Dev Genes Evol (1998) 208:421430

Springer-Verlag 1998

O R I G I NA L A RT I C L E

&roles:M.D. Candia Carnevali F. Bonasoro M. Patruno

M.C. Thorndyke

Cellular and molecular mechanisms of arm regeneration

in crinoid echinoderms: the potential of arm explants

&misc:Received: 9 March 1998 / Accepted: 5 June 1998

&p.1:Abstract Crinoid echinoderms can provide a valuable

experimental model for studying all aspects of regenerative processes from molecular to macroscopic level. Recently we carried out a detailed study into the overall
process of arm regeneration in the crinoid Antedon mediterranea and provided an interpretation of its basic
mechanisms. However, the problem of the subsequent
fate of the amputated arm segment (explant) once isolated from the animal body and of its possible regenerative
potential have never been investigated before. The arm
explant in fact represents a simplified and controlled regenerating system which may be very useful in regeneration experiments by providing a valuable test of our hypotheses in terms of mechanisms and processes. In the
present study we carried out a comprehensive analysis of
double-amputated arm explants (i.e. explants reamputated at their distal end immediately after the first proximal
amputation) subjected to the same experimental conditions as the regenerating donor animals. Our results
showed that the explants undergo similar regenerative
processes but with some significant differences to those
mechanisms described for normal regenerating arms. For
example, whilst the proximal-distal axis of arm growth is
maintained, there are differences in terms of the recruitment of cells which contribute to the regenerating tissue.
As with normal regenerating arms, the present work focuses on (1) timing and modality of regeneration in the
explant; (2) proliferation, migration and contribution of
undifferentiated and/or dedifferentiated/transdifferentiated cells; (3) putative role of neural growth factors. These
problems were addressed by employing a combination of
conventional microscopy and immunocytochemistry.
Comparison between arm explants and regenerating
Edited by D. Tautz
M.D. Candia Carnevali () F. Bonasoro
Dipartimento di Biologia, Via Celoria 26, I-20133 Milano,
Italy
M. Patruno M.C. Thorndyke
Department of Biology, Royal Holloway University of London,
Egham, Surrey TW20 OEX, UK&/fn-block:

arms of normal donor adults indicates an extraordinary

potential and regenerative autonomy of crinoid tissues
and the cellular plasticity of the phenomenon.&bdy:

Introduction
Regeneration is widespread in the animal kingdom but in
spite of the infinite choice of models, this phenomenon
has been explored in detail only in a few taxa. Even in
the many groups well known for their regenerative capabilities there is a substantial gap in our understanding not
only concerning regenerative mechanisms at the cellular
and molecular level but also more fundamental aspects
such as temporal and spatial conditions. This is certainly
the case for echinoderms whose spectacular regenerative
phenomena often related to asexual reproduction
(Mladenov 1996; Mladenov and Burke 1994) have attracted developmental biologists in the past but, save for
a few notable exceptions, have been disregarded in more
recent times.
Among echinoderms, crinoids are well known for
their striking regenerative potential (Perrier 1873; Reichensperger 1912; Amemiya and Oji 1992). In the past few
years we have studied in detail the overall process of arm
regeneration in the crinoid Antedon mediterranea by employing experimentally-induced regeneration of different
stages (Candia Carnevali et al. 1989, 1993, 1995, 1996,
1997, 1998; Bonasoro et al. 1995, 1997, 1998; Candia
Carnevali and Bonasoro 1994, 1995). The majority of
our data so far highlight the fundamental aspects of the
phenomena and suggest that this still largely unexplored
echinoderm model could benefit from a more modern
developmental biology approach.
The process of arm regeneration covers a period of
about 4 weeks and can be divided schematically into
three main phases: an initial repair phase, including the
first 24 h post-amputation period, an early regenerative
phase, including the 24 h72 h post-amputation period,
and an advanced regenerative phase, covering the 72 h4
weeks post-amputation period. These phases have been

