Gut Regeneration in Holothurians A Snapshot of

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 outer-most complex

Gut Regeneration in Holothurians: A Snapshot of

Recent Developments

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

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

(sur-rounded 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 re-search For a more detailed account of earlier publications in the field, the reader is referred to Garcı´a-Arrara´s 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 as-pects of visceral regeneration have been studied in one or a few species only; therefore, little is known about interspe-cific variation in the regeneration response in sea cucum-bers Although there are still many gaps in our knowledge,

we have attempted to combine the existing data and hypoth-eses into a cohesive story, which represents current achieve-ments 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 (somato-coel) There is no generally accepted nomenclature for the different parts of the alimentary canal, since different au-thors use different names when referring to the same struc-ture (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

se-quence tag; LRC, label-retaining cell; SLS, spindle-like structure; TUNEL,

deoxynucleotidyl transferase-mediated dUTP nick end labeling.

93

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

ac-cording to the direction of its axis or to morphological

features; thus, in some species, distinctions are made

be-tween the ascending and descending portions of the

intes-tine 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 outer-most complex muscular mesothelium (gut coelomic epithe-lium), 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 se-cretion, 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

sep-tate junctions) The cell body of each peritoneocyte contin-ues 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 interme-diate filaments The basal half of the mesothelium is occu-pied by myoepithelial cells, whose contractile processes are organized into longitudinal, circular, or oblique gut muscu-lature The mesothelium also contains a prominent nervous plexus Cell bodies and processes of nerve cells are also occasionally observed in the luminal epithelium and con-nective 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 prolif-erating cells in mammals, which are restricted to narrow compartments at the bottom of the crypts of the intestinal

Figure 1. Diagram illustrating visceral anatomy of a sea cucumber.

The animal positioned with the anterior (oral) end to the top

Abbrevia-tions: 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.)

epithelium (Crosnier et al., 2006), mitotic cells of the sea

cucumber gut are scattered all along the digestive

epithe-lium, without any preferential localization to the basal

re-gion of the luminal epithelium or to the bottom of epithelial

folds (Leibson, 1986, 1989; Mashanov et al., 2004)

An-other interesting feature of cellular division in sea

cucum-bers 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 obser-vation is that some myoepithelial cells of the normal meso-thelium show condensation of their myofilaments into com-pact spindle-like structures (SLSs) It has previously been shown that SLS formation is a hallmark of myocyte dedif-ferentiation in the regenerating body wall and visceral mus-culature (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

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.

The Regenerating Gut

The nature of the injury

Many sea cucumber species of the orders Aspidochirota

and Dendrochirota are capable of autotomizing their

inter-nal 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

compo-nents along predetermined breakage zones (Wilkie, 2001;

Byrne, 2001) Therefore, evisceration occurs in a very

con-sistent and repeatable manner, which minimizes the

varia-tion 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

at-tractive model systems to study various aspects of

post-traumatic recovery

There are two types of evisceration in sea cucumbers

(Fig 3) Posterior evisceration occurs mainly in the

Aspi-dochirota 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

evis-ceration, is a characteristic feature of the order

Dendrochi-rota It results in a more extensive tissue loss than the

posterior evisceration (Fig 3C, D), since not only the

in-testine 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

eviscer-ation 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

poste-rior 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

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 Abbrevia-tions: cl, cloaca; es, esophagus; in, intestine; mes, mesentery; phb,

pha-ryngeal 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 mesentery involves extensive dedifferentiation of the

mesothelium, which initially starts at the distal free margin

(Mashanov et al., 2005; Candelaria et al., 2006;

Garcı´a-Arrara´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

concomi-tantly with the remodeling of their cytoskeleton The

dedif-ferentiated 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

interme-diate filaments can remain within the cytoplasm and

there-fore can serve as natural markers helping to trace the

de-velopmental 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

pseu-dopodium-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

de-composition 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

con-nective 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,

dedif-ferentiation spreads to other areas of the mesentery in a

distal (free margin) to proximal (body wall) gradient

No-tably, 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

disap-pearance 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 dedif-ferentiation 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 (esoph-agus) 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 (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 (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;

Od-intsova et al., 2005) The dedifferentiated enterocytes

be-come 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 holothu-rians 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-lial lining of the folds eventually detaches from the

meso-thelium on the rudiment surface and reorganizes itself to

form a single blind lumen lined with a newly formed

di-gestive epithelium derived from the mesothelium (Fig 4C)

These morphogenetic movements are accompanied by

di-rect transformation of the mesothelial cells into typical

enterocytes The SLSs and bundles of intermediate

fila-ments 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 aspi-dochirotids—that is, the endodermally derived luminal ep-ithelium of the cloaca grows along the connective tissue thickening of the mesentery

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

mesothe-lium 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.

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 stud-ied in only one sea cucumber species, the aspidochirotid

Holothuria glaberrima (Garcı´a-Arrara´s et al., 1998; Masha-nov et al., 2010) As shown above, physiological cell

turn-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.)

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-proxi-mal 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

dedifferen-tiated 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

post-traumatic regeneration and could be an absolute

require-ment 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

embryogen-esis, 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

develop-mental programs, since regeneration always involves

unique processes, such as wound healing and

dedifferenti-ation, which have no counterpart in embryonic

develop-ment The question of the degree to which regeneration

recapitulates embryonic development has been debated in

the literature for decades (Carlson, 2007; Brockes and Ku-mar, 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 devel-oping 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 develop-mental 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; Dolma-tov and Yushin, 1993) It is worth mentioning here that the

primary blastopore does not close in the holothurian Cu-cumaria 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 devel-opment 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 regen-eration 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 dif-ferentiate into myoepithelial cells (Dolmatov and Ivantey, 1993) Interestingly, regeneration of the longitudinal muscle bands after transection employs a similar mechanism (Dol-matov and Ginanova, 2001; Garcı´a-Arrara´s and Dol(Dol-matov, 2010) Therefore, it can be hypothesized that mechanisms of generation of muscle bundles in development and

regener-ation through infolding and detachment of stretches of the

mesothelium are partly co-opted by gut regeneration

Another interesting aspect of relationships between

nor-mal development and regeneration is how regenerative

ca-pacities change at different times throughout the life cycle

Post-traumatic regeneration is usually studied in adult

or-ganisms, when most of the processes of normal

develop-ment 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

(Dol-matov, 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

mesothe-lium 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,

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

re-generate 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

regen-eration After the initial phase of wound closure and

histol-ysis 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

transdifferentia-tion 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 Sa´n-chez 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 hypoth-esis 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 mor-phogenesis either by the expansion of the luminal epithe-lium of the stump or by transdifferentiation of the

mesothe-lium (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 dedif-ferentiation 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 di-vision phase In contrast, deep dedifferentiation in the me-sothelium results in drastic simplification of the epithelial organization and the complete loss of phenotypic character-istics in the peritoneal and myoepithelial cells It can be hypothesized, therefore, that this deep level of dedifferen-tiation 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 entero-cytes 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 Unfortu-nately, 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-rian 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

ex-ample) 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

theo-retical 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

individu-als every 12 h for 7 days The animindividu-als 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

oc-curred (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

sug-gests 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

Unfortu-nately, no label-retaining experiments have yet been

per-formed in regenerating sea cucumbers Nevertheless,

al-though 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 mesothe-lium (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.