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Gut regeneration in holothurians: a snapshot of recent developments.

Introduction

Sea cucumbers, or holothurians, are exclusively marine invertebrates classified in the phylum Echinodermata, class Holothuroidea. They are characterized by a soft, orally-aborally elongated cylindrical body with the mouth (surrounded by a crown of tentacles) and the anus located at opposite ends of the body (see Fig. 1). These creatures are able to regenerate various parts of the body after injury, autotomy, or in some cases, asexual reproduction. They are also known to practice one of the most impressive forms of regeneration in the animal kingdom--they can completely discard most of their internal organs and then rapidly re-grow them.

The present review is mostly focused on the current developments in the field of visceral regeneration in sea cucumbers and therefore is largely based on data that have been obtained during the last 10 years or so. The old works are referenced only when used as a basis for further research. For a more detailed account of earlier publications in the field, the reader is referred to Garcia-Arraras and Green-berg (2001) and Hyman (1955). A new review is timely because new data on cellular mechanisms and underlying molecular processes have been accumulated over the last decade. Not all the phenomena contributing to successful regeneration have been studied deeply enough. Most aspects of visceral regeneration have been studied in one or a few species only; therefore, little is known about interspecific variation in the regeneration response in sea cucumbers. Although there are still many gaps in our knowledge, we have attempted to combine the existing data and hypotheses into a cohesive story, which represents current achievements in the field.

The Normal Gut

Anatomy and histology

The holothurian digestive tube is usually very long and looped, occupying most of the main body cavity (somatocoel). There is no generally accepted nomenclature for the different parts of the alimentary canal, since different authors use different names when referring to the same structure (Feral and Massin, 1982). Moreover, there are also some differences in the organization of the digestive tube among different holothurian taxa, which reflect differences in feeding mode (suspension vs. detritus feeders) and food composition (Hyman, 1955; Feral and Massin, 1982). Nevertheless, on the basis of anatomical and histological data, it is possible to distinguish the following anatomical regions of the digestive tube in many sea cucumbers: a pharynx lying within the pharyngeal bulb (a complex anatomical structure that unifies the radial branches of the nervous, water-vascular, and hemal systems), a short esophagus that connects the pharynx to a long looping intestine, which eventually opens into a large thick-walled muscular cloaca (Fig. 1). The intestine itself can be further subdivided according to the direction of its axis or to morphological features; thus, in some species, distinctions are made between the ascending and descending portions of the intestine or between what has been named the small and large intestine. The loops of the digestive tube within the body cavity are suspended by the mesentery, which anchors them to the body wall and which, as will be shown below, plays the crucial role in visceral regeneration.

[FIGURE 1 OMITTED]

In all regions of the digestive tube, the wall of the gut consists of three layers--a folded innermost luminal (digestive) epithelium, which is endodermal in origin; an outermost complex muscular mesothelium (gut coelomic epithelium), which derives from the mesoderm; and a connective tissue layer, sandwiched between the two epithelia and delimited by their basal laminae. The luminal epithelium is a simple pseudostratified columnar epithelium (Fig. 2A), which is composed mainly of tall and slender cells called enterocytes. Each of those cells is thought both to reach the apical (luminal) surface and to make contact with the basal lamina (that is why the epithelium is classified as simple), but their nuclei can occupy varying positions along the apical-basal layer (hence, pseudostratified). The enterocytes seem to be multifunctional cells that play multiple roles in digestive physiology, including mucus production and secretion, nutrient absorption, synthesis of digestive enzymes, phagocytosis of food particles, accumulation of nutrients, and transport of the latter to hemal lacunae of the connective tissue layer (Feral and Massin, 1982; Mashanov et al., 2004). These enterocytes constitute the vast majority of luminal cells; however, other cell types have also been identified within the luminal epithelia. including specialized mucus-producing cells and neuroendocrine cells.

The gut mesothelium is usually a tall pseudostratified epithelium that shows a very complex organization (Fig. 2B). Its apical surface is occupied by cell bodies of perito-neocytes, monociliated epithelial cells, connected to each other via intercelluar junctions (zonula adhaerense and septate junctions). The cell body of each peritoneocyte continues into a long slender process that passes though the thickness of the epithelium and attaches to the basal lamina. Those processes usually contain thick bundles of intermediate filaments. The basal half of the mesothelium is occupied by myoepithelial cells, whose contractile processes are organized into longitudinal, circular, or oblique gut musculature. The mesothelium also contains a prominent nervous plexus. Cell bodies and processes of nerve cells are also occasionally observed in the luminal epithelium and connective tissue of the gut, but they are much less abundant there than in the mesothelium (Feral and Massin, 1982; Garcia-Arraras et al., 2001; Mashanov et ai, 2004).

Physiological regeneration of digestive tube: maintaining tissue homeostasis

The normal functioning of the digestive tube is associated with cells being damaged and worn out. Cells undergoing programmed cell death occur in all regions of the digestive tube and are more numerous in the luminal epithelium than in other tissue layers of the gut wall (Mashanov et ai, 2010). Like many other animals, holothurians are capable of replacing the cells that get lost or worn out in the course of normal functioning of the digestive tube. Unlike the proliferating cells in mammals, which are restricted to narrow compartments at the bottom of the crypts of the intestinal epithelium (Crosnier et ai, 2006), mitotic cells of the sea cucumber gut are scattered all along the digestive epithelium, without any preferential localization to the basal region of the luminal epithelium or to the bottom of epithelial folds (Leibson, 1986, 1989; Mashanov et al, 2004). Another interesting feature of cellular division in sea cucumbers is that the mitotic cells show morphological features of vesicular enterocytes, the major cell type in the gut luminal epithelium (Mashanov et al, 2004).

