epimorphosis (growth from the wound surface). However, no blastema (which is the major characteristic of epimorphic regeneration) is formed. On the other hand, the entire digestive system of echinoderms can be removed and still regenerate quite quickly. For example, after evisceration in the crinoid Antedon mediterranea the anlage of the intestine appears by day 3, while differentiation of the enterocytes begins by day 5 (Mozzi et al, 2006). In this case, no remnant of the digestive system is available, and so no mechanisms of morphallaxis can be involved. Moreover, despite the presence of dividing cells, no blastema arises and the intestine develops as a result of epithelial morphogenesis; that is, the major characteristics of epimorphosis are also absent.

The problem of mode of regeneration is also related to the problem of cell sources for organ restoration. Leibson (1980) suggested the possibility that undifferentiated cells (stem cells) participate in the formation of lost organs in echinoderms. Candia Carnevali et al. (2009) advanced the hypothesis that crinoids have two types of stem cells, resident stem cells and circulating stem cells; however, only one type, which gives rise to certain coelomocytes, has been identified (Eliseikina et al., 2010).

Thus, further investigation of different aspects of regeneration in echinoderms is required at both the cellular and molecular levels. Brittlestars are convenient models for such studies, having a high capacity for regeneration (Hyman, 1955; Dolmatov, 1999; Candia Carnevali, 2006). Several brittlestar species have been used to study autotomy mechanisms (Dobson, 1988; Charlina and Dolmatov, 2008; Charlina et al., 2009) and the effects of different factors on regeneration (Hall, 2002; McAlister and Stancyk, 2003; Biressi et al., 2010). For example, some members of the family Amphiuridae rapidly regenerate autotomized central disks (Hyman, 1955; Emson and Wilkie, 1980). During such autotomy the animals lose almost the entire digestive system, gonads, and bursae. Despite this significant loss, regeneration proceeds very quickly and takes only 10-15 days, at which point the brittlestars are able to feed again (Litvinova and Zharkova, 1977; Dobson, 1988). However, the mechanisms of autotomy and regeneration of the central disk in brittlestars are not well understood. Although a few papers have dealt with specific aspects in several species (Litvinova and Zharkova, 1977; Dobson, 1988; Dobson and Turner, 1989; Charlina et al., 2009), no detailed studies of the formation of the internal organs have been published, the ultrastructure of the regenerative process is not known, and the sources of cells for the restoration of the digestive lining have not been identified.

Previously, we presented a brief description of stomach lining regeneration after central disk autotomy in the brittlestar Amphipholis kochii (Frolova and Dolmatov, 2006). The present paper provides a full ultrastructural account of the regeneration of the digestive system of A. kochii after autotomy. It includes a detailed description of the ultrastructure of the intact digestive system, since the only other published ultrastructural accounts of the intact ophiuroid digestive system have been based on species from a single family--the Ophiodermatidae (Schechter and Lucero, 1968; Deschuyteneer and Jangoux, 1978; Byrne, 1994)--the feeding biology of which is probably very different from that of the Amphiuridae. to which A. kochii belongs.

Materials and Methods

Adults of the brittlestar Amphipholis kochii (Lutken) (Ophiuroidea, Echinodermata) were collected in Peter the Great Bay, Sea of Japan. To study the microanatomy of the normal digestive system, entire animals were placed in fixative, and after fixation the arms were cut off, so that only the central disk remained. For light microscopy we used three animals. After several days in Bouin's fixative at 4 [degrees]C. the samples were dehydrated in an ethanol series and embedded in paraffin according to standard methods. The disks of the brittlestars were sectioned transversely, parallel to the oral-aboral axis. Sections (5 [micro]m thick) were stained with hematoxylin. For transmission electron microscopy we used four animals. The material was fixed in 2.5% glutaraldehyde in 0-05 mol [1.sup.-1] cacodylate buffer at pH 7.6 for several days at 4 [degrees]C and postfixed in 1% Os[O.sub.4] for 1 h. Then specimens were decalcified with ascorbic acid for 24 h (Dietrich and Fontaine, 1975), dehydrated in a graded series of ethanol and acetone, and embedded in a mixture of araldite and Epon 812. Sections were cut parallel to the oral-aboral axis of the animal, using an LKB ultracut microtome. Semithin sections (1-2 [micro]m thick) were stained with 1% methylene blue. Ultrathin sections were consequently contrasted in uranyl acetate and lead citrate solutions and examined in a Carl Zeiss Libra 120 transmission electron microscope.

