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Transdifferentiation in holothurian gut regeneration.

Introduction

There are two hypotheses for the origin of progenitor cells involved in regeneration (Brockes, 1998). The first possibility is that differentiated cells in the vicinity of the wound undergo a reversal of their differentiated state, reenter the cell cycle, and then re-differentiate or transdifferentiate. On the other hand, a growing body of reports suggests the existence of undifferentiated reserve cells, or stem cells, that can produce the whole spectrum of cell types (Brockes, 1998; Carlson, 1998; Weissman, 2000; Filip et al., 2004). However, in spite of the presence of stem cells in the tissues, mammals, for instance, are not capable of regenerating most of their organs. Thus, understanding the mechanisms that underlie cell plasticity and transdifferentiation will open up promising possibilities for the development of new approaches in biology and medicine (Reik and Dean, 2003). The best way to study transdifferentiation is to find a model in which this process takes place naturally.

Most echinoderms have a remarkable capacity for regeneration of lost body parts (Vickery and McClintock; 1998; Dolmatov, 1999; Candia Carnevali and Bonasoro, 2001a, b), and many can reproduce asexually (Emson and Wilkie, 1980; Eaves and Palmer, 2003). They therefore show histogenetic plasticity, exemplified by the capability of completely differentiated tissues to re-enter a new developmental process at any life stage. One of the best known cases of echinoderm regeneration is the regrowth of new internal organs in holothurians following evisceration or experimental transection (Garcia-Arraras and Greenberg, 2001). Two distinct mechanisms of gut regeneration have been described in holothurian species studied so far. When regeneration is accomplished through the first mechanism, the luminal epithelium appears to originate from the endodermally derived lining epithelia of gut remnants that have not been eliminated by evisceration or experimental ablation (Bertolini, 1932; Smith, 1971a; Gibson and Burke, 1983; Garcia-Arraras et al., 1998; Shukalyuk and Dolmatov, 2001). In some species, however, endodermally derived tissues are either completely lost by evisceration or, if still present in the gut remnants, do not contribute to gut regeneration. It is generally believed that in these animals the luminal epithelium of the gut originates directly from mesodermally derived cells (Bertolini, 1930; Dawbin, 1949; Mosher, 1956; Leibson, 1992). Although it has been proposed that the process of regeneration involves mesodermally derived cells from the lining epithelium of the water vascular system or perivisceral coelomic epithelium (mesothelium), or even from neoblast-like reserve cells, the exact source of the progenitor cells has not been identified so far because no specific techniques to follow and reconstruct the developmental pathways of cells have been employed, and even electron microscopy has rarely been used in studies of visceral regeneration in holothurians (Garcia-Arraras and Greenberg, 2001).

Of particular interest for studying mechanisms of gut regeneration are dendrochirotid holothurians, since they, in contrast to aspidochirotids and apodids, lose almost all endodermal tissues during evisceration (Kille, 1935; Tracey, 1972; Leibson and Dolmatov, 1989; Dolmatov, 1999). The dendrochirotid holothurian Eupentacta fraudatrix has been extensively used as a model organism in studies of morphology (Dolmatov, 1986a, b, 1995; Mashanov et al., 2004; Zueva et al., 2004), development (Dolmatov and Ivantey, 1993; Dolmatov and Yushin, 1993; Mashanov and Dolmatov, 2001a, 2004), and regeneration of various organs (Leibson and Dolmatov, 1989; Dolmatov, 1992, 1994; Leibson, 1992; Dolmatov and Eliseikina, 1998; Mashanov and Dolmatov, 2001b). Like many other dendrochirotids studied so far, adult individuals of E. fraudatrix can expel the entire digestive tube (except the cloaca) along with the pharyngeal complex through the rupture of the anterior body wall (Leibson and Dolmatov, 1989). Leibson and Dolmatov (1989) and Leibson (1992) provided a detailed description of the microscopic aspects of evisceration and visceral regeneration in E. fraudatrix. They also gave a brief account of the histological structure of the regenerating gut tissues. Since the bulk of endodermal tissues (except the cloaca) were expelled during evisceration, the authors proposed that non-endodermal cells might contribute to the regrowth of a new luminal epithelium; however, the source of these cells was not identified.