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The mesothelium of the digestive tube also shows signs of physiological cell turnover under normal conditions. As in the luminal epithelium, the mitotic cells are rare and seem to be evenly distributed without forming distinguishable clusters (proliferative zones). There is no direct evidence as to the identity of the dividing cells. However, most of the mesothelial cell division occurs in the apical half of the epithelium, which is known to be predominantly occupied by cell bodies of peritoneocytes. Another interesting observation is that some myoepithelial cells of the normal mesothelium show condensation of their myofilaments into compact spindle-like structures (SLSs). It has previously been shown that SLS formation is a hallmark of myocyte dedifferentiation in the regenerating body wall and visceral musculature (Dolmatov and Ginanova, 2001; Mashanov and Dolmatov, 2001; Mashanov et ai, 2005; San Miguel-Ruiz and Garcia-Arraras, 2007; Garcia-Arraras and Dolmatov, 2010). Dedifferentiated myoepithelial cells were shown to be capable of migration and cell division. Therefore, under normal conditions, myoepithelial cells of the mesothelium can undergo dedifferentiation, which could probably reflect involvement of this cell type in some form of plasticity of the mesothelium of the uninjured gut.

The Regenerating Gut

The nature of the injury

Many sea cucumber species of the orders Aspidochirota and Dendrochirota are capable of autotomizing their internal organs in response to certain stimuli (Emson and Wilkie, 1980). This visceral autotomy (evisceration) is an active process that proceeds under the control of the nervous system and involves separation of the anatomical components along predetermined breakage zones (Wilkie, 2001; Byrne, 2001). Therefore, evisceration occurs in a very consistent and repeatable manner, which minimizes the variation among individuals in the extent and severity of the trauma. This high reproducibility of starting conditions at the onset of regeneration makes the re-growing visceral organs of sea cucumbers a particularly convenient and attractive model systems to study various aspects of posttraumatic recovery.

There are two types of evisceration in sea cucumbers (Fig. 3). Posterior evisceration occurs mainly in the Aspidochirota and involves the detachment of the intestine from the esophagus and the cloaca, and also from the supporting mesenteria (Fig. 3A, B). The autotomized region of the digestive tube, along with associated visceral organs such as hemal vessels, gonads, and one or both respiratory trees, is then expelled though the rupture in the cloacal wall (Emson and Wilkie, 1980; Garcia-Arraras et al., 1998; Wilkie. 2001). The second type of visceral autotomy, anterior evis-ceration, is a characteristic feature of the order Dendrochirota. It results in a more extensive tissue loss than the posterior evisceration (Fig. 3C, D), since not only the intestine but the whole anterior body end of the animal is discarded, including the tentacles and the pharyngeal bulb (Dolmatov, 1992; Byrne, 2001; Mashanov et al.. 2005). Eviscerated animals, if kept under good conditions, almost invariably survive and completely regenerate their viscera. For a more detailed account on the mechanisms of evisceration and associated structural changes in the involved tissues, the reader is referred to Byrne (1986, 2001) and Wilkie (2001).

Early regenerate

After evisceration, the animals are left with the mesentery attached to the body wall and one (Dendrochirota) or both (Aspidochirota) terminal fragments of the digestive tube (Fig. 3B, D). The earliest response to injury within the first few days involves wound closure at the anterior and posterior ends of the body and a remarkable reorganization of the mesentery. The latter undergoes significant extension in width, especially in those regions where it angles and loops. The net result of this differential growth is the strengthening and shortening of the free margin of the mesentery. This obviously enables the animal to regenerate the lost segment of the gut between the ends of the body much faster and to commence feeding much earlier than if the digestive tube had to regenerate along its original curved path (Dawbin, 1949: Mosher, 1956). At the tissue level, the reorganization of the mesentery involves extensive dedifferentiation of the mesothelium, which initially starts at the distal free margin (Mashanov et al., 2005; Candelaria et al., 2006; Garcia-Arraras et al., unpubl.). The dedifferentiating mesothelium undergoes drastic simplification in its architecture, with both peritoneal and myoepithelial cells forming a simple epithelial layer of irregularly shaped cells (Fig. 2D). The change in shape of the mcsothelial cells occurs concomitantly with the remodeling of their cytoskeleton. The dedifferentiated myoepithelial cells undergo condensation of their myofilaments into SLSs. The peritoneal cells cleave their long bundles of intermediate filaments into smaller fragments. Both SLSs and fragmented bundles of intermediate filaments can remain within the cytoplasm and there-fore can serve as natural markers helping to trace the developmental pathways of the coelomic epithelial cells. Alternatively, the SLSs are occasionally discarded by the dedifferentiated myoepithelial cells into the coelom or the underlying connective tissue, where they are scavenged by wandering phagocytes.

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The dedifferentiated cells remain connected to each other by intercellular junctions, but the underlying basal lamina is often discontinuous or not visible at all. The mcsothelial cells covering the free edge of the mesentery develop pseudopodium-like protrusions, detach from the epithelium, and invade the underlying connective tissue. As the immigrating cells accumulate below the epithelial surface (Fig. 4A), collagen fibers start to disappear from the connective tissue near the distal margin of the mesentery, suggesting that some of the ingressing cells are involved in collagen decomposition by phagocytosis or matrix metalloproteinase activity (Garcfa-Arraras et al., unpubl.). Concomitantly, the width of the connective tissue layer at the free edge of the mesentery increases, resulting in the formation of the early gut primordium, which develops as a solid thread-like connective tissue thickening in the free edge of the mesentery and is covered by dedifferentiated coelomic epithelium. Depending on the species, this swelling either appears along the entire length of the mesenteric margin at once (Dawbin, 1949; Mosher, 1956; Bai. 1971) or initially originates as two separate rudiments at the anterior and posterior terminal regions of the mesentery adjacent to the healed autotomy breakage points (Kille, 1935; Leibson, 1992; Garcfa-Arraras et ai, 1998; Mashanov et ai, 2005).