Autotomy was stimulated mechanically by gripping the disk edge in a radial area with forceps until the disk was detached. After autotomy, brittlestars were kept in an aquarium with flowing water and aeration. Animals on days 1, 2, 3, 4, 5, and 7 after autotomy were used for the study. Three samples were used for ultrastructural analysis, and two to three samples were used for light microscopy at each regenerative stage. This material was treated in the same way as were the intact samples. For scanning electron microscopy, the samples were fixed in 2.5% glutaraldehyde in seawater. They were then dehydrated in increasing concentrations of ethanol and acetone and dried in C[O.sub.2] The dried specimens were mounted on SEM pin stubs, coated with carbon, and analyzed in a Leo 430 scanning electron microscope.

The number of cells of different types was calculated in paraffin sections, as the ratio between each individual cell type and the total number of cells. For these calculations we used three specimens, and the total number of cells in each case was 1000. The size of granules was calculated in TEM micrographs for three cells; for each cell we measured five granules.


Morphology of the digestive system

The brittlestar Amphipholis kochii has a very simple digestive system, which is composed of only two parts: a very short esophagus and a sac-like stomach (Fig. 1A). There is no cloaca or anal opening. The wall of the esophagus and stomach consists of coelomic epithelium, a connective tissue layer with hemal lacunae, and digestive epithelium.

The internal surface of the esophagus has transverse folds and is lined with a thick cuticular epithelium (Fig. 1B, C). The latter consists of two cell types: enterocytes, which will be called "type-1 enterocytes" to distinguish them from enterocytes of the stomach; and mucocytes, which will be called "type-1 mucocytes" for the same reason. Type-1 enterocytes are the most abundant cell type in this epithelium. Their apical surfaces bear numerous microvilli, which are embedded in a cuticle. The cells are connected to each other by septate junctions (Fig. 1C). The subapical cytoplasm is filled with small vesicles. There are prominent secretory granules with electron-dense content in the apical half of the cell. The mean granule diameter is 0.54 [+ or -] 0.05 [micro]m. The cytoplasm contains a well-developed RER (rough endoplasmic reticulum) and GA (Golgi apparatus), numerous mitochondria, as well as phagosomes, multivesicular bodies, and lipid droplets. An oval nucleus with prominent nucleolus and large heterochromatin particles is located in the middle part of the cell.

Type-1 mucocytes are less abundant. They constitute about 20.3% of the total number of cells in the epithelium of the esophagus. A distinguishing cytological characteristic of these cells is the presence of a prominent GA and spherical granules filled with mucus (mean diameter 0.87 [+ or -] 0.07 [micro]m) (Fig. 1D). The latter has a heterogeneous appearance with moderate electron density. Mucus-filled granules enlarge, fuse with each other, and occupy almost the entire volume of the cell. The cytoplasm also contains well-developed RER and mitochondria. There are nerve cells and bundles of axons in the middle and basal parts of esophageal epithelium (Fig. 1E).

The digestive epithelium of the stomach has no cuticle. It is composed of four cell types: type-2 enterocytes, two types of granular secretory cells, and type-2 mucocytes. Type-2 enterocytes are the commonest cell type (Fig. 1F, G). These cells are cylindrical with an oval nucleus. Their apical surface bears numerous long microvilli forming a conspicuous brush border. The cytoplasm contains large secretory granules of varying electron density, which obviously correspond to different stages of the secretory cycle. The mean granule diameter is 1.34 [+ or -] 0.03 [micro]m. The apical cytoplasmic membrane forms pinocytotic pits, and the subapical cytoplasm contains numerous pinocytotic vesicles. RER is well developed in the cytoplasm, GA is located in the supranucleus zone, and lipid droplets are concentrated in the middle and basal parts.