422

formulated by reference to a standard model of regeneration resulting from self-induced amputation (Candia
Carnevali et al. 1989, 1993). In all the phases a key role is
performed by the brachial nerve and the coelomic canals,
all of which are involved in cell proliferation and migration. Different populations of migratory undifferentiated
(amoebocytes and coelomocytes) and differentiated elements (phagocytes and granule cells) can be distinguished and are employed extensively in both the repair
and regenerative processes. Our findings have shown arm
regeneration in Antedon to be a typical blastemal phenomenon, that is an epimorphic process comprising two
important components: (1) source and proliferation sites
of new cells, and (2) intervention of putative growth factors. Both these points have been explored at different
levels and significant results have been recently obtained
(Holland 1994; Candia Carnevali et al. 1995, 1996, 1997,
1998; Bonasoro et al. 1995, 1998).
In this study we have explored the problem of the fate
and possible regenerative potential of the amputated arm
segments isolated from the animal body. In other echinoderms amputated arm segments can display striking regenerative capabilities (a famous case is represented by
the comet forms of the starfish Linckia guildingi;
Mladenov and Burke 1994): this point has never before
been taken into account in crinoids and seems to be a
crucial test of the regenerative potential of these animals,
in general, and the regenerative autonomy of their arm
system, in particular. Taking advantage of the exceptional amenability of feather stars from an experimental
point of view, we have carried out a parallel analysis on
both the regenerating arms and the respective amputated
arm segments (explants). The isolated explants can be
easily maintained in living condition for a relatively long
time (more than 2 weeks) and unexpectedly undergo regenerative processes comparable to those already described for donor regenerating arms which remain insitu. This paper focuses in particular on double-amputated arm explants (i.e. arm segments reamputated at their
distal end immediately after the first proximal amputation) and on their regenerative mechanisms. Uniquely,
the explant represents a simplified, controlled and tractable experimental model system which can provide indespensable correlative information as well as confirmation of the regenerative mechanisms and autonomy of
this system in terms of its cellular and molecular potential. In particular, those specific aspects already explored
in standard arm regeneration (point 1 and 2, above) have
now been specifically addressed in explants by employing a combination of conventional microscopy (light,
transmission electron and confocal) and immunocytochemistry. The crucial problem of identification of cell
lineages responsible for both repair and regenerative processes has been approached by employing specific methods for monitoring cell proliferation which we established in standard regenerating samples (Candia Carnevali et al. 1995, 1997; Bonasoro et al. 1998). In parallel,
we have also carried out a series of tests focusing on the
presence and pattern distribution of regulative molecules

in the regenerating arm explants. It is relevant to point

out that our previous studies have revealed the presence
and respective pattern distribution of three different
classes of regulatory molecules in standard regenerating
arms: monoamine neurotransmitters (Bonasoro et al.
1995; Candia Carnevali et al. 1996), neuropeptides (Bonasoro et al. 1995; Candia Carnevali et al. 1998) and, finally, growth factors (Candia Carnevali et al. 1998). This
problem has only been approached in a preliminary fashion in the regenerating explants by employing a series of
tests selected on the basis of the quality of the results
previously obtained in standard regenerating arms. In
particular the present experiments have been carried out
on neuropeptides (S1 and S2 SALMFamides; Elphick et
al. 1991a, b) and a growth factor (TGF-); this choice
was determined by the wide and well-defined distribution of these substances seen in both normal non-regenerating and regenerating arms.
Our results highlight some significant differences in
terms of basic mechanisms and the elements involved
compared to those described for the normal regenerating
adult.

Materials and methods

Experimental animals and explant culture
Specimens of Antedon mediterranea collected by scuba divers
from the southern coast of Italy (Gulf of Taranto) were maintained
in aquaria with a closed artificial seawater system at 14C. Experimental regeneration of arms was induced by mimicking the conditions of natural autotomy (see Candia Carnevali et al. 1993). The
amputated arm segments (explants) were immediately reamputated at their distal end and maintained in little compartments of the
aquaria together with the respective donor animals and under the
experimental conditions described above. The artificial seawater
employed was not sterilized. The isolated explants could be easily
maintained in a living condition for 2 weeks or more and underwent regenerative processes in parallel with their donor regenerating arms. The mortality of the explants was very low but slightly
increased according to the culture time. Regeneration was monitored at fixed times [24, 48, 72 h, 7 and 15 days post amputation
(p.a.)] in both the explants and the donor arms. These samples
were prepared according to the procedures outlined below.
Light microscopy (LM) and electron microscopy (TEM)
Explants and regenerating donor arm were prefixed with 2% glutaraldehyde in 0.1 M cacodylate buffer for 45 h, then, after an
overnight washing in the same buffer, postfixed with 1% osmic acid in the same buffer. After standard dehydration in an ethanol series, the samples were embedded in Epon-Araldite 812. The semithin and thin sections, cut with a Reichert Ultracut E (diamond
knife), were stained by conventional methods (crystal violet-basic
fuchsin for LM, uranyl acetate and lead citrate for TEM) and then
observed in a Jenaval light microscope and Jeol 100 SX electron
microscope respectively.
Immunocytochemistry (ICC)
The samples were fixed in paraformaldehyde 4% - glutaraldehyde
0.1% in 0.1 M phosphate buffer for 2 h. Following an overnight
wash in the same buffer, the samples were dehydrated and embedded in Epon-Araldite (see above). This fixation and embedding