In the present study, the cellular and tissue processes involved in gut regeneration were investigated by employing light and electron microscopic analysis to trace the origin of the luminal epithelial cells in the regenerating gut of the holothurian Eupentacta fraudatrix (Djakonov et Baranova, 1958).

Materials and Methods

Adult individuals of Eupentacta fraudatrix (Holothuroidea, Dendrochirota) were collected from Vostok Bay, Sea of Japan. Evisceration was induced by injecting a few milliliters of distilled water into the coelomic cavity. Eviscerated animals were kept in well-aerated seawater aquaria. At various stages of regeneration, the animals were fixed in 2.5% glutaraldehyde in seawater. A small amount of the fixative was injected directly into the coelomic cavity. The animals were then dissected, and the whole body cavity was exposed for a preliminary examination under a stereomicroscope. The rudiments of the digestive tube were isolated from the rest of the body and placed in fresh fixative for 1-7 days at 4 [degrees]C. They were then rinsed for 8 h at 4 [degrees]C in artificial seawater (ASW) adjusted to 1090 mOsm, post-fixed in 1% Os[O.sub.4] in ASW for 1 h at the same temperature, dehydrated in a graded series of ethanol and acetone, and embedded in Araldite. Sections were cut with glass knives, using an Ultracut E (Reichert) ultratome. Semi-thin (0.8 [micro]m) serial sections were collected on gelatin-coated slides, stained with 1% toluidine blue in 1% aqueous sodium borate, and mounted in DPX (Fluka). The slides were examined and photographed with a Jenamed 2 (Carl Zeiss Jena) light microscope equipped with a Leica DC 150 digital camera. Ultrathin (50-70 nm) sections were stained, using an Ultrastainer (Reichert), with aqueous uranyl acetate and lead citrate and examined in a Zeiss EM 10 transmission electron microscope.

Results

Macroscopic observations

Evisceration in the holothurian Eupentacta fraudatrix involves rupture of the introvert (eversible anterior portion of the body wall), the junctions between the pharyngeal retractor muscles and longitudinal muscle bands, and the junction between the intestine and cloaca (Fig. 1A). These ruptures result in the loss of the anterior end of the body, including the tentacles and aquapharyngeal bulb, together with the digestive tube. For a more detailed description of the evisceration in dendrochirotes, see Byrne (2001). Experimental specimens examined immediately after evisceration consisted of the body wall, a cloaca with attached respiratory trees, and the mesentery that served to anchor the viscera to the body wall (Fig. 1B). The following stages of regeneration were established arbitrarily on the basis of observed changes in gross anatomy and histological structure.

Stage 1. Five to seven days after evisceration, an anterior gut rudiment appears in the free edge of the dorsal mesentery at the healed oral end of the body (Fig. 2A). The rudiment has the shape of an inverted cone with a thickened anterior part and a tapering posterior part. The anterior region gives rise to the aquapharyngeal bulb, while the thinner posterior part is the area where the lumen of the new gut starts to develop. The wound at the anterior end of the cloaca appears to be healed, although no sign of a developing posterior rudiment can be seen at this stage.

Stage 2. By day 10 after evisceration, the anterior rudiment increases in diameter and elongates posteriorly in the edge of the dorsal mesentery. At this stage, a posterior rudiment can be clearly detected as a thickening growing anteriorly from the cloaca along the free margin of the ventral mesentery (Fig. 2B).