Granular cells in the digestive epithelium of the stomach of A. kochii make up about 13.2% of the total number of cells of the luminal epithelium. Two types of such cells could be distinguished in transmission electron micrographs. One of these, which we designated as a "type-1 granular secretory cell," contains rounded electron-dense membrane-bounded granules (Fig. 1H, I). The mean granule diameter is 0.95 [+ or -] 0.03 [micro]m. These cells have numerous long microvilli. The apical cytoplasmic membrane has pi-nocytotic pits, and the apical cytoplasm contains a GA and pinocytotic vesicles. An oval nucleus with a large nucleolus is located in the middle of the cell. In the cytoplasm there are elongated narrow cisternae of RER, mitochondria, and many free ribosomes and vesicles.

Type-2 granular secretory cells (Fig. 1I) are similar to type-1. Type-2 cells are characterized by rounded membrane-bounded granules of medium electron density. However, the mean diameter of the granules is somewhat smaller than that of type-1 granular secretory cells and is 0.66 [+ or -] 0.04 [micro]m. The cytoplasm contains well-developed GA located in the perinuclear space, numerous expanded elongated or rounded RER cisterns, and free ribosomes. The cytoplasm of these cells is more electron-dense than that of other cell types revealed in stomach lining.

The fourth cell type--type-2 mucocytes (Fig. 1J)--constitute about 12.5% of the total number of cells in the luminal epithelium. They are characterized by numerous small granules (mean diameter 0.41 [+ or -] 0.02 [micro]m) that are electron-lucent or of moderate electron density, often with a dense core. The granules fuse with each other in the apical cytoplasm to form large mucous droplets. There are scarce bundles of axons in the basal part of stomach epithelium (Fig. 1K).

The coelomic epithelium of both the esophagus and the stomach has a typical structure and consists of two cell types--peritoneocytes and myoepithelial cells (Fig. 2). Between these cells there are nerve cell bodies and bundles of axons of the basiepithelial nerve plexus. Peritoneocytes are ciliated cells with rare microvilli; they are connected to each other by zonulae adhaerentes (Fig. 2A). Their flattened apical parts form a continuous apical sheet. The thin basal processes of these cells span the height of the epithelium and are linked to the basal lamina by hemidesmosomes. The nucleus has an oval to irregular shape and contains a nucleolus and numerous heterochromatin particles. RER and GA are moderately developed. The cytoplasm also contains mitochondria, scarce large phagosomes, free ribosomes, and lipid droplets.


Myoepithelial cells are situated among the basal parts of the peritoneocytes and do not reach the apical surface of the epithelium. Bundles of myofilaments occupy most of the cytoplasm. The myoepithelial cells are connected to each other by desmosomes and to the basal lamina by hemidesmosomes (Fig. 2A, B). Their processes make up the three layers of musculature in the stomach wall (Fig. 2B). The outer and inner layers are made of processes of myoepithelial cells expanded in the meridional direction; the middle layer is constructed of processes arranged in latitudinal direction.

The connective tissue layer located between the digestive and coelomic epithelia consists of collagen fibrils and unstructured extracellular matrix. It contains different coelo-mocytes (Fig. 2B-D) and bundles of axons (Fig. 2D). Haemal lacunae also are rather common in this layer (Fig. 2C).

Regeneration of the digestive system

During autotomy most of the disk is detached and discarded. The brittlestar loses almost the entire digestive system, as well as aboral parts of the bursae and the gonads. The stomach wall is broken close to the mouth opening (Fig. 1A). After the autotomy the animal consists of an oral frame with attached arms (Fig. 3A). The removal of the disk uncovers basal arm segments and the skeletal elements of the oral frame. As a result, the coelom and remaining internal organs come into contact with the external environment. Remnants of the digestive system can be observed around the mouth opening. The esophageal lining and the adjacent part of the stomach epithelium remain (Fig. 3B, C). The latter comprises mostly enterocytes; other cell types are found only very rarely.