423
protocol maintains good tissue integrity and provides good preservation of antigenicity (see Candia Carnevali et al. 1995, 1997). It
also allows preparation of both semi-thin sections for LM and ultra-thin sections for TEM. Semi-thin sagittal sections cut with an
LKB Ultratome V and Reichert Ultracut E were processed for immunocytochemistry (see below).
BrdU labelling
Cell proliferation was monitored using in vivo incorporation of the
substituted nucleotide, 5-bromodeoxyuridine (BrdU), then later revealed by a monoclonal antibody against BrdU (Amersham: Cell
proliferation kit). Animals were immersed in BrdU dissolved in
artificial seawater at a final concentration of 0.05% for the final
2 h of the prefixed regeneration periods. This incubation protocol
allowed detection of cells actively proliferating immediately before fixation (Gratzner 1982; Candia Carnevali et al. 1995, 1997;
Bonasoro et al. 1998). Control samples were obtained from nonregenerating arms of the same animals. For use with semi-thin
Epon-Araldite sections, the standard BrdU-immunocytochemistry
protocol for paraffin sections was modified as described in detail
elsewhere (Candia Carnevali et al. 1995, 1997). After a brief treatment (2 min) with a resin-remover mixture (methanol, propylene
oxide and KOH), the sections were rinsed with methanol, then
with phosphate-buffered saline (PBS) and incubated overnight at
4C with anti-BrdU serum diluted 1:100 with nuclease (Amersham: Cell proliferation kit). A pre-treatment of 20 min with 0.3%
H2O2 in PBS was performed to exclude the potential activity of
endogenous peroxidases. After several washings in PBS the specimens were incubated (3 h) with peroxidase anti-mouse IgG
(Amersham: Cell proliferation kit) at room temperature and, after
a further washing in PBS, incubated (5 min) with 0.05% 3,3-diaminobenzidine and 0.03% H2O2 in PBS, and then washed in distilled water. To amplify the peroxidase reaction product, the experiments included use of the cobalt and nickel intensifier supplied
with the kit. Control reactions were carried out by omitting the
primary antiserum.
The same fixation/embedding protocol described for LM was
employed for TEM. Ultra-thin sections were cut with a Reichert
Ultracut E and collected on gold grids. After rapid etching with
NaOH 0.15% in absolute methanol (1 min), sections were carefully soaked on drops of methanol (30 min). The grids were washed
repeatedly with distilled water (30 min) and soaked on drops of
TRIS-buffered saline (TBS) containing 1% bovine serum albumin
(BSA) (pH 7.2, 30 min). Incubation with the anti-BrdU murine antibody diluted 1:100 with nuclease (Amersham: Cell proliferation

Fig. 1 Schematic drawing illustrating the experimental

preparation of the explant. On
the left is shown the donor arm,
on the right the respective arm
explant. The explant, immediately reamputated at its distal
end, undergoes regenerative
processes in a distal direction
in parallel with its donor arm&ig.c:/f

kit) was performed for 1 h at room temperature. After repeated

washing with TBS (pH 7.2, 30 min), with TBS/BSA 0.2%
(pH 7.2, 1 min) and TBS/BSA 1% (pH 8.2, 5 min) sections were
incubated with goat anti-mouse IgG conjugated to 10-nm colloidal
gold particles (Sigma; 1:30) in TBS/BSA 1% (pH 8.2) for 45 min,
at room temperature. After repeated washing with TBS/BSA 0.2%
(pH 7.2), TBS (pH 7.2) and distilled water the specimens were
post-fixed with glutaraldehyde 2.5% in distilled water for a few
minutes and then washed carefully in distilled water. Finally the
sections were stained with aqueous uranyl acetate and observed
with a Jeol 100 SX electron microscope.
Peptide and TGF- labelling
ICC tests were carried out on resin sections according to the specific immunoperoxidase ABC system (Vector) or immunofluorescence methods. For neuropeptide ICC-specific polyclonal antibodies anti-S1 (BLV) and anti-S2 (BGII; Elphick et al. 1991a, b) were
used. For growth factors ICC commercial mouse anti-TGF- (Serotec) was used. For single labelling, standard ICC methods using
either fluorescein or Texas red-conjugated secondary antibody
were employed (Candia Carnevali et al. 1995; Moss et al. 1998).
For double labelling both BrdU and S1- or S2- SALMFamide
neuropeptide, the ABC avidin-biotin fluorescence system was
used with some modification. Processing for the first antiserum
was as normal, using fluorescein-conjugated avidin to visualize
the reaction. The second antibody was diluted in PBS and 0.1%
Tween to block further any crossreactivity between the antisera,
followed by visualization with Texas red-conjugated avidin D. Anti-BrdU was applied after anti-S1 to avoid any possibility of the
DNase reagent affecting peptide antigenicity. The sections were
observed in a Leica TCS NT confocal microscope.
ICC controls were: (1) preabsorption with the appropriate antigen, (2) replacing the primary antiserum with non-immune sera of
the animal in which the primary was raised, or (3) omitting the primary antibody and incubating in PBS and 1% normal goat serum.