Stage 3. During this stage (days 10 to 27 after evisceration), the two rudiments grow toward one another. The anterior rudiment occupies the entire length of the dorsal mesentery and extends farther along the free edge of the lateral mesentery (Fig. 2C). At the anterior end of the body, the aquapharyngeal bulb begins to develop all its components: the water-vascular ring, polian vesicle, and minute plates of the calcareous ring can be identified at this stage.

Stage 4. By day 27 after evisceration, the anterior and posterior rudiments have come into contact and eventually fuse, forming a continuous digestive tube (Fig. 2D). The further changes in gut development are represented by less significant events, such as elongation, looping, increase in diameter, and differentiation.

Microscopic anatomy of the regenerating gut

Stage 1. The rudiment that appears at the healed oral end of the body on days 5-7 after evisceration consists of a solid rod of connective tissue covered by the coelomic epithelium (Fig. 3A) that seems to have migrated from the adjacent regions of the body wall (Dolmatov, 1992). In non-eviscerated individuals, this epithelial layer is composed of peritoneal and myoepithelial cells and includes a basiepithelial nervous plexus (Mashanov et al., 2004). Prior to migration, the cells seem to dedifferentiate. The primitive lumen of the new alimentary canal initially arises in a short region of the anterior rudiment lying just posterior to the regrowing aquapharyngeal bulb. In this region, the ventral side of the rudiment is covered by a completely dedifferentiated mesothelium composed of flattened irregularly shaped cells that rest on a ruptured basal lamina (Fig. 4A, B). These cells bear a cilium and rare microvilli. A large nucleus with a prominent nucleolus occupies most of the cell volume. The perinuclear cytoplasm contains 1-3 Golgi bodies, residual bodies, granules of medium electron density, rare mitochondria, and cisternae of rough endoplasmic reticulum (RER). The cytological indicator of dedifferentiation in myoepithelial cells is the grouping of myofilaments into dense spindle-like structures (SLS) (Fig. 4A). In peritoneal cells, bundles of intermediate filaments appear to be broken into short fragments (Fig. 4B). Both SLSs and fragmented bundles of intermediate filaments often remain within the cytoplasm of dedifferentiated cells and therefore can serve as natural markers that may help to trace the developmental pathways of the coelomic epithelial cells. The dedifferentiated mesothelium that covers the ventral side of the rudiment develops deep longitudinal folds that penetrate the underlying connective tissue (Fig. 3A). It is worth noting that although the coelothelial cells have lost their differentiated phenotype, they remain connected to one another via typical intercellular junctions. Moreover, dividing cells are also connected to their neighbors with intercellular junctions and retain SLSs or short bundles of tonofilaments in their cytoplasm (Fig. 4C).

On the lateral sides of the rudiment, the coelomic epithelium recovers its normal histological structure (Fig. 4D, E). This epithelium rests on a continuous basal lamina, consists of clearly distinguishable peritoneal and myoepithelial cells, and includes basiepithelial nerve processes. Peritoneocytes are tall columnar cells whose apical surface lines the coelomic cavity (Fig. 4D) and bears a cilium and well-developed microvilli. The subspherical nucleus shows a prominent nucleolus and occupies the subapical to middle portion of the cell. A Golgi complex lies above the nucleus. The basal cytoplasm contains bundles of intracellular filaments (Fig. 4E). Mitochondria, RER cisternae, residual bodies, and granules of medium electron density are evenly distributed in the cytoplasm. Myoepithelial cells occur at the base of the mesothelium (Fig. 4E). The basal region of these cells is occupied by the myofilaments of the contractile apparatus, whereas the rest of the cytoplasm contains a nucleus, mitochondria, RER cisternae, and granules of medium electron density.

The connective tissue of the rudiment is invaded by masses of various free cells (amoebocytes, spherule cells, and presumptive fibroblasts), which are particularly abundant just underneath the dedifferentiated coelomic epithelium, in the ventral surface of the rudiment (Fig. 4F). Some of these cells contain granules of medium electron density and SLSs in their cytoplasm. Few dividing cells occur within the connective tissue rod.