On days 1-2 after autotomy the active migration of epidermal cells is noticeable on the surface of the animal. The cells move from the oral to the aboral side of the arm bases and oral frame (Fig. 4A-C). By this time the epithelia of the esophagus and stomach have lost their normal organization. The cells of the esophageal lining flatten out and migrate to the aboral side, forming, together with epidermal cells, a cuticular epithelium in the wounded area (Fig. 4D-G). Under the cuticle there are bacteria that have presumably arrived from the external environment.

The number of microvilli on the cells of the esophageal lining decreases. The cell nuclei are oval and contain large chromatin granules. The numbers of lysosomes and phagosomes in the cells increase (Fig. 4F). The mucous cells lose most of their secretory granules. The cytoplasm of esophageal cells contains numerous mitochondria and a well-developed GA, and their intercellular junctions persist (Fig. 4F).

The stomach anlage is represented by an amorphous aggregation of cells encircling the mouth opening (Fig. 4D, E, G). The cells lose their connections to the basal lamina and perhaps to each other, since we found no intercellular junctions. On the other hand, the enterocytes obviously retain polarity, as their apical regions with microvilli are directed toward the interior of the aggregation (Fig. 4G). The nuclei of the enterocytes become rounded and contain condensed chromatin. Their cytoplasm is filled with small RER cisternae, lysosomes, and phagosomes. Moreover, many secretory granules are retained in the cytoplasm. As well as type-2 enterocytes, we found in the aggregation type-1 enterocytes of the esophagus, containing characteristic secretory granules (Fig. 4E). At the periphery of the stomach anlage there are coelomocytes and, presumably, fibroblasts.

On day 3 after autotomy the whole aboral surface of the oral frame is covered with epidermis (Fig. 5A). The latter fuses with the esophageal lining, thus forming a thin epidermis around the entire oral frame, which separates the coelom from the external environment (Fig. 5B). The aboral side of the disk consists of three layers: epidermis, layer of coelomocytes, and aboral coelomic epithelium, which lines the coelomic cavity (Fig. 5C).


The esophagus is composed of cuboidal cells (Fig. 5D), each containing a large rounded nucleus with a well-developed nucleolus. The chromatin is decondensed. The cytoplasm of the cells contains small RER cisternae, a well-developed GA, and numerous mitochondria. The apical surface of the cells is complicated in shape and has sparse branching microvilli that pass through the cuticle.

The diameter of the stomach anlage increases and almost equals that of the oral frame (Fig. 5B). A lumen appears inside the anlage, and its walls have an epithelial structure (Fig. 5C, E, F). The walls consist of coelomic and luminal epithelia separated by a layer of connective tissue. The luminal epithelium consists of irregularly shaped dedifferentiated cells connected by apically located zonulae adhae-rentes (Fig. 5E, F). The nuclei contain decondensed chromatin and a large nucleolus. The cytoplasm retains many phagosomes and sometimes secretory granules (Fig. 5G). The GA is well-developed and located in the apical part of the cell (Fig. 5F, G). The basal lamina is absent. At this stage the first mitotically dividing cells could be distinguished within the luminal epithelium of the anlage (Fig. 5C, E).

The coelomic epithelium develops on the external side of the stomach anlage (Fig. 5C, E). It consists of flattened dedifferentiated cells arranged on a well-developed basal lamina and connected to each other by zonulae adhaerentes. Their cytoplasm contains expanded RER cisternae, a well-developed GA, mitochondria, and small phagosomes, and they have an apical cilium.

The space between the coelomic and digestive epithelia is filled with amorphous extracellular matrix of low electron density and includes coelomocytes, fibroblasts, and small bundles of collagen fibrils (Fig. 5E).

