Reference: Biol. Bull. 221: 93–109. (August 2011)
© 2011 Marine Biological Laboratory

Gut Regeneration in Holothurians: A Snapshot of
Recent Developments
´ S*
V. S. MASHANOV AND J. E. GARCI´A-ARRARA
Department of Biology, University of Puerto Rico, PO Box 70377, San Juan, Puerto Rico 00936-8377

Abstract. Visceral regeneration in sea cucumbers has
been studied since early last century; however, it is only
within the last 15 years that real progress has been made in
understanding the cellular and molecular events involved.
In the present review, we bring together these recent studies,
providing readers with basic information on the anatomy
and histology of the normal gut and detailing the changes in
tissue organization and gene expression that occur during
the regenerative process. We discuss the nature and possible
sources of cells involved in the formation of the intestinal
regenerate as well as the role of cell death and proliferation
in this process. In addition, we compare gut formation
during regeneration and during embryogenesis. Finally, we
describe the molecular studies that have helped advance
regenerative studies in holothurians and integrate the gene
expression information with data on cellular events. Studies
on visceral regeneration in these echinoderms provide a
unique view that complements regeneration studies in other
animal phyla, which are mainly focused on whole-animal
regeneration or appendage regeneration.

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 regrow 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 Garcı´a-Arrara´s and Greenberg (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.

Introduction
Sea cucumbers, or holothurians, are exclusively marine
invertebrates classified in the phylum Echinodermata, class
Holothuroidea. They are characterized by a soft, orallyaborally 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

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). Nev-

Received 18 January 2011; accepted 25 February 2011.
* To whom correspondence should be addressed. E-mail: jegarcia@
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.
93


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V. S. MASHANOV AND J. E. GARCIA-ARRARAS

Figure 1. Diagram illustrating visceral anatomy of a sea cucumber.
The animal positioned with the anterior (oral) end to the top. Abbreviations: 1di, first descending intestine; 2di, second descending intestine; ai,
ascending intestine; cl, cloaca; e, esophagus; g, gonad; phb, pharyngeal
bulb; pv, Polian vesicle (part of the water-vascular system); rt, respiratory
tree. Not to scale. (Modified from Diaz-Miranda et al., 1995.)

ertheless, 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.
In all regions of the digestive tube, the wall of the gut
consists of three layers—a folded innermost luminal (diges-

tive) 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 peritoneocytes, 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;
Garcı´a-Arrara´s et al., 2001; Mashanov et al., 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 al.,
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


GUT REGENERATION IN HOLOTHURIANS

95

Figure 2. Organization of the luminal epithelium and mesothelium of the sea cucumber gut wall in
non-eviscerated (A and B, respectively) and regenerating (C and D, respectively) animals. Abbreviations: bl,
basal lamina; if, bundles of intermediate filaments in peritoneocytes; m, myoepithelial cell; mc, mucocyte; mi,
mitotic cell; n, neuron; ns, neurosecretory cell; np, nervous plexus; pc, peritoneal cell (peritoneocyte); sls, spindle-like
structure composed of condensed myofilaments; sv, secretory vacuoles; ve, vesicular enterocyte. Not to scale.


epithelium (Crosnier et al., 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).
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 al., 2005; San Miguel-Ruiz
and Garcı´a-Arrara´s, 2007; Garcı´a-Arrara´s 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.


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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; Garcı´a-Arrara´s et al., 1998; Wilkie,
2001). The second type of visceral autotomy, anterior evisceration, 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

Figure 3. Two types of evisceration in sea cucumbers. (A and B)
Posterior evisceration in aspidochirotids results in a loss of the digestive
tube between the esophagus and cloaca. (C and D) Anterior evisceration in
dendrochirotids leaves the animal with only the cloacal stump. Abbreviations: cl, cloaca; es, esophagus; in, intestine; mes, mesentery; phb, pharyngeal bulb. The mesentery is not shown on the diagrams showing
non-eviscerated animals, and arrowheads indicate the anterior and posterior
planes of autotomy (A and C). Not to scale. (Modified from Mashanov et
al., 2005, and Mashanov et al., 2010.)

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


GUT REGENERATION IN HOLOTHURIANS

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; Garcı´aArrara´s 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 mesothelial 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 therefore 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.
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 mesothelial
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 (Garcı´a-Arrara´s 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; Garcı´a-Arrara´s
et al., 1998; Mashanov et al., 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

97

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 alimentary 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 (Garcı´a-Arrara´s 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 (although 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; Garcı´a-Arrara´s et
al., 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 epithe-


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V. S. MASHANOV AND J. E. GARCIA-ARRARAS

Figure 4. Plasticity of the gut mesothelium in regeneration. (A). Early rudiment formation (5 days after
evisceration) in Holothuria glaberrima. The mesothelial cells at the free margin of the mesentery undergo
epithelial-to-mesenchymal transition, and ingress into the underlying connective tissue. Note that the ingressed
cells are positively labeled with Meso1 antibody (green), which has been shown to be a specific marker of
mesothelial cells in sea cucumbers (Garcı´a-Arrara´s et al., unpubl.). Cell nuclei are labeled with DAPI nuclear
marker (blue). The inset shows a low-magnification view of the gut rudiment with the boxed area corresponding
to the main image. (B) In the dendrochirotid holothurian Eupentacta fraudatrix, the dedifferentiated mesothelium at the free margin of the anterior mesentery develops deep epithelial folds (arrows) that protrude into the
underlying connective tissue. The epithelium of these folds detaches from the rest of the mesothelium and
re-organizes itself to form the luminal epithelium (C). (From Mashanov et al., 2005.) Abbreviations: de,
digestive (luminal) epithelium; me, mesothelium.

lial 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 epithelium of the cloaca grows along the connective tissue
thickening of the mesentery.