As formation of the intestinal rudiment continues, dedifferentiation spreads to other areas of the mesentery in a distal (free margin) to proximal (body wall) gradient. Notably, as regeneration progresses, the area of the mesentery devoid of differentiated myoepithelial cells increases, and the SLSs appear in the mesothelial cells closer and closer to the body wall. Collagen degradation, evidenced by disappearance of the fibers from the mesenteric connective tissue, occurs in a similar distal-to-proximal pattern. Nerve fibers in the mesentery appear disorganized, and those within the developing early rudiment seem to undergo degeneration following the same gradient seen in the mesothelial dedifferentiation and collagen degradation (Tossas, unpubl.).

Origin of the luminal epithelium

The regeneration mechanisms of the luminal epithelium are largely determined by the mode of evisceration. Species of the family Aspidochirota retain the most anterior (esophagus) and the most posterior (cloaca) segments of the ali-mentary canal after autotomy (Fig. 3B). Therefore, after wound closure separates the remnants of the gut lumen from the coelomic cavity and the solid rod-like swelling develops in the free edge of the mesentery (Fig. 4A, Fig. 5A), the two stumps of the digestive tube retain the typical trilaminar organization of the gut wall and give rise to the anterior and posterior blind tubular outgrowths (Garcfa-Arraras et al,. 1998). The enterocytes of the luminal epithelium undergo partial dedifferentiation: they detach from the basal lamina, become shorter and irregularly shaped, and lose most (al-though not all) of their characteristic secretory vacuoles. Nevertheless, the luminal epithelium always maintains its integrity, since the dedifferentiated cells remain joined by intercellular junctions (Shukalyuk and Dolmatov, 2001; Odintsova et al., 2005). The dedifferentiated enterocytes become capable of active cell division. The mitotic cells are dispersed throughout the luminal epithelium at the tip of the blind gut rudiments and do not form distinct proliferative zones (Marushkina and Gracheva, 1978; Garcfa-Arraras et ai, 1998; Shukalyuk and Dolmatov, 2001; Odintsova et al., 2005). The two tubular rudiments, as they grow along the free edge of the mesentery, invade the amorphous matrix of the connective tissue thickening until they eventually fuse together to form a new continuous digestive tube (Fig. 5B-D). After regeneration has completed, the basal lamina of the luminal epithelium is restored and the epithelial cells return to their characteristic enterocyte phenotype.

Regeneration of the luminal epithelium in adult holothurians of the family Dendrochirota involves a more complex set of events and recruits cells from two different sources. The anterior mode of evisceration results in the loss of the whole anterior end of the animal, including the pharyngeal bulb and the entire digestive tube except for the most posterior terminal part (Fig. 3D). Therefore, the animal loses all its endodermally derived tissues, with the only exception being the luminal epithelium of the cloaca. Shortly after evisceration, a cone-shaped rudiment appears in the free edge of the mesentery attached to the healed oral end of the body. This early primordium consists of a solid rod of connective tissue covered by the mesothelium and it develops through the mechanisms described above. The dedifferentiated mesothelium on the anti-mesenterial side of the rudiment folds to form deep invaginations into the amorphous interior connective tissue (Fig. 4B). The epithelial lining of the folds eventually detaches from the mesothelium on the rudiment surface and reorganizes itself to form a single blind lumen lined with a newly formed digestive epithelium derived from the mesothelium (Fig. 4C). These morphogenetic movements are accompanied by direct transformation of the mesothelial cells into typical enterocytes. The SLSs and bundles of intermediate filaments disappear from the cytoplasm of dedifferentiated myoepithelial and peritoneal cells, respectively. The cells become columnar in shape and develop a prominent Golgi complex and secretory vacuoles. Concomitantly with the transdifferentiation events, cell division continues, and the newly created anterior rudiment grows along the free edge of the mesentery (Mashanov et al., 2005). The posterior regions of the gut regenerate in the same way as in aspidochirotids--that is, the endodermally derived luminal ep-ithelium of the cloaca grows along the connective tissue thickening of the mesentery.

[FIGURE 4 OMITTED]

Role of cell division and cell death

Cell division and cell death are the two processes that control the number of cells in a multicellular organism. Although the balance between them is known to be always under tight control, it can be shifted to meet the needs of tissue homeostasis, growth, and regeneration. Until now, cell proliferation and apoptosis have been extensively studied in only one sea cucumber species, the aspidochirotid Holothuria glaberrima (Garcfa-Arraras et al., 1998; Mashanov et al., 2010). As shown above, physiological cell turn over occurs constantly in the normal digestive tube, and therefore, some dividing and apoptotic cells are always present in the tissues of noneviscerated animals. In the luminal epithelium, both the mitotic and apoptotic cells are more abundant in the anterior regions (esophagus) of the digestive tube. Evisceration triggers a burst of cell division in both the digestive epithelium and mesothelium. which reaches its maximum at the stage of growth of the anterior and posterior rudiments toward each other before returning to the normal values (Garcfa-Arraras et ai, 1998). Cell division also takes place in the mesentery that attaches the regenerate to the body wall. And, as shown for other events, the distribution of dividing cells follows a distal-to-proximal pattern. That is, the levels of cell division are much higher in the mesothelium of the mesentery closer to the growing rudiment than in the mesothelium closer to the body wall. There is also an increase in cell division in the cells within the connective tissue layer of the mesentery; these dividing cells are probably the progeny of dedifferentiated mesothelial myoepithelial cells (Garcfa-Arraras et al., unpubl.).