On days 4-5, only a small opening is visible on the aboral side of the animal (Fig. 6A, B). The layer of coelomocytes and fibroblasts in the aboral wall of the disk thickens (Fig. 6B). The stomach anlage expands and its diameter increases. At the same time, the aboral wall of the stomach grows toward the center, so that the opening in the upper part becomes narrower. A thin wound epidermis is clearly visible interconnecting the oral and aboral parts of the disk (Fig. 6B). It encloses the lumina of the stomach and coe-loms and isolates the internal organs from the external environment.

At this stage the transformation of the cells of the luminal epithelium continues (Fig. 6C, D). The cells of the epithelium lengthen and become cuboidal or cylindrical. Microvilli appear on the apical surfaces of the cells (Fig. 6C). The cytoplasm shows a well-developed GA, numerous free ribosomes, and small RER cisternae. An oval nucleus is located in the basal part of the cell and contains one or two large nucleoli and small chromatin granules. The cytoplasm of the cells contains many phagosomes of varying size, as was the case in the previous stage.

On the aboral side, the luminal epithelium gradually becomes thinner; at the opening it is folded inward and blind-ended (Fig. 6B, D). In this area the coelomic epithelium is absent and the luminal lining is separated from epidermis only by the aggregation of coelomocytes.

Mitotically dividing cells could still be seen in the luminal epithelium (Fig. 6D). The remnants of secretory granules could still be observed in the cytoplasm of the cells of the epithelium. Some of them are similar in morphology to the secretory granules of esophageal mucocytes and enterocytes (Fig. 6E, F). Moreover, in some cells there are vacuoles filled with fibrillar material that resembles myofilaments (Fig. 6G).

The basal lamina of the luminal epithelium is first visible in animals fixed on day 5 after autotomy, although no hemidesmosomes were found in these specimens (Fig. 6H). At the base of the epithelium are small bundles of axons (Fig. 61).

The structure of the coelomic epithelium is similar to that of the previous stage and consists of flattened epithelial cells (Fig. 6D, J), between which are small bundles of axons. No myoepithelial cells are present.

On the day 7 the opening in the aboral side of the disk is closed and the wound epidermis disappears (Fig. 7A). The stomach cavity now communicates with the environment only through the mouth opening. The digestive lining consists mostly of tall cells with long and numerous microvilli (Fig. 7B, C), and its surface has small folds. The cytoplasm of the ceils contains large phagosomes, a well-developed GA, small RER cisternae, and many free ribosomes. At this stage some cells showing signs of specialization are found in the luminal epithelium (Fig. 7D). They contain numerous rounded RER cisternae and free ribosomes. In the apical part of the cell there are well-developed GA and small lysosomes. The nucleus is rounded, has a large nucleolus, and is located in the middle part of the cell. The perinuclear area is expanded. In terms of morphology, these cells are similar to the granular secretory cells. Myoepithelial cells containing bundles of myofilaments appear in the coelomic epithelium (Fig. 7E).


The closure of the opening in the aboral side of the disk and the beginning of cell differentiation in the stomach lining show that the regeneration of the digestive system is almost complete. Later, the cells of the digestive lining complete their differentiation and the animal begins feeding.


Morphology of the digestive system

Our study shows that Amphipholis kochii has a typical ophiuroid digestive system, which comprises only two parts--esophagus and stomach (Hyman, 1955; Filimonova, 1979; Jangoux, 1982). As in other members of the class Ophiuroidea, the esophageal epithelium of A. kochii is covered with a cuticle. The latter is synthesized by the main cell type making up the esophageal lining, namely type-1 en-terocytes. The formation of the cuticle is obviously one of the primary functions of this cell type in echinoderms (Eli-seikina and Leibson, 1996; Mashanov et al., 2004). The presence of microvilli and microvesicles in the apical cytoplasm suggests that type-1 enterocytes are also able to take up some organic nutrients that can be sequestered without being digested in the gut lumen.

The mucocytes of the esophagus and stomach demonstrate a structure typical for brittlestars (Byrne, 1994). They produce mucus to agglutinate food particles and facilitate their movement through the digestive system. The secretory granules in the cells of the esophagus and stomach differ, and we therefore distinguish in A. kochii two types of mucous cells, namely types 1 and 2.