Results
The isolated explant, immediately reamputated at its distal end and maintained in living conditions for 1 or 2
weeks, underwent regenerative processes similar to those
of its respective donor arm. However, since it was char-

424

425

Fig. 3ad Microscopic anatomy of the arm explant at 1 week p.a.

TEM details of the migrating cells involved in repair and regeneration. a amoebocyte (bar 2 m); b phagocyte (bar 2 m); c granule
cells (bar 3 m); d coelomocytes (bar 4 m)&ig.c:/f

acterized by two symmetrical amputation surfaces, proximal and distal (Fig. 1), these two amputation surfaces
were analysed in parallel at different stages in terms of
processes and cellular elements involved.

Distal amputation

Fig. 2af Microscopic anatomy of the arm explant at 1 week post

amputation (p.a). a Light microscope (LM) semi-thin sagittal section at the level of the proximal amputation. The amputation surface is covered by a complete and thick cicatricial layer (arrows).
There is no sign of a regrowing blastema (ae ambulacral epithelium, cc coelomic canals, m muscle, n brachial nerve, bar 200 m).
b LM semi-thin sagittal section at the level of the distal amputation. A prominent regenerating bud (rb) is recognizable on the amputation surface. Its developmental stage corresponds to that of a
standard regenerating arm of 45 days. The regenerative regrowth
of the coelomic canals and the brachial nerve is outlined by a
marked cellular flux towards the bud region (ae ambulacral epithelium, cc coelomic canals, m muscle, n brachial nerve, bar
200 m). c LM view of the proximal amputation surface in semithin sagittal section. Detail of the cicatricial layer. The unusual
thickness of this layer mainly is due to the overlapping of confluent flows of migrating cells originating from the brachial nerve
(arrows) and the coelomic canals (double arrowheads) respectively (bar 50 m). d LM view of the distal amputation. Detail of the
regenerative bud. The regenerate largely comprises a blastema of
undifferentiated elements (rb) inside which some elements are differentiating. The coelomic system is developing as solid cords of
proliferating coelomocytes which split to produce the internal cavity (ae ambulacral epithelium, hc hydrocoelic canal, sc somatocoelic canal, bar 50 m). e LM sagittal semi-thin section of the intermediate stump. Detail of a muscle. All the muscle bundle is involved in a massive histological and cellular rearrangement. Its peripheral regions, in particular, are characterized by the presence of
apparently dedifferentiating myocytes at different stages (arrows)
intermingled with a number of coelomocytes and phagocytes
which form areas of intense cell proliferation/migration (double
arrowheads) in the adjacent coelomic canal (cc) (bar 100 m).
f Transmission electron microscope (TEM) section of the intermediate stump at the level of the rearranging muscle. Detail of a presumptive dedifferentiating myocyte (dm). A phagocyte (ph) and a
coelomocyte (c) are also shown (bar 2 m).&ig.c:/f

In terms of regenerative stages and general histology the

results were comparable to those obtained for the standard regenerating arm. The initial repair phase of
2448 h p.a. was followed by an early regenerative phase
characterized by the growth of the typical regenerative
blastema (72 h p.a.). At 1 week p.a. (Fig. 2b) the regenerative bud was well developed although its growth was
slightly delayed when compared with a standard regenerating arm at the same age p.a. In the bud (Fig. 2d) typical
histological components, such as the ambulacral epithelium, skeletal elements, coelomic compartments (hydrocoelic and somatocoelic) and the brachial nerve, were developing and differentiating according to the modalities
previously described for normal regenerating arms (Candia Carnevali et al. 1993). All these structures are continuous with those of the stump: in particular brachial nerve
regrowth was characterized by its appreciable bending
towards and inside the regenerative bud (Fig. 2b,d). At
the same time the regenerating coelomic canals grew distally and developed in the bud in the form of solid cords
of coelomocytes. The internal cavity was seen later following the appearance of a split in the coelomocyte cord.
The brachial nerve and coelomic canals are preferential
pathways for extensive cell migration in a distal direction. This took place both in the distal regenerate itself
and the intermediate stump. The various cell types identified (Fig. 3a,b,c,d) were the same as those already described for standard arm regeneration: amoebocytes, coelomocytes, phagocytes and granule cells (Smith 1981).
The amoebocytes (Fig. 3a) are apparently undifferentiated non-neural cells, normally located around the brachial