[FIGURE 5 OMITTED]

Although it can be intuitively perceived that regeneration will shift the balance between cell division and cell death in favor of cell division, induction of apoptosis has been shown to be equally important for the success of posttraumatic regeneration and could be an absolute requirement for tissue regrowth to be initiated (Tseng et al., 2007; Li et al.. 2010). Interestingly, it is the mesothelium of the regenerating digestive tube that shows the most significant changes in the number of apoptotic cells (at least in H. glaberrima) (Mashanov et al., 2010). These alterations are time-dependent and roughly follow the dynamics of cell proliferation; that is, evisceration triggers a sharp increase in the percentage of TUNEL-positive cells, which remains high during the stages of dedifferentiation and growth of the anterior and posterior rudiments and then starts to return to normal values. Surprisingly, no significant time-dependent variations in the rate of cell death were observed in the luminal epithelium of the regenerates.

Regeneration and development

Regeneration is defined as a process of secondary (postembryonic) development of an injured or autotomized organ or structure. Therefore, since the same structure is created as an outcome of both regeneration and embryogenesis, regeneration is often stated to involve a reactivation of developmental mechanisms. On the other hand, one cannot expect regeneration to be an exact reproduction of developmental programs, since regeneration always involves unique processes, such as wound healing and dedifferentiation, which have no counterpart in embryonic development. The question of the degree to which regeneration recapitulates embryonic development has been debated in the literature for decades (Carlson, 2007; Brockes and Kumar, 2008), and the answer seems to be different in each particular case. As to the digestive tube, there are interesting parallels between normal development and regeneration of this organ in sea cucumbers.

The free-swimming auricularia larva of indirectly developing holothurians possesses a well-differentiated digestive tube consisting of esophagus, muscular stomach, intestine, and rectum. During metamorphosis, the larval anus (the derivative of the blastopore) closes, and the larval rectum and intestine undergo resorption. The new intestine begins to form at the posterior end of the stomach and then fuses with the ectoderm to form a new anus (Smiley, 1986; Malakhov and Cherkasova, 1992). In most holothurians with accelerated metamorphosis (i.e., in species that do not form a feeding auricularia larva) the process of developmental degeneration of the primary posterior intestine is much less drastic, but still involves the closure of the primary anus and later fusion of the growing primordial intestine with the body wall to form the secondary anus (Chia and Buchanan. 1969; Ivanova-Kazas, 1978; Dolmatov and Yushin, 1993). It is worth mentioning here that the primary blastopore does not close in the holothurian Cucumaria japonica and becomes the anus of the adult animal (Mashanov and Dolmatov, 2000). Intriguingly this species is not capable of gut regeneration at any of the stages of its life cycle (Dolmatov, 1994). Therefore, there is a certain degree of similarity in gut morphogenesis between larval metamorphosis and adult regeneration. In both cases, a part of the old digestive tube is lost and the new gut is formed by the outgrowth of the stump. Unfortunately, neither cell sources nor molecular mechanisms are known for the larval gut transformation during metamorphosis. Therefore, its direct comparison with regeneration is not yet possible.

Dendrochirotid holothurians provide an example of the most extreme deviation of gut regeneration from the development of this organ. In eviscerated adult individuals, the mesodermally derived mesothelium crosses germ-layer boundaries to give rise to the luminal epithelium of the anterior regenerate, which, in embryogenesis, develops from the endoderm. Nevertheless, even in this case regeneration is somewhat redolent of development, although not of the digestive tube itself, but of the longitudinal muscles of the body wall. In developing muscle, the mesothelium also develops deep invaginations, which penetrate far into the underlying connective tissue and then detach from the epithelium covering the surface of the rudiment to form blind cavities delimited by epithelial cells, which later differentiate into myoepithelial cells (Dolmatov and Ivantey, 1993). Interestingly, regeneration of the longitudinal muscle bands after transection employs a similar mechanism (Dolmatov and Ginanova. 2001; Garcfa-Arraras and Dolmatov, 2010). Therefore, it can be hypothesized that mechanisms of generation of muscle bundles in development and regeneration through infolding and detachment of stretches of the mesothelium are partly co-opted by gut regeneration.

Another interesting aspect of relationships between normal development and regeneration is how regenerative capacities change at different times throughout the life cycle. Post-traumatic regeneration is usually studied in adult organisms, when most of the processes of normal development have been completed. Few researchers have compared this adult pattern of repair with recovery at earlier stages, when regeneration per se is accompanied by continuation of embryonic mechanisms. Overall, the regenerative capacity in most organisms is thought to decline with increasing age (Carlson, 2007), but this is not necessarily the case for sea cucumbers, which show a great diversity of species-specific relationships between regenerative capacities and age (Dolmatov, 1994; Dolmatov and Mashanov, 2007). The dendro chirotid holothurian Eupentacta fraudatrix provides one of the most interesting examples of differences in regenerative response between stages of its life cycle. Those differences involve not only quantitative aspects, such as completeness and the rate of recovery, but also variations in the types of morphogenic processes involved and the nature of cell sources recruited during the repair (Dolmatov, 1994; Mashanov and Dolmatov, 2001). Adults of this species undergo anterior evisceration and regenerate their luminal epithelium in the "dendrochirotid way" as described above--that is, through transdifferentiation of the mesothelium in the anterior rudiment and through proliferation of the luminal epithelium of the cloacal stump in the posterior part of the body (Mashanov et ai, 2005). The 5-month-old juveniles of this species already show the typical adult body plan, but they are much smaller (1-2 mm in length, com-pared with the adult body size of several centimeters) and are not capable of evisceration (Dolmatov and Yushin, 1993). At this developmental stage, regeneration can be triggered by transverse bisection at about the mid-body level. All the posterior halves eventually die in a few days, while most of the anterior halves survive and quickly regenerate the missing posterior structures, including the lost regions of the digestive tube (Mashanov and Dolmatov, 2001). Surprisingly, unlike adult individuals, juveniles of E. fraudatrix show a typical "aspidochirotid mode" of regeneration. After the initial phase of wound closure and histolysis of the most posterior region of the stump, the luminal epithelium and the mesothelium of the remaining anterior segments of the gut undergo typical dedifferentiation and give rise to the corresponding tissue layers of the missing parts of the alimentary canal, without any transdifferentiation events (Mashanov and Dolmatov, 2001).