Stage 2. By day 10 after evisceration, the epithelial lining of the folds detaches from the surface of the anterior rudiment, forming a number of lumina that fuse into a single blind lumen lined with a newly formed luminal epithelium derived from the external mesothelium of the rudiment (Fig. 3B). By that time, SLSs and bundles of tonofilaments disappear from the cytoplasm of the cells. The newly formed digestive epithelium shows the first signs of differentiation: it rests on a continuous basal lamina and the cells become columnar in shape and develop microvilli on their apical surface (Fig. 5A, B). An elliptical nucleus with a prominent nucleolus occupies the central, or basal, region of the cell. A Golgi complex lies above the nucleus. Granules of moderate electron density, phagosomes, mitochondria, and RER cisternae are evenly scattered in the cytoplasm.

At this second stage, a posterior rudiment appears as a rod of connective tissue extending anteriorly from the cloaca along the free edge of the mesentery (Figs. 2B, 3C). The endodermally derived luminal epithelium of the cloaca invades the connective tissue, thereby giving rise to a tubular outgrowth lined with an epithelium (Fig. 3C). This epithelium is composed of vesicular enterocytes whose ultrastructure is only slightly different from that seen in non-eviscerated animals (Mashanov et al., 2004). A nucleus with a prominent nucleolus occupies the basal portion of the cell. The supranuclear cytoplasm contains a Golgi complex, electron-lucent secretory vacuoles, mitochondria, RER cisternae, and residual bodies. The adjacent cells are joined to each other by intercellular junctions. Despite their differentiated phenotype, these cells are capable of mitotic division (Fig. 5C).

Stage 3. The luminal epithelium of the anterior gut rudiment appears to be at an advanced stage of differentiation. This differentiation is morphologically expressed by the appearance of electron-lucent secretory vacuoles in the apical cytoplasm of epithelial cells, indicating the progressive development of these cells into vesicular enterocytes of the new gut (Fig. 5D). Simultaneously with the differentiation, cell division continues and the organ grows in size. The dividing cells are joined to their neighbors by desmosomes and retain secretory vacuoles in their cytoplasm.

Stage 4. At this stage, the morphology of the gut tissues is very similar to that found in non-eviscerated individuals (Figs. 3D, 5E). The luminal epithelium develops longitudinal folds. In vesicular enterocytes, secretory vacuoles are much more abundant than at the previous stage. These vacuoles are densely packed in the apical cytoplasm and often fuse together (Fig. 5E). The subspherical to elliptical nuclei occupy the basal, or central, part of the cells. The nucleoli are smaller and less prominent than at the previous stages. A Golgi complex lies above the nucleus. The granules of medium electron density are no longer present in the vesicular enterocytes.

Goblet cells are the second recognizable differentiated cell type in the luminal epithelium. They are filled with large electron-lucent secretory vacuoles with a fine reticular substructure (Fig. 5E).

Discussion

The wall of the holothurian gut consists of three distinct histological layers--an inner luminal (digestive) epithelium and an outer coelomic epithelium (mesothelium) separated by a layer of connective tissue (Feral and Massin, 1982; Mashanov et al., 2004). Therefore, a major goal of any study of gut regeneration is to reveal the origin of the progenitor cells that give rise to the two epithelia after evisceration.