426

nerve, whereas the coelomocytes (Fig. 3d) are also apparently undifferentiated elements, but move freely in
the coelomic canals (Endean 1966). These two types of
cell migrate towards the amputation site and are employed extensively in both repair and regenerative processes. Phagocytes (Fig. 3b) and granule cells (Fig. 3c)
are separate classes of well-differentiated migratory
cells, the first characterized by a number of phagosomes,
the second by large chromatophilic granules. These latter
cells are associated with both the brachial nerve and the
coelom and are employed specifically during the repair
phase.
Proximal amputation
Here, regeneration stopped at the first repair stage. At
2448 h p.a. wound healing was completed and the amputation surface was covered by a well-developed cicatricial layer (Fig. 2a). The brachial nerve and the coelomic canals of the stump appeared to be involved in substantial cell migration in a proximal direction. This resulted in progressive symmetrical cellular fluxes that in
the brachial nerve tended to diverge towards the ambulacral region, whereas in the coelom their direction was
towards the brachial nerve (Fig. 2a). These phenomena
became even more evident at more advanced stages
(1 week, Fig. 2c) with particular reference to the severed
ends of the coelom which proliferated as prominent
dense rods of cells curved towards the brachial nerve.
This massive invasion of migrating cells produced a very
thick and multistratified cicatrial layer (Fig. 2c) giving
rise sometimes to local hyper-thickenings. The cells involved in these phenomena were the same migrating elements described above. There was no blastema growing
in the distal-proximal direction.
Although the general tissue histology and ultrastructure was well preserved for the entire explant length, in
the intermediate tract of the stump some tissues showed
a degree of rearrangement, particularly the connective
tissue, the ambulacral epithelium and the muscles
(Fig. 2e). The latter appeared to undergo significant reorganization and perhaps dedifferentiation (Fig. 2f) which
involved all muscle bundles, particularly in their peripheral regions, where the presence of elements at different
degrees of rearrangement was evident at both histological and ultrastructural levels. These same regions were
characterized by large numbers of migrating coelomocytes and phagocytes (Fig. 2e). Masses of coelomocytes
were also seen gathering and accumulating locally in the
coelomic canals of areas adjacent to the rearranging
muscles (Fig. 2e).
In order to identify the source, proliferation sites and
recruitment times of the new cells employed in both repair and regenerative phases, cell proliferation was monitored by employing the well-established BrdU method
already used successfully in normal regeneration stages
(Candia Carnevali et al. 1995, 1997; Bonasoro et al.
1998). At the proximal amputation site, in whichever

stage was analysed (from 24 h to 1 week p.a.) strong labelling of nuclei was localized to the cicatricial layer
(Fig. 4a) particularly in association with the proximal
ends of the brachial nerve and the coelom. As expected,
a comparable distribution of labelling could be seen in
the distal amputation zone during the repair phase
(2448 h p.a.). As in normal regenerating arms, during
the following regenerative phases (72 h and 1 week p.a.)
a particularly intense reaction could be found in the apical blastema and in the regrowing coelomic compartments (Fig. 4b). In advanced stages, a marked labelling
was also still detectable in the stump, even far from the
amputation site, at the level of both the coelomic epithelium and the brachial nerve (Fig. 4d). It is relevant that
the labelling involved migrating amoebocytes specifically and only rarely phagocytes and granule cells. The employment of specific methods of immunogold labelling
for BrdU in TEM is useful for identifying the various
cell types involved in proliferation. As expected, an intense BrdU reaction could be detected in the nuclei of
blastemal cells, migrating amoebocytes, coelomocytes
and coelothelial cells (Fig. 4e). In contrast to normal regeneration, many strongly labelled nuclei were also noted at the level of the muscle bundles of the stump
(Fig. 4c) corresponding to the areas of extensive cell rearrangement. Labelled nuclei involved both migrating
coelomocytes and presumptive dedifferentiating myocytes (Fig. 4f).
Taking into account the primary role of the nervous
system widely demonstrated in normal regenerating
arms, particular attention has been given to the presence
of neurally-derived factors in the explants. Preliminary
results were obtained for the native echinoderm S1- and
S2-SALMFamide peptides. These peptides are normally
present in non-regenerating arms in various components

Fig. 4af Cell proliferation in the arm explant at 1week p.a. BrdU
immunocytochemistry. a LM semi-thin sagittal section at the level
of the proximal amputation. A marked labelling is detectable in
the cicatricial layer and at the level of the brachial nerve, the coelomic lining and the ambulacral epithelium. The muscle bundles
are also involved in the reaction. ABC immunocytochemistry (ae
ambulacral epithelium, cc coelomic canals, m muscle, n brachial
nerve, bar 200 m). b LM semi-thin sagittal section at the level of
the distal amputation. Detail of the regenerating blastema (rb). A
strong immunoreaction involves the blastemal cells and the epithelium of the regrowing coelomic canal (cc). ABC immunocytochemistry (bar 100 m). c LM semi-thin sagittal section at the
level of the intermediate stump. Detail of the muscle. A marked labelling can be seen in the entire bundle, with particular reference
to its peripheral regions. The epithelium of the adjacent coelomic
canal (cc) is also strongly reactive. ABC immunocytochemistry (n
brachial nerve, bar 50 m). d LM semi-thin sagittal section at the
level of the intermediate stump. Detail of the brachial nerve. The
labelling involves many cells of the cortex. ABC immunocytochemistry (bar 50 m). e TEM detail of the coelomic epithelium
of the stump showing the strong specific reaction for BrdU at the
level of the nuclei. Immunogold labelling (cl cilium, bar 1 m).
f TEM detail of a rearranging muscle in the stump showing the
BrdU reaction in the nucleus of a presumptive dedifferentiating
myocyte. The remains of the contractile apparatus are still recognizable (arrows). Immunogold labelling (bar 1 m)&ig.c:/f