The digestive epithelium of the ophiuroid stomach includes four cell types: enterocytes, two types of granular cells, and mucocytes (Jangoux, 1982; Byrne, 1994). The same cell types were distinguished in A. kochii. Type-2 enterocytes are the most abundant cells in the epithelium. Their distinguishing characteristic is the presence of numerous secretory granules in the cytoplasm. On the apical surface arc numerous microvilli that make up a typical brush border. The presence of pinocytotic vesicles in the apical cytoplasm is evidence that these cells are involved in the absorption of nutrients (Byrne, 1994), while the presence of phagosomes indicates that intracellular digestion may be one of their functions. Moreover, type-2 enterocytes obviously also perform a storage function, as there are numerous lipid droplets in their basal regions. According to Jangoux (1982) and Byrne (1994). each of these cells bears a cilium. However, we did not find any cilia in the type-2 enterocytes of A. kochii.

Using the electron microscope, we distinguished two types of granular cells in A. kochii: type-1 and type-2. They differ in the structure of their cytoplasm and granules. Type-1 granular secretory cells resemble granular cells described in other species of brittlestars (Byrne, 1994). Their cytoplasm contains large electron-dense granules and elongated RER cisternae. Type-2 granular secretory cells have not been described in other species of brittlestars. In comparison with type-1, their granules have lower electron density and smaller diameter, they contain numerous rounded RER cisternae, and their cytoplasm is more electron-dense. It is possible that these represent different stages of the secretory cycle in a single cell type. As in other brittlestars. they are probably exocrine cells synthesizing and releasing enzymes for extracellular digestion (Pen-treath, 1969; Jangoux, 1982; Byrne. 1994).

Regeneration of the digestive system

Our studies showed that the regeneration of the aboral part of the disk and the stomach in the brittlestar A. kochii is a very rapid process that is completed within 7 days after autotomy. This rapid replacement may be related to the great importance of the digestive system for the survival of the animal. Such rapid regeneration has also been reported for the disk of other brittlestars (Dobson, 1988) and for the intestine of the crinoid Antedon mediterranea (Mozzi et al., 2006).

It is common practice to distinguish several successive stages within the process of regeneration (Needham, 1965). Describing disk regeneration in Microphiopholis gracil-lima, Dobson (1988) distinguished three stages: wound healing, dedifferentiation, and growth. Similar stages could also be distinguished in A. kochii, although, as regeneration in this species is very fast, these stages overlap in time and take place almost simultaneously. As is the case in many other animals (Brockes and Kumar, 2008), the first noticeable event in the regeneration of brittlestars is migration of the epidermal cells and coelomocytes. Already on days 1-2 after autotomy the wound epithelium covers the entire damaged area. Coelomocytes gather under the epidermis, on the aboral side of the disk, where they probably clean up the wound and remove damaged cells. These cells continue to aggregate in the aboral wall of the disk where they form a rather thick layer by day 5 after autotomy. Simultaneously with wound healing, dedifferentiation of cells in the lining of the esophagus and the remaining part of stomach begins. The major signs of dedifferentiation are activation of the nuclear apparatus, reorganization of the internal structure of the cells, and changes in intercellular interactions. Cell nuclei increase in size and assume a regular spherical shape. The chromatin decondenses, the heterochromatin particles become smaller, and a nucleolus appears. The processes of synthesis characteristic of digestive cells are arrested; thus, microvilli shorten, pinocytotic vesicles disappear, and the number of secretory granules decreases. The latter enter phagosomes and are presumably re-utilized. The number of phagosomes increases in the first days after autotomy, which is an indication that cellular activity is being reorganized. Moreover, organelles involved in synthesis are activated in the cytoplasm: the GA becomes more pronounced and the numbers of RER cisternae and ribosomes increase. The cells become detached from the basal lamina and undergo migration. As a result of all these processes, on day 3 it is already very hard to identify the cell types in the digestive lining from the appearance of their secretory granules. Dedifferentiation allows the cells to enter the mitotic cycle and begin to divide. First mitoses appear in the stomach anlage as early as 3 days after autotomy, and as a result it begins to grow.