In echinoderm species studied so far, it seems to be a general rule that the coelomic epithelium regenerates from the mesothelial cells of the stump (Dolmatov, 1992; Garcia-Arraras et al., 1998; VandenSpiegel et al., 2000; Mashanov and Dolmatov, 2001b; Garcia-Arraras and Greenberg, 2001; Shukalyuk and Dolmatov, 2001). Although the histological structure of the mesothelium varies to some extent with the organ examined, in almost all species studied so far it is composed of two major cell types--peritoneal cells (peritoneocytes) and myoepithelial cells (Byrne, 1994; Chia and Koss, 1994; Smiley, 1994; Mashanov et al., 2004). At the early stages of regeneration, these two cell types dedifferentiate. As myoepithelial cells dedifferentiate, their myofilaments form dense spindle-like structures (SLSs) (Dolmatov, 1992; Dolmatov et al., 1996). In the cytoplasm of dedifferentiating peritoneal cells, bundles of intermediate filament split into short fragments. Since both SLSs and fragmented bundles of filaments persist in the cytoplasm for a certain period of time, they can be used as natural markers for dedifferentiated myoepithelial and peritoneal cells, respectively. During and following these dedifferentiation processes, the cells are capable of migration and mitotic division. It is worth noting that the dedifferentiated coelomic epithelial cells are joined together by intercellular junctions and migrate as a continuous epithelial sheet rather than as individual cells.

With regard to regeneration of the holothurian gut, the problem of the origin of the luminal epithelium has generated more controversy than any other fundamental topic. Various hypotheses for regeneration of this epithelium were extensively discussed in recent reviews (Dolmatov, 1999; Garcia-Arraras and Greenberg, 2001). In adult apodids, some aspidochirotids, and juvenile dendrochirotid holothurians, the digestive epithelium has been shown to originate from the cells of the endodermally derived luminal epithelium of the remnants of the digestive tube (Bertolini, 1932; Smith, 1971a, b; Gibson and Burke, 1983; Garcia-Arraras et al., 1998; Shukalyuk and Dolmatov, 2001; Mashanov and Dolmatov, 2001b). In apodids, gut regeneration involves a morphallactic remodeling of the remaining intestinal tissues to form a functionally complete organ without cell proliferation, whereas in aspidochirotids and juveniles of dendrochirotids, mitotic division in the cells of the luminal epithelium plays a significant role in the development of the new intestine.

In adult dendrochirotids, the regeneration mechanism is much more complicated. The anterior evisceration results in expulsion of most of the endodermally derived tissues, the only exception being the luminal epithelium of the cloaca (Kille, 1935; Hyman, 1955; Leibson and Dolmatov, 1989). At the early stages of regeneration, the rudiments develop at both the anterior and posterior ends of the body. Apart from the body wall with five ambulacra, only the mesentery is present in the anterior region after evisceration. One can therefore hypothesize that the luminal epithelium of the anterior rudiment could originate from either ectodermally or mesodermally derived tissues, or from a combination of both. Our data suggest, however, that the tissues of ectodermal origin do not contribute to regeneration. The analysis of serial transverse sections through the anterior gut rudiment of Eupentacta fraudatrix shows that the dedifferentiated coelomic epithelium migrates from the surface of the rudiment into the underlying connective tissue at a level just posterior to the rudiment of the aquapharyngeal complex, thereby giving rise to the luminal epithelium of the gut. The presence of SLSs and bundles of intermediate filaments in the precursors of the luminal epithelial cells provides further evidence that the digestive epithelium can originate from the mesothelium. After 10 days of regeneration, the newly formed luminal epithelium detaches from the mesothelium and progresses in both directions parallel to the axis of the rudiment. Subsequently, the epithelial cells differentiate into all the cell types that normally constitute the digestive epithelium.

The hypothesis that the luminal epithelium could originate from mesodermally derived cells has already been discussed in the literature (Bertolini, 1930; Kille, 1935; Dawbin, 1949; Mosher, 1956; Tracey, 1972; Leibson, 1992). The epithelial lining of the water vascular system, perivisceral mesothelium, and even neoblast-like cells have been proposed as possible sources of cells that form the new luminal epithelium. Among the available reports, our data seem to coincide most closely with Mosher's (1956) observations on gut regeneration in the aspidochirotid holothurian Actinopyga agassizi. In this species, the coelomic epithelium develops a series of longitudinal folds on the surface of the gut rudiment. These folds deepen into the underlying connective tissue and are closed over at the periphery, leaving small cavities lined with epithelial cells derived from the external epithelium of the rudiment. These cavities then fuse to form a continuous lumen lined with the digestive epithelium. Thus, as early as the middle of the last century, the coelomic epithelium was shown to be able to give rise to the luminal epithelium of the regenerating holothurian gut. However, as mentioned above, the holothurian mesothelium is a complex tissue composed of peritoneocytes, myoepithelial cells, and a basiepithelial nerve plexus. Is it really possible for a highly differentiated tissue to undergo such a fundamental transformation?