427

428

Fig. 5ac Growth factors in arm explants at 12 weeks p.a. S1and S2- SALMFamide-neuroptides and TGF- immunocytochemistry. a Confocal fluorescence image of semi-thin sagittal section
of the intermediate stump (1 week p.a.). Detail of the brachial
nerve. A positive immunoreaction for S2-neuropeptide specifically
involves neural cells and processes. Secondary antibodies conjugated with Texas red (bar 20 m). b Semi-thin sagittal section of
the distal regenerating bud (1 week p.a.). Detail of the coelothelium (ce) and of the ambulacral epithelium (ae). Double staining for
S1-neuropeptide (Texas red-conjugated secondary antibody) and
BrdU (FITC-conjugated secondary antibody). The immunoreaction for the peptide involves scattered cells and processes of the
basiepithelial nerve plexuses (orange) of both the coelothelium
and the ambulacral epithelium. Many cells of the coelomic epithelium are also strongly reactive for BrdU (green) (bar 20 m). c
LM semi-thin sagittal section at the level of the distal amputation
(2 weeks p.a.). ABC immunocytochemistry for TGF-. The detail
shows some cells positive for TGF- (arrows) at the level of the
regenerating blastema (rb) and the brachial nerve (n, cc coelomic
canals, bar 50 m)&ig.c:/f

of the nervous system (brachial nerve and basiepithelial

nerve plexuses of both the ambulacral epithelium and the
coelothelium) with only a few minor differences. During
regeneration the pattern of reactivity of these peptides is
re-established in parallel with the regeneration of the
nervous system (Bonasoro et al. 1995; Candia Carnevali
et al. 1998). This also occurred in the explants where the
reaction for both peptides was more or less comparable
to that shown by the respective regenerating donor arms.
The immunoreaction was particularly evident during the
advanced regenerative stages when the nerve began to regrow in the distal regenerating bud. In the explants of 1
week (Fig. 5a,b) labelling for both S1 and S2 was particularly strong in the brachial nerve and was also appreciably intense in the basiepithelial nerve plexuses of both
the ambulacral epithelium and the coelothelium, especially in the regenerate. The employment of double
staining techniques for S1 and BrdU (Fig. 5b) allowed us

to discriminate neural elements from proliferating cells

in the nervous system. Whichever method was employed, there was no significant immunoreactivity in the
blastema for peptides. The role of putative growth factors was explored in a preliminary fashion with particular reference to TGF-. According to our previous results
TGF-, or at least an antigen which cross-reacts positively with specific antisera against this factor, is not only
normally present in the different components of the nervous system in normal non-regenerating arms, but is significantly involved in regeneration, with a markedly enhanced and diffuse reaction in both cells and processes
of the nervous system and at the level of the blastema itself (Candia Carnevali et al. 1998). In the explants, although the reaction for TGF-, even in the advanced regenerative stages, was generally weaker and less diffuse
than in normal regenerating arms, a significant labelling
(Fig. 5c) could be observed at the level of the distal amputation, especially in the advanced regenerative stages
(2 weeks) and involved both nervous system components
(nerve processes and granule cells) and the distal regenerative blastema.

Discussion
Our present results show that the crinoid explant is potentially a valuable model for studying regenerative
mechanisms at many levels. It is a system endowed with
striking autonomy and is able to manage and control extensive repair and regenerative processes utilizing bidirectional phenomena of cell proliferation and migration.
BrdU incorporation confirms that in explants the overall
repair/regeneration process is due to extensive cell proliferation at preferential sites. As in normal regenerating
arms, these are the terminal blastema and, even distant