The nature of sources of new cells in regeneration.

The central question in any study of animal regeneration is the nature of the cells that are recruited to repair the injury. The usual dichotomy is between involvement of some kind of undifferentiated reserve/progenitor cells as opposed to local plasticity of differentiated cells. Reparative processes of both kinds are known to occur without any particular correlation with taxonomic position and even within the same organism (Carlson, 2007; Gurley and Sanchez Alvarado, 2008; Brockes and Kumar, 2008). Initially, the rapidity of visceral regeneration in sea cucumbers and the accumulation of mesenchyme-like cells in the early connective-tissue primordium led researchers to the hypothesis of involvement of wandering pluripotent neoblast-like cells in the formation of the luminal epithelium of the regenerate (Kille, 1935; Leibson, 1980, 1992). However, upon extensive reexamination, it was shown that the lumen of the regenerating gut is always formed by epithelial morphogenesis either by the expansion of the luminal epithelium of the stump or by transdifferentiation of the mesothelium (Garcfa-Arraras etal., 1998; Shukalyuk and Dolmatov, 2001; Mashanov et ai, 2005). Electron microscopy studies (Shukalyuk and Dolmatov, 2001; Mashanov and Dolmatov, 2001; Mashanov et al, 2005; Odintsova et ai, 2005) showed that the regenerative capacities of the gut wall epithelia are largely based on the plasticity of the epithelial cells. These cells perform specialized functions within the epithelia, but their differentiated state, although stable, is not irreversible, since they are capable of undergoing dedifferentiation by losing their specialized features and entering the cell cycle. Nevertheless, the integrity of the epithelial sheets is always retained, because the dedifferentiated cells remain connected to each other by intercellular junctions. The enterocytes of the luminal epithelium undergo only partial dedifferentiation: they often retain some of their microvilli and secretory vacuoles, even during the cell division phase. In contrast, deep dedifferentiation in the mesothelium results in drastic simplification of the epithelial organization and the complete loss of phenotypic characteristics in the peritoneal and myoepithelial cells. It can be hypothesized, therefore, that this deep level of dedifferentiation is one of the key factors that allows the mesothelium not only to regenerate itself, but also to reprogram its cells into myocytes of the longitudinal muscle band and enterocytes of the digestive epithelium in dendrochirotids.

Although available microscopic data identify the dividing cells in the holothurian gut as specialized cells (enterocytes in the luminal epithelium and peritoneal and myoepithelial cells in the mesothelium), it is currently unknown whether all differentiated epithelial cells are capable of entering the cell cycle or whether those cells that can proliferate are all equal in their potential. There is also a possibility that at least some of those mitotic cells could represent dividing progeny of rare and more quiescent stem cells. Unfortunately, lineage relationships in the tissues of echinoderms have never been a subject of rigorous study, and no attempts have been made to establish cell fate maps in the holothurian digestive tube. One of the most basic techniques, which can be tried to tentatively explore the histogenetic relation-ships in the gut wall epithelia, is the label-retaining cell (LRC) approach. This method involves labeling of DNA-synthesizing cells with a thymidine analog (BrdU, for example) followed by a long chase period, and it is based on two assumptions. First, the stem cells are expected to divide much less often than their progeny, which eventually give rise to differentiated ceils of the tissue. Therefore, the stem cells will retain the DNA synthesis marker (will remain strongly BrdU-positive), while the differentiating progeny will, by dividing more frequently, dilute the labeling beyond the detection limit over the chase period. The second theoretical concept behind the label-retaining approach is the "immortal strand hypothesis," which predicts that, because a stem cell divides asymmetrically, the renewing daughter stem cell inherits the chromatids with the older DNA strands, while the newer template strands are segregated to the differentiating progenitor daughter cell (Cairns, 1975; Conboy et al., 2007). Stem cells also occasionally undergo symmetric cell division. If a thymidine analog is available during the S-phase preceding such a division, both daughter stem cells will be labeled, and they will remain strongly labeled regardless of how many cell divisions they go through. In our experiments (Mashanov et al., unpubl.), in order to label potential slow-cycling cells in the normal digestive tube, we injected BrdU (50 mg/kg body weight) into the coelomic cavity of adult non-eviscerated individuals every 12 h for 7 days. The animals were sacrificed 4 h, 2 weeks, and 5 weeks after the last injection. The saturating BrdU injections resulted in strong labeling of many cells in the digestive epithelium (Fig. 6A). However, after 2-5 weeks of the chase period, very few BrdU-positive cells remained in the luminal epithelium. All these cells were strongly labeled, suggesting that no labeling dilution occurred (Fig. 6B). In the gut mesothelium, the initial (4 h after the last injection) number of BrdU-incorporating cells was much smaller than in the luminal epithelium of the esophagus, but 2-5 weeks after the last injection, scattered strongly labeled BrdU positive cells were still found (Fig. 6). Therefore, the presence of the label-retaining cells suggests that although the specialized cells of the holothurian gut wall epithelium are known for their plasticity and the ability to differentiate and enter the mitotic cycle, one cannot rule out the possibility that resident stem cells are involved in tissue homeostasis of normal animals. Unfortunately, no label-retaining experiments have yet been performed in regenerating sea cucumbers. Nevertheless, although the available data suggest that the regeneration of the holothurian gut wall epithelia occurs mostly or entirely due to remarkable plasticity of the differentiated epithelial cells and that participation of any kind of reserve or stem cells seems unnecessary, involvement of resident stem cells in the regrowth of the gut tissue layers remains a theoretical possibility and cannot be ruled out completely, unless the issue is studied directly.