Simultaneously with migration and proliferation, the cells of the stomach lining continue to dedifferentiate. As a result, by day 7 after autotomy the hole on the aboral side of the disk is closed and the digestive system is replaced. When the stomach forms, its lining initially consists mostly of dedifferentiated cells. In contrast to this, in the crinoid A. mediterranea specialized intestinal cells appear on day 5 after gut removal (Mozzi et al., 2006).

In relation to the formation of the wound epidermis, another peculiarity of disk regeneration in brittlestars should be noted. After autotomy only the oral frame with connected arms remains. During disk regeneration, the wound epithelium develops over the entire damaged area, including the sides of the central opening. When the stomach develops, its lumen expands and the oral and aboral walls separate from each other. Since they are not joined together at the area of the central opening, the stomach lumen should open into the environment at the mouth area. However, this is not the case, as the internal end of the central opening is covered with epidermis. This provisional structure, the wound epidermis, is characteristic not only of A. kochii, but also of M. gracillima (Dobson, 1988). It is obviously needed to isolate the internal tissues of the regenerating disk from the environment and prevent bacterial infection of the wounded area.

A major problem in regeneration studies is the origin of the cells that form the developing organ. In echinoderms, gut regeneration depends mostly on dedifferentiated cells from remnants of the digestive epithelium (Dolmatov and Mashanov, 2007; Dolmatov, 2009). However, when the entire digestive system is lost, as in some species of ho-lothurians and crinoids, its replacement is derived from the coelomic epithelium (Mashanov et al., 2005; Mozzi et al., 2006). Dobson (1988) pointed out that in M. gracillima, undifferentiated coelomocyte-like cells can take part in the restoration of the stomach. The participation of such cells in the regeneration of arms in crinoids and brittlestars has also been suggested (Candia Carnevali et al., 2009; Biressi et al., 2010). However, none of the cited papers above has demonstrated the presence of stem cells in echinoderms. Proceeding from an analysis of light microscopic sections, Dobson (1988) mentioned that a blastema-like aggregation of undifferentiated cells arises in the early stages of regeneration, which later gives rise to stomach. Using transmission electron microscopy techniques, we have shown that in A. kochii this aggregation comprises dedifferentiated en-terocytes from the stomach lining and coelomocytes. Candia Carnevali et al. (2009) described in crinoids two types of stem cells, namely resident stem cells and circulating stem cells, which form a blastema at the wounded area. However, the authors supposed these cells to arise from cells of the coelomic epithelium, the peritoneocytes. This means that the described cell types are dedifferentiated peritoneocytes rather than stem cells.

Our studies demonstrated that in A. kochii it is the epithelial lining of the esophagus and the remaining region of the stomach that is the main source of cells for regeneration of the digestive epithelium. We did not find evidence for the participation of other cell types, in particular coelomocytes and coelomic epithelial cells, in the formation of the stomach lining. In the early stages of restoration, on days 1-2 after autotomy, we found no intercellular junctions between the cells of the stomach remnant. The anlage of the digestive system at this stage seems to represent a mesenchyma-like structure. However, at this stage, enterocytes are only at the very start of dedifferentiation and contain specific secretory granules (Fig. 4F, G); thus they could be easily identified using the transmission electronic microscope. On day 3 the anlage already shows an epithelial structure, its cells being connected to each other by intercellular junctions. The cytoplasm of these cells still contains the remnants of specific secretory granules. Later, the cells begin to divide and migrate as a single epithelium. The intercellular junctions are not disrupted even during the process of mitosis.