429

from the amputation zone, the coelomic epithelium and

brachial nerve. Apart from the blastema, the primary
sources of new cells are, therefore, the major continuous
structures along the arm. The two main cell components
which contribute to the regenerate seem to have a different derivation: the blastemal cells (and all blastemal-derived cells) from amoebocytes, whereas the coelomic
cells arise from the migratory coelomocytes which in
their turn derive from proliferation of the coelomic epithelium. In contrast to normal regenerating arms, however, the recruitment of new cells contributing to the regenerating tissues also involves the rearrangement of differentiated tissues of the stump, in particular the muscles. It
is not clear at present if this represents direct dedifferentiation of myocytes to give new migrating coelomocytes
or if it is a more complex phenomenon mediated by
phagocytes, in which the myocytes are only passively involved as sources of raw materials for the production of
new coelomocytes from the coelomic epithelium. Whatever mechanism is involved, it is clear that in explants
morphallactic mechanisms play a significant role. Thus,
blastemal regeneration of Antedon arm seems to be a
phenomenon much more plastic than expected and involving a variety of cell recruitment mechanisms. It normally invokes the intervention of presumptive stem cells
and trans-differentiation of differentiated elements from
the coelomic epithelium, but, in extreme cases, it can
also involve a significant contribution of highly differentiated tissues via extensive cell rearrangement and dedifferentiation.
Utilizing a suitable combination of these fundamental
mechanisms the explant can undergo (1) complete repair
at the proximal amputation site, without subsequent regenerative phenomena, that is without developing a regenerative blastema in the distal-proximal direction; (2)
complete repair and subsequent regeneration at the distal
amputation site, by developing a typical regenerative
blastema in the proximal-distal direction. This continues
to grow up to at least 2 weeks p.a. following the times,
modalities and developmental processes comparable to
those described previously in regenerating donor arms.
Therefore, although the basic mechanisms of cell proliferation/migration occur in both distal-proximal and proximal-distal directions, the normal developmental processes in terms of growth, morphogenesis and differentation appear to be strictly directional and, even in the
double-amputated explants, maintains a strict proximaldistal axis. This significant difference in terms of regenerative potential of the two symmetrical amputations
thus appears not to be due to differential capacities of
cell proliferation/migration, but must be genetically programmed and is possibly regulated by the differential expression of signal molecules and growth factors. The
strictly directional blastemal regeneration of the explant
is an important basis to exclude the possibility that crinoids are spontaneously able to perform reconstructive
mechanisms comparable to strategies of asexual reproduction. On the other hand, it is relevant to point out
that, since crinoid arms normally undergo continuous

apical growth, developmental processes in the arms have

to be always maintained, though slowly, throughout life.
Their acceleration during regeneration is possibly due to
the stimulatory action of specific factors and growth regulators. As pointed out above, current work is focusing
on the intervention of such presumptive growth factors in
regenerative processes. The contribution of the brachial
nerve and the coelom during regeneration potentially invokes not only a prompt cell supply but also the release
of presumptive growth factors. With regard to neuropeptides, our previous and present results suggest, in particular, a basic physiological, neurotrophic and modulatory
role for S1 and S2 peptides at the level of the nervous
tissue perhaps without a significant involvement in regeneration itself. On the other hand, with respect to
TGF-, we can confirm the hypothesis that also in
echinoderms this factor can be considered not only as a
constitutive neurotrophic factor playing an important
fundamental role in development, repair, maintainance
and regulation of neuronal function, but possibly also as
a broad-spectrum multifunctional regulator of cell proliferation and differentiation which plays a key role during
regenerative developmental processes (Logan et al.
1994). The results obtained for the explants suggest, in
particular, that arm regeneration in crinoids is largely dependent on a remarkable functional autonomy of the arm
system not only in terms of cells and tissues involved,
but also in terms of regulative mechanisms and molecules implied. In fact, apart from possible quantitative
variations which have still to be demonstrated, these
seem to be controlled by the same type of substances on
the basis of a comparable pattern of specific tissue distribution.
In conclusion, our studies of explants show clearly
that the regenerative potential of crinoid echinoderms is
much wider than expected and suggests a remarkable
flexibility of mechanisms. In the light of these results,
epimorphic and morphallactic mechanisms, which are
traditionally considered contrasting strategies of regeneration (Bonasoro et al. 1998), lose their defined boundaries. In other words, the direct recruitment of undifferentiated stem cells, or the indirect recruitment of differentiated elements, previously dedifferentiated and/or
transdifferentiated, seem to be alternative mechanisms
which can be employed by the same organism and the
same structure according to local conditions. Both, however, produce an identical result: the development of a
regenerative blastema formed by undifferentiated cells
which proliferate actively under the control of regulatory
molecules.
&p.2:Acknowledgements The present work has received financial support from: (1) Consiglio Nazionale delle Ricerche, (CNR), Roma;
(2) MURST Research Project 1997-98: Biologia ed Evoluzione
del riconoscimento e delle interazioni nelle cellule animali; (3)
British-Italian Collaboration Grant for Research and Higher Education (The British Council/MURST) 1996-97.