[FIGURE 6 OMITTED]

An interesting aspect of regenerative biology highlighted by the examples of sea cucumber visceral regeneration is that some animals exhibit a certain kind of redundancy in their regeneration mechanisms. Regeneration of the same structure can be accomplished in more than one way, from different sources, and through different mechanisms. In adult individuals of the dendrochirotid Eupentacta fraudatrix, once regeneration is complete the luminal epithelia of the anterior and posterior intestine are indistinguishable in their histological organization despite the different origins of the cells (Mashanov et al., 2005). Moreover, there are fundamental differences in regeneration mechanisms of the luminal epithelium between the anterior gut rudiment of the adult animals and the corresponding part of the digestive tube in 5-month-old juveniles (Mashanov and Dolmatov, 2001; Mashanov et al., 2004). Such a redundancy of regeneration mechanisms seems to be necessary for successful regeneration at different life stages, in different starting conditions, from different types of injury. This phenomenon is not unique to sea cucumbers but is observed even in higher vertebrates. In adult mammals, the liver has the most prominent regenerative capacity, and the way it regenerates depends on the type and severity of injury (Michalopoulos, 2009; Kung et al., 2010). After acute injury, such as partial hepatectomy, the tissue mass is restored via division of mature hepatocytes, the major functional cells of the liver. However, if the proliferative potential of hepatocytes is not sufficient to restore the organ subjected to massive or chronic injury, or when proliferation of hepatocytes is inhibited, the facultative liver progenitor cells, called oval cells, are recruited in the re-growth process.

Gene expression studies

Efforts to characterize the genetic basis of regenerative processes in echinoderms began during the last decade. The initial strategy focused on a gene-by-gene analysis, where the targeted genes were usually candidate genes known to be associated with regenerative or developmental processes in other organisms. By using this approach, members of the Bmp, TGF-beta, and Hox gene families were found to be associated with arm regeneration in the crinoids Antedon bifida and Antedon mediterranea (Thorndyke et al., 2001; Patruno et al., 2002), and eventually a novel Bmp member was identified in regenerating arms of the brittle star Amphiura filiformis (Bannister et al., 2005, 2008). In the sea cucumber Holothuria glaberrima, two genes associated with intestinal regeneration--namely serum amyloid protein A (Santiago et al, 2000) and ependymin (Suarez-Castillo et al, 2004)--were identified with the aid of similar approaches. However, for a long time the molecular basis of echinoderm regeneration was limited to the characterization and study of this handful of genes.

In recent years expressed sequence tag (ESTY genomic techniques have been increasingly applied to nontraditional model systems, in some cases even before a complete genome became available. Hundreds of genes associated with regenerative responses have been identified in model systems. Thus, the molecular basis of regeneration could be studied in amphibians (Habermann et al., 2004; Putta et al., 2004; Smith et al, 2005; Monaghan et al, 2007, 2009; Pearl et al, 2008), zebrafish (Lien et al, 2006; Andreasen et al, 2006; Cameron et al, 2005; Nakatani et al, 2007; Sleep et al, 2010), ascidians (Azumi et al, 2003, 2007; Rinkevich et al, 2007, 2009), planarians (Sanchez Alvarado and New-mark, 1999; Nakazawa et al, 2003; Sanchez Alvarado and Tsonis, 2006; Rossi et al, 2007; Said et al, 2009) and Hydra (Stout et al, 2007; Chera et al, 2006; Galliot et al., 2006, 2007; Chapman et al, 2010). However, echinoderm molecular studies first targeted the sea urchin (Sea Urchin Genome Sequencing Consortium, 2006), which ironically is the echinoderm group with the least regenerative capacities (Dubois and Ameye, 2001; Candia Carnevali, 2006).

At present, the only echinoderm in which EST and genomic techniques have been applied to characterize the molecular basis of regeneration is the sea cucumber H. glaberrima. Most of the gene sequences described originate from three cDNA libraries of two stages of regenerating intestine (3-dpe and 7-dpe) and normal line viscerated intestine (Rojas-Cartagena et al, 2007). Over 7000 ESTs were obtained from this effort, many of which were identified by their similarities to gene sequences in databases. In addition, sequences were found that apparently codified for novel, previously undescribed, genes. Moreover, a comparison among the sequences in the three libraries produced a listing of genes that were differentially expressed at certain regenerative stages, most of which were validated using PCR.

In a subsequent series of experiments, the EST sequences were tested in a custom-made microchip to compare the gene expression profile at three regeneration stages (3-, 7-and 14-days post-evisceration) and normal, uneviscerated intestines (Ortiz-Pineda et al, 2009). The results from the microarray experiments were surprising in view of the sheer number of sequences found to be differentially expressed. Depending on the statistical rigor used to analyze the differences in gene expression, the percentage of differentially expressed genes ranged from 39% (at P < 0.001) to 73% (at P < 0.05). These results caused a drastic change in the field of echinoderm regeneration; from having a few genes associated with regeneration, we now have a plethora of genes that show differential expression during regeneration of the holothurian intestine.