GUT REGENERATION IN HOLOTHURIANS


99

Figure 5. Anatomical features of the regenerating digestive tube in a holothurian, as exemplified by the
aspidochirotid Holothuria glaberrima, at different times points after evisceration. Diagrams to the right of each
of the anatomical drawings show organization of the regenerating digestive tube, as seen on cross sections,
whose position is shown by horizontal bars. Abbreviations: ar, anterior rudiment; cl, cloaca; de, digestive
(luminal) epithelium; es, esophagus; me, mesothelium; mes, mesentery; phb, pharyngeal bulb; pr, posterior
rudiment. Not to scale. (Modified from Mashanov et al., 2010.)

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 (Garcı´a-Arrara´s et al., 1998; Mashanov et al., 2010). As shown above, physiological cell turn-


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V. S. MASHANOV AND J. E. GARCIA-ARRARAS

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 (Garcı´a-Arrara´s et al., 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 (Garcı´a-Arrara´s et al.,
unpubl.).
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; Garcı´a-Arrara´s and Dolmatov,
2010). Therefore, it can be hypothesized that mechanisms of
generation of muscle bundles in development and regener-


GUT REGENERATION IN HOLOTHURIANS

ation 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 dendrochirotid 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 al., 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, compared 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

101

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 Sa´nchez 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 (Garcı´a-Arrara´s et al., 1998; Shukalyuk and Dolmatov,
2001; Mashanov et al., 2005). Electron microscopy studies
(Shukalyuk and Dolmatov, 2001; Mashanov and Dolmatov,
2001; Mashanov et al., 2005; Odintsova et al., 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 holothu-


102

V. S. MASHANOV AND J. E. GARCIA-ARRARAS


rian digestive tube. One of the most basic techniques, which
can be tried to tentatively explore the histogenetic relationships in the gut wall epithelia, is the label-retaining cell
(LRC) approach. This method involves labeling of DNAsynthesizing 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 cells 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 unecessary, involvement of resident stem cells
in the regrowth of the gut tissue layers remains a theoretical

Figure 6. Label-retaining (BrdU-positive) cells in the esophagus of
non-eviscerated individuals of Holothuria glaberrima. (A) Two to four
hours after the last BrdU injection (BrdU was injected twice a day for
7 days at a dose of 50 mg/kg) a large number of cells were labeled in
the luminal epithelium. The inset shows a labeled cell in the mesothelium. (B) Fourteen days after the last injection some few strongly
labeled cells are present in both luminal epithelium and mesothelium.
Abbreviations: ctl, connective-tissue layer of the gut wall; de, digestive
(luminal) epithelium; me, mesothelium.


GUT REGENERATION IN HOLOTHURIANS

possibility and cannot be ruled out completely, unless the
issue is studied directly.
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 (Sua´rez-Castillo et al., 2004)—were identified with the aid of similar
approaches. However, for a long time the molecular basis of

103

echinoderm regeneration was limited to the characterization
and study of this handful of genes.
In recent years expressed sequence tag (EST)/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 (Sa´nchez Alvarado and Newmark, 1999; Nakazawa et al., 2003; Sa´nchez Alvarado and
Tsonis, 2006; Rossi et al., 2007; Salo´ 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 uneviscerated 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-, 7and 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 immunohistochem-


104

V. S. MASHANOV AND J. E. GARCIA-ARRARAS

istry 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 (Quin˜ones
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.


GUT REGENERATION IN HOLOTHURIANS

105

Figure 7. Double labeling with antisense riboprobe for (A) survivin and (B) TUNEL assay on the posterior
gut regenerate of Holothuria glaberrima on day 7 after evisceration. (C) Note the negative correlation between
the localization of survivin in situ hybridization signal and the density of the TUNEL-positive cells, which
suggest an anti-apoptotic role for survivin in visceral regeneration. (From Mashanov et al., 2010.) Abbreviations:
vm, ventral mesentary.

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 Garcı´a-Arrara´s, 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-Lo´pez 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 (RoigLo´pez, 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 overexpressed 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


106

V. S. MASHANOV AND J. E. GARCIA-ARRARAS

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 post-tramatic repair (Marconi et al., 2007; Li
et al., 2010). Taken together, these data suggest that the
cells of the holothurian mesothelium can temporarily acquire 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; Garcı´a-Arrara´s
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: 1SC1GM08477001), NSF (grant number: IOS-0842870), and the University
of Puerto Rico.
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