430

References
Amemiya S, Oji T (1992) Regeneration in sea lilies. Nature
357:546547
Bonasoro F, Candia Carnevali MD, Thorndyke MC, Welsch U
(1995) Neural factors in crinoid arm regeneration. In: Emson
R, Smith AB, Campbell AC (eds) Echinoderm research 1995.
Balkema, Rotterdam, pp 237243
Bonasoro F, Candia Carnevali MD, Patruno M, Sala F (1997) Potenzialit rigenerative in espianti di braccia di crinoidei (Antedon mediterranea). Proceedings of the 58th Congresso U.Z.I.,
Cattolica, Italy 1997, p 67
Bonasoro F, Candia Carnevali MD, Moss C, Thorndyke MC (1998)
Epimorphic versus morphallactic mechanisms in arm regeneration of crinoids and asteroids: pattern of cell proliferation/differentiation and cell lineage. In: Mooi R, Telford M (eds)
Echinoderms: San Francisco. Balkema, Rotterdam, pp 1318
Candia Carnevali MD, Bonasoro F (1994) Mechanisms of arm regeneration in Antedon mediterranea (Echinodermata, Crinoidea). Anim Biol 3:8388
Candia Carnevali MD, Bonasoro F (1995) Arm regeneration and
pattern formation in crinoids. In: Emson R, Smith AB, Campbell AC (eds) Echinoderm research 1995. Balkema, Rotterdam, pp 245253
Candia Carnevali MD, Bruno L, Denis Donini S, Melone G (1989)
Regeneration and morphogenesis in the feather star arm. In:
Kiortsis V, Koussoulakos S, Wallace H (eds) Recent trends in
regeneration research. (Nato Asi Series, vol 172) Plenum
Press, New York London, pp 447460
Candia Carnevali MD, Lucca E, Bonasoro F (1993) Mechanisms
of arm regeneration in the feather star Antedon mediterranea:
healing of wound and early stages of development. J Exp Zool
267:299317
Candia Carnevali MD, Bonasoro F, Lucca E, Thorndyke MC
(1995) Pattern of cell proliferation in the feather star Antedon
mediterranea. J Exp Zool 272:464474
Candia Carnevali MD, Bonasoro F, Invernizzi R, Lucca E, Welsch
U, Thorndyke MC (1996) Tissue distribution of monoamine
neurotransmitters in normal and regenerating arms of the feather star Antedon mediterranea. Cell Tissue Res 285:341352
Candia Carnevali MD, Bonasoro F, Biale A (1997) Pattern of bromodeoxyuridine incorporation in the advanced stages of arm

regeneration in the feather star Antedon mediterranea. Cell

Tissue Res 289:363374
Candia Carnevali MD, Bonasoro F, Welsch U, Thorndyke MC
(1998) Arm regeneration and growth factors in crinoids. In:
Mooi R, Telford M (eds) Echinoderms: San Francisco. Balkema, Rotterdam, pp 145150
Elphick MR, Price DA, Lee TD, Thorndyke MC (1991a) The
SALMFamides: a new family of neuropeptides isolated from
an echinoderm. Proc R Soc London ser B 243:121127
Elphick MR, Reeve JR Jr, Burke RD, Thorndyke MC (1991b) Isolation of the neuropeptide SALMFamide-1 from starfish using
a new antiserum. Peptides 12:455459
Endean R (1966) The coelomocytes and coelomic fluids. In: Boolootian RA (ed) Physiology of Echinodermata. John Wiley,
London New York, pp 301328
Gratzner GH (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: a new reagent for detection of DNA replication.
Science 218:474475
Holland ND (1994) Cell cycle subdivisions in regenerating arm
blastema of a feather star (Antedon mediterranea). In: David
B, Guille A, Feral JP (eds) Echinoderm through time. Balkema, Rotterdam, pp 217220
Logan A, Oliver JJ, Martin B (1994) Growth factors in CNS repair
and regeneration. Prog Growth Factor Res 5:379405
Mladenov PV (1996) Environmental factors influencing asexual
reproductive processes in echinoderms. Oceanologica Acta
19:227235
Mladenov PV, Burke RD (1994) Echinodermata: asexual propagation. In: Adiyodi KG, Adiyodi RG (eds). Asexual propagation
and reproductive strategies. (Reproductive biology of Invertebrates, vol VI B) Oxford and IBH (Put), New Delhi Bombay
Calcutta, pp 339383
Moss C, Hunter AJ, Thorndyke MC (1998) Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity
in the regenerating arm of the starfish, Asterias rubens. Philos
Trans R Soc London ser B, 353:421436
Perrier E (1873) Lanatomie et la rgnration des bras de la comatula. Arch Zool Exp Genet 2:2886
Reichensperger A (1912) Beitrge zur Histologie und zum Verlauf
der Regeneration bei Crinoiden. Z Wiss Zool 101:169
Smith VJ (1981) The echinoderms In: Ratcliffe NA, Rowley AF
(eds) Invertebrate blood cells, vol 2. Academic Press, London
pp 513562