Two main hurdles remain. First, one needs to determine where and when the genes are expressed. This question is being addressed with techniques such as immunohisto chemistry and in situ hybridization, which will bring the relatively large amount of information on the cellular events that occur during regeneration together with the newly obtained molecular data. It would help to clearly establish the cell types and cellular events that are associated with regeneration-specific changes in gene expression, as exemplified in recent experiments focusing on two cancer-related genes, survivin and mortalin (discussed below). Second, knowing the spatiotemporal expression pattern is not enough, since the function of the gene product must be probed directly to determine its role in the regeneration process. Some information has been obtained by using pharmacological tools targeted at some of the gene products. For example, to explore the possible role of matrix metalloproteases in intestinal regeneration, enzyme inhibitors were administered to regenerating animals, resulting in a disruption of the intestinal regeneration process (Quinones et al, 2002). Nonetheless, this type of experiment is limited to those genes or gene products for which activators or inhibitors can be obtained to modulate their activity. Thus, efforts focused on developing an RNA interference procedure for echinoderms, where specific gene sequences can be targeted and their functional role determined, should be encouraged and will be essential to identify genes involved in intestinal regeneration.

In spite of the problems that still need to be solved, enormous progress has been made in establishing the molecular basis of intestinal regeneration. Some of the differentially expressed ESTs are homologs of genes known to be involved in regeneration-related processes--wound healing, cell proliferation, differentiation, morphological plasticity, cell survival, stress response, pathogenic insult, and neoplastic transformation. At the same time, some of these genes can be linked with some known cellular events. The limits imposed by this review prevent us from providing a complete list of the genes and gene pathways associated with intestinal regeneration. Instead, we present a few examples of genes associated with intestinal regeneration that are being explored in depth using the holothurian model. The three examples shown below integrate the information available on some gene expression patterns and the cellular events with which they have been associated, thus providing a cellular/molecular scenario that can be explored in future studies.

Genes associated with intestinal rudiment formation

Several developmental genes have been found to be implicated in the formation of the intestinal rudiment early in the regeneration process. Principal among these are those associated with the Wnt and BMP signaling pathways. A Wnt 9/14 homolog was identified from our EST library and shown by both microarray and PCR to be overexpressed very early during intestinal regeneration (Ortiz-Pineda et al., 2009). Recent unpublished in situ hybridization results (Mashanov et al, unpubl. data) show that the Wnt transcript is expressed in cells of the coelomic epithelium of the intestinal rudiment. Wnt expression has been increasingly associated with regenerative phenomena in a multitude of species. Wnt has been found to be involved in blastema formation in the regenerating limbs and tails of tadpoles (Yokoyama et al, 2007, Lin and Slack, 2008), in lens regeneration in newts (Hayashi et al, 2006), and in zebrafish fin regeneration (Tal et al, 2010). In mammals, Wnt has been studied in bone (Kim et al., 2007), hair follicle (Ito et al, 2007), and deer antler regeneration (Mount et al, 2006) among others. Liver regeneration was retarded in the absence of beta-catenin, one of the proteins in its signaling pathway (Tan et al, 2006). Wnt apparently plays a key role in the control of intestinal stem cell proliferation and differentiation (Yen and Wright, 2006; Clarke and Meniel, 2006). Wnt pathways have also been involved in invertebrate regeneration models. In planarians, Wnt is necessary for proper brain pattern formation (Kobayashi et al, 2007) and beta-catenin for anteroposterior axis formation during regeneration (Gurley et al, 2008; Petersen and Reddien, 2008). Finally, in Hydra, Wnt has been associated with head regeneration (Galliot and Chera, 2010). In the formation of the intestinal rudiment of H. glaberrima, our working hypothesis is that Wnt is involved in the ingression of cells from the overlying coelomic epithelium to form mesenchymal cells of the underlying connective tissue in the growing rudiment. Such a role is consonant with the role that Wnt has been shown to play in epithelium-mesenchyme transitions both during normal development and during pathological transformations.

A Bmp homolog has also been identified in our EST library and shown by both microarray and PCR to be overexpressed during intestinal regeneration (Ortiz-Pineda et al, 2009). Preliminary in situ hybridization data (Mashanov et al, unpubl.) show that Bmp-1 transcripts are expressed in the mesothelium of both the gut rudiment and the supporting mesentery in an asymmetric way; that is, they are present on one side of the rudiment but not on the other. The functional significance of this expression is not yet known. Although BMPs have received less attention than the Wnts in regenerative biology, they have been found to be active in tail regeneration in tadpoles (Beck et al, 2003), limb regeneration in axolotl (Guimond et al, 2010), fin and liver regeneration in fish (Smith et al. 2006), digit regeneration in mice (Han et al, 2005), lens regeneration in newt (Grogg et al, 2005), and regenerative processes in planaria (Adell et al, 2010). It is well known that in developing embryos there are interactions between Wnt and BMP pathways (Brandhorst and Klein, 2002; Rubin, 2007), thus suggesting that these interactions can also be important in regenerating tissues and organs.

[FIGURE 7 OMITTED]

Genes associated with muscle dedifferentiation and myogenesis

Early events in gut regeneration leading to the formation of the intestinal rudiment are characterized by the dedifferentiation of the mesothelial myoepithelial cells. On the other hand, once the intestinal rudiment is formed, myogenesis begins and the circular and longitudinal muscle layers of the new intestine are formed (Murray and Garcfa-Arraras, 2004). Pivotal to muscle dedifferentiation and formation is the modulation of the cytoskeletal filaments that compose its contractile apparatus. Thus, the finding that two actin and three myosin isoforms are differentially expressed during intestinal regeneration becomes highly relevant (Ortiz-Pineda et al, 2009). Changes in the abundance of actin and myosin isoforms have been shown in developing organisms (Buckingham et al, 1986; Eddinger and Murphy, 1991), including differential isoform expression in the smooth muscle of the digestive tract (Ayas et al, 1995). Therefore, a transition in actin and myosin isoforms also appears to occur during intestinal regeneration. This finding is supported by previous results from our group where an actin isoform was identified in the regenerating intestine by using a differential display technique (Roig-Lopez et al., 2001). Northern blots experiments demonstrated that transcripts for this actin showed a preferential increase during the later stages of regeneration, when the intestinal muscle is forming (Roig-Lopez, 2002).