Recently, a growing body of observations proved that stem cells, under suitable conditions, can produce a whole spectrum of cell types, regardless of whether these tissues are derived from the same germ layer (Weissman, 2000; Lagasse et al., 2001; Filip et al., 2004). Accordingly, the possibility has been considered that the echinoderm mesothelium might contain precursor cells for the luminal epithelial cells (Garcia-Arraras and Greenberg, 2001). Indeed, our data suggest that in the holothurian E. fraudatrix the mesodermally derived coelomic epithelium plays a key role in regeneration of the gut luminal epithelium (normally endodermal in origin). Similarly, it has recently been proposed that the coelomic epithelium plays a crucial role in gut regeneration in the crinoid Antedon mediterranea. (Dolmatov et al., 2003). However, what makes regeneration in E. fraudatrix particularly remarkable is that it is differentiated peritoneal and myoepithelial cells of the mesothelium that give rise to the luminal epithelial cells in the anterior gut rudiment. At no stage of regeneration could we observe a contribution of stem or reserve cells. This absence of stem cells suggests that differentiated cells of echinoderms can transdifferentiate into other cell types and can even cross the boundaries of the germ layer.

Interestingly, in all holothurian species studied so far, the most posterior portion of the digestive tube remains in the body after evisceration (Dolmatov, 1996; Garcia-Arraras and Greenberg, 2001). Our observations suggest that endodermally derived cells of the cloacal luminal epithelium migrate into the connective tissue thickening of the ventral mesentery, thereby giving rise to the digestive epithelium of the posterior rudiment. Thus, the luminal epithelia of the anterior and posterior gut rudiments in E. fraudatrix develop from two different cell sources, namely from the mesodermally derived mesothelium and from the endodermally derived cloacal lining epithelium, respectively. However, in spite of this difference in origin, the luminal epithelial cells of the two rudiments are indistinguishable after the new continuous digestive tube has developed.

In conclusion, our study suggests that the holothurian E. fraudatrix is a good model for studying transdifferentiation and factors that may influence this process. Further experiments on this animal are expected to improve our understanding of morphogenetic mechanisms.

Acknowledgments

We are grateful to the anonymous journal reviewers and to I. C. Wilkie and O. R. Zueva for valuable comments, which enabled us to improve the quality of the manuscript. We thank B. Aschauer (LMU, Munich) and D. V. Fomin (IMB, Vladivostok) for technical assistance. This work was supported by a grant from FEB RAS (project no. 05-I-P10-007) to I.Yu.D. and by a DAAD fellowship to V.S.M.

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VLADIMIR S. MASHANOV (1,*), IGOR YU. DOLMATOV (1), AND THOMAS HEINZELLER (2)

(1) Institute of Marine Biology FEB RAS, Palchevsky 17, 690041 Vladivostok, Russia; and (2) Ludwig-Maximilians-Universitat Munchen, Pettenkoferstrasse 11, D-80336 Munchen, Germany

Received 7 July 2005; accepted 18 October 2005.

* To whom correspondence should be addressed. E-mail: mashanovvlad@yahoo.co.uk

Abbreviations: RER, rough endoplasmic reticulum; SLS, spindle-like structure composed of densely packed myofilaments.
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Author:Mashanov, Vladimir S.; Dolmatov, Igor Yu.; Heinzeller, Thomas
Publication:The Biological Bulletin
Date:Dec 1, 2005
Words:4925
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