On days 4-5 after autotomy, vacuoles with fibrillar contents resembling myofilaments were observed in some cells of the stomach lining (Fig. 6G). From this it might be interpreted that coelomic epithelium cells participate in the restoration of stomach lining, as in the holothurian Eupen-tacta fraudatrix (Mashanov et al., 2005). However, it is more likely that the myofilaments arrive in the enterocytes as a result of phagocytosis. Like amoebocytes, different cells in echinoderms are capable of active phagocytosis of damaged or dead cells and their fragments. In particular, myofilament-containing fragments of cytoplasm arising during destruction or dedifferentiation of myoepithelial cells can be phagocytosed by peritoneocytes of the coelomic epithelium (Dolmatov and Ginanova, 2009) or glial cells of the radial nerve cords (Mashanov et al., 2008). It is highly likely that the enterocytes of A. kochii are also able to phagocytose fragments of myoepithelial cells.

An important characteristic of regeneration is the mode of formation of the lost organ. The mode of regeneration refers to the general pattern of restorative processes, and to the relationships between the old and new parts of the organism or organ and between growth and differentiation (Liosner, 1982). Describing regeneration in invertebrates, usually the terms "epimorphosis" and "morphallaxis" are used, these having been introduced by Morgan as far back as 1901 (Morgan, 1901). Recently some attempts have been made to reconsider the entire concept of "mode of regeneration" (Agata et al., 2007; Dolmatov and Ginanova, 2009).

In echinoderms, restoration depends mostly on the reorganization of a small part of the organ around the wounded area, migration of cells, and moderate cell proliferation (for reviews, see Dolmatov, 1999, 2009; Candia Carnevali, 2006; Dolmatov and Mashanov, 2007). Only in rare cases does regeneration take place without any cell proliferation or with only the minimum contribution of cell division (Gibson and Burke, 1983; Dolmatov and Ginanova, 2001). In such cases it is usually concluded that regeneration involves morphallaxis with some contribution from epimorphosis, or vice versa (Dolmatov, 1992, 1999; VandenSpie-gel et al., 2000; Candia Carnevali, 2006; Biressi et al., 2010). However, in some cases such a conclusion would be wrong, as the major characteristics of neither epimorphosis (formation of a blastema) nor morphallaxis (reorganization of the remnant of the organ without proliferation) are at all pronounced. The regeneration of many internal organs, like muscles, the nervous system, and intestine, of echinoderms in general and respiratory trees of holothurians in particular, involves the migration of entire epithelia rather than some isolated cells, therefore representing typical epithelial morphogenesis (Dolmatov, 1999, 2009; Dolmatov and Mashanov, 2007; Mashanov et al., 2008; Dolmatov and Ginanova, 2009; Garcia-Arraras and Dolmatov, 2010). Such a mode of regeneration differs from both epimorphosis and morphallaxis.

The regeneration of the stomach in the brittlestar A. kochii is also an example of epithelial morphogenesis. No blastema forms. The clump of cells in the developing disk that looks like a blastema at the light microscopic level (Fig. 4D, E) actually represents an aggregation of dedifferentiated enterocytes (Fig. 4G) or coelomocytes and fibroblasts between the epithelia (Figs. 5C, 6D). In almost all stages of regeneration, the cells of the coelomic and digestive linings retain intercellular junctions and migrate only within their respective epithelia. Mitosis first appears in the stomach anlage on day 3 after autotomy, when the latter shows an epithelial structure. It is thus obvious that the problem of the mode of regeneration in echinoderms needs to be revisited and thoroughly investigated.


We are grateful to the anonymous reviewers, whose valuable critical comments enabled us to improve the quality of the manuscript. Our special thanks are extended to Dr. I.C. Wilkie (Glasgow Caledonian University, Scotland) for suggestions and critical reading of the manuscript. This work was funded by a grant from the Russian Foundation for Basic Research (project no. 08-04-00284) to I.Y.D. and by a grant from the Far Eastern Branch of the Russian Academy of Sciences and the Russian Foundation for Basic Research (project no. 09-04-98547) to L.T.F.

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Received 21 September 2009; accepted 24 April 2010.

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Abbreviations: GA, Golgi apparatus; RER. rough endoplasmic reticulum.


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Author:Frolova, Lidia T.; Dolmatov, Igor Yu.
Publication:The Biological Bulletin
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Geographic Code:4EXRU
Date:Jun 1, 2010
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