Cancer-related genes

Analysis of the EST database showed the presence of a large number of cancer-related genes in the regenerating gut tissues, many of which were found to be significantly over-expressed during regeneration in the microarray experiments (Ortiz-Pineda et al 2009). Among these genes are TCTP, NM23, melanotransferin, survivin, and mortalin. In fact, two of the cancer-related genes, survivin and mortalin, were found to be expressed by most of the dedifferentiated mesothelial cells (Fig. 7A) and by some cells in the regenerating digestive epithelium (Mashanov et al, 2010). These genes are known to have a strong anti-apoptotic effect (Fig. 7) (Wadhwa et al, 2002; Marusawa et al, 2003; Mashanov et al, 2010), and their high expression in human cancers usually correlates with more aggressive tumor phenotypes (Li, 2003; Wadhwa et al, 2006; Kaul et al, 2007; Mita et al, 2008; Yi et al, 2008).

The question is, why are cancer-related genes expressed during regeneration? Although this finding may seem surprising at first glance, in retrospect it should have been expected, since many of the hallmarks of cancer, including extensive cell division, resistance to apoptosis, increased cell motility, and ability to invade other tissues (Hanahan and Weinberg, 2000), are processes that also occur during intestinal regeneration. During gut regeneration, the specialized epithelial cells dedifferentiate, re-enter the cell cycle, and migrate either as an epithelial sheet or as single cells.

Mortalin and survivin are mostly absent from the adult tissues, but are extensively expressed in embryonic development and, notably, in stem cells (Adida et al, 1998; Ma et al, 2007; Marconi et al, 2007; Delvaeye et al, 2009; Li et al, 2010). For instance, mortalin was shown to be constitutively expressed in planarian neoblasts, and its experimental silencing resulted in inability to regenerate and maintain the normal cell turnover (Conte et al., 2009). Survivin expression was documented in stem cells of mammalian tissues undergoing extensive physiological cell replacement or posttramatic repair (Marconi et al., 2007; Li et al, 2010). Taken together, these data suggest that the cells of the holothurian mesothelium can temporarily ac-quire some stem cell properties through reversible dedifferentiation. Those properties include the absence or reduction of specialized cytoplasmic features, self-renewal through cell divisions, and expression of survivin and mortalin. It is worth mentioning here that in vitro studies of the cells derived from sea cucumber visceral regenerates showed that only the cells obtained during the phase of extensive dedifferentiation and proliferation were capable of sustained growth in culture (Odintsova et al., 2005).

Since regeneration shares certain similarities with cancer both in morphology and gene expression, why is it that tumor formation has never been reported in studies of visceral regeneration in holothurians or documented in animals captured in the wild? Sea cucumbers are characterized by a long life span, estimated at about 4 to 10 years; they constantly renew cells in their adult tissues, including the digestive tube; and, most interestingly, they can quickly regrow most of their tissues after traumatic injury, autotomy, or seasonal atrophy (Hyman, 1955; Garcfa-Arraras and Greenberg, 2001; Candia Carnevali, 2006) and also regenerate the same structure multiple times over their lifetime. These are all factors that provide opportunities for carcinogenic changes to occur. Nevertheless, visceral regeneration in holothurians always results in a perfect redevelopment of the lost organ without formation of any apparent abnormalities. Therefore, sea cucumbers, and echinoderms in general, must have evolved a particularly strong set of anti-tumor mechanisms, further studies of which could improve our understanding of relationships between embryogenesis, cancer, and regeneration, and might help us to devise more effective strategies for cancer treatment. The absence of tumors in the holothurian gut may be part of a more general phenomenon of resistance of regenerative tissues to tumor formation. For instance, the tissues involved in lens and limb regeneration in urodele amphibians are particularly unlikely (in comparison to non-regenerating parts of the body) to form cancerous abnormalities even when the regrowing structures are treated with chemical carcinogens (Oviedo and Beane, 2009).

In summary, visceral regeneration in sea cucumbers provides a promising system in which to seek answers to the most fundamental questions of regenerative biology, such as relationships between regeneration on one hand and embryogenesis, normal cell turnover, and carcinogenesis on the other; changes in regenerative abilities with age; and the nature of the cell sources for regeneration. In this review we have summarized the recent data that help us better understand the above phenomena. Taken together, the available data suggest that the extraordinary regenerative potential of holothurian visceral organs is mostly due to the ability of specialized cells to dedifferentiate and rebuild the lost structures through proliferation and migration. Analysis of molecular mechanisms underlying the regenerative response revealed involvement of Wnt and Bmp pathways in the formation of gut rudiment, as well as extensive expression of cancer-related genes.

Acknowledgments

The authors are grateful to Olga R. Zueva for technical help and critical reading of the manuscript. They also thank the many members of the JEGA laboratory (past and present) whose labor and ideas have been critical in advancing our knowledge on the holothurian model system. The work was supported by NIH (grant number: 1SC1GM084770-01), NSF (grant number: IOS-0842870), and the University of Puerto Rico.

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V. S. MASHANOV AND J. E. GARCIA-ARRARAS (*)

Department of Biology, University of Puerto Rico, PO Box 70377, San Juan, Puerto Rico 00936-8377

Received 18 January 2011; accepted 25 February 2011.

* To whom correspondence should be addressed. E-mail: jegarcia At hpcf.upr.edu

Abbreviations: BrdU, 5-bromo-2-deoxyuridine; EST, expression sequence tag; LRC, label-retaining cell; SLS, spindle-like structure; TUNEL, deoxynucleotidyl transferase-mediated dUTP nick end labeling.
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Author:Mashanov, V.S.; Garcia-arraras, J.E.
Publication:The Biological Bulletin
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Geographic Code:1U0PR
Date:Aug 1, 2011
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