The regenerated RNC
Our results clearly show that the RNC of holothurians can regenerate
within a month following a transection. The regenerated RNC looks
amazingly similar to the non-injured RNC as judged by several
histological and cellular parameters: It is well organized into
ectoneural and hyponeural components, there is no scar or fibrotic
tissue, the number of cells is similar to those found in uninjured
tissue, neurons are localized to the nerve periphery, and
spherule-containing cells that have infiltrated the injured cord have
all but dissapeared. This is an amazing regeneration feat for an adult
deuterostome, particularly if we consider that the RNC conforms the
echinoderm CNS [24,29,30] and has been proposed to be evolutionary related to the vertebrate CNS [36-38].
Although we lack direct proof, a large amount of circumstantial
evidence suggest that regeneration is not solely the joining back of
the severed nerve stumps but that actually a new portion of the RNC is
generated. First, from the moment it is cut, the wound forms a V-shaped
structure where the nerve stumps are initially separated by a gap, and
then by connective tissue. Second, most of the ongoing cellular
division occurs within the regenerating RNC. Third, other tissues that
surround the nerve and that also regenerate, do so by forming new
tissues. This is particularly true of the surrounding muscle and
connective tissues, which regenerate in parallel to the RNC and where
very small differences in organization, cell density and texture
distinguish the "new" tissues from the adjacent "old" .
Fourth, the initial and most visible regeneration event is the
outgrowth of fibers from the RNC stumps. These outgrowths increase with
time and eventually completely fill the space between the stumps. The
latter retain their original morphology throughout regeneration, and it
is the space in between that grows and becomes organized. Finally,
recent work in another holothurian, Eupentacta fraudatrix, also shows regeneration of the RNC to be newly formed tissue .
In some ways, the formation of the new RNC can be compared to the
"new" RNC segments that grow when the tip of the arm is severed in
starfish or crinoids [19,20,22].
In all these cases the regenerated arm tip eventually grows to form new
segments that include all the components found in the RNCs.
Experimental evidence for the formation of a new RNC also comes from
other closely related deuterostomes, the ascidians, that have an
impressive ability to regenerate main components of their nervous
system as has been shown by Bollner and colleagues [17,18]
in studies where the main neural ganglion regenerates afer ablation.
Similarly, lower vertebrates are able to regenerate a lost segment of
the spinal cord as has been shown by nerve cord injury in amphibians [39,40] and fish .
Phylogenetic analysis of regeneration following transection
How does the regeneration of the holothurian RNC compares to that
shown by other animals? To answer this question we will focus on those
experiments where injury to the CNS is done by transection of the main
nerve cord (or spinal cord). This includes studies in protostomes, such
as mollusks and annelids and lower vertebrates, such as fish and
urodele amphibians. It also includes developing vertebrates, since
experiments have shown that spinal cord regeneration can occur at
particular developmental stages of some higher vertebrates, such as
chicks or marsupials, and then becomes restricted as development
RNC regeneration in H. glaberrima shows strong similarities
to other animal species known to be capable of axonal regeneration
and/or formation of a missing part of their CNS. In most species where
CNS regeneration takes place, two events can be highlighed to occur:
the outgrowth of fibers from the nerve stumps and the absence of
fibrotic tissue or glial scar. The growth of fibers into the lessioned
area provides the first indication of the regeneration process. As has
been reported by TEM in the sea cucumber E. fraudatrix  and by immunohistochemistry in the starfish, Asterias rubens ,
these fibers originate from the lesioned stumps and move across the
lesion eventually forming a bridge between the two nerve stumps.
Similarly, in urodele larvae, fibers extending from each of the cut
ends grow across the gap and eventually form a bridge connecting the
two nerve ends .
New fibers are mainly attributed to the re-growth of fibers from
lesioned cells as has been shown by retrograde labeling at the time of
Another common observation in regenerating animals, including
holothurians, is the fact that there seems to be a directionality to
the process, since fibers extending from one stump are directed toward
the opposite stump and are rarely observed to be traveling toward
another target, suggesting the participation of soluble factors
originating from the severed stumps and/or guidance molecules in the
The absence of a glial scar or fibrotic tissue in regenerating cords
is another characteristic of those animals capable of regenerating
their main nervous system. No scar tissue is detected or described in
studies showing regeneration in urodeles, fish or echinoderms.
Similarly, chick embryos that are capable of regeneration do not form
scar tissue .
In fact, what investigators regularly show to occur during the
regenerative response is an increase in cells or cell activity
associated with the removal of the injury debris and of apoptotic cells.
There are also differences in the regeneration process among
animals. One of these is the level or extent at which regeneration
occurs. In holothurians, the regenerated RNC appears almost indistinct
from the non-injured RNC. However, in other organisms where it has been
studied, the regenerated cord is not fully equivalent to the normal one
in spite of fibers crossing the lesion site. For example, in the
regenerating chick embryo, fibers do cross the transection site and
functional regeneration occurs, but the site of transection shows a
slight disorganization of the gray matter .
Another example is the regenerating spinal cord of bullfrogs that have
been transected as tadpoles, where the transection site is still
visible 8 months after injury (and 6 after metamorphosis), showing a
considerable constriction in size .
The differences can be striking, as occurs in the lizard where it has
been shown that the regenerated spinal cord in the tail is considerably
different from the uninjured cord and that it only contains descending
fiber tracts from the old cord and scattered glial cells .
Other species show subtle differences, as in the case of regenerating 1
year-old goldfish, where the regenerated spinal cord appeared normal
after 90 days post injury, but there were no motor horn cells in the
regenerated area . In H. glaberrima,
the regenerated RNC has NURR 1 and GFS expressing neurons (albeit in
lower numbers) and appears to be normal according to all anatomical
parameters used. Nonetheless, more detailed experiments are needed to
fully determine that all cell populations and fiber connections are
Finally, it is important to acknowledge that structural differences
do exist in the regenerating nervous systems of animals. The echinoderm
ganglionated nerve cord differs greatly from the regenerating nerves of
some invertebrates where only fibers are found, and from the complex
cellular and fiber organization of the vertebrate spinal cord. Thus, it
remains unclear whether similar events occur during the regeneration of
the different structures. Nonetheless, it will be by performing
comparative studies, such as this one, that the commonalities and
differences in nervous system regeneration among animals will be
Role of Cell division and apoptosis
Two related and at the same time, opposite processes are occuring
within the RNC during regeneration; cell proliferation and cell death.
The two show a spatial and temporal profile that seems perplexing
because they seem to be occurring simultaneously; both show a peak in
the nerve stumps at 6 dpi and a peak within the regenerating RNC at 12
In vertebrates, apoptosis is associated with
neurons that have been injured at the time of transection, but also
with a period of secondary injury where the lesion size increases due
to inflammation, immune-related events and hemorrhage [47-49].
Therefore, in our system, apoptosis within the nerve stumps seems to
occur in cells whose fibers were damaged by the transection and that
eventually failed to survive, undergoing apoptosis during the first
week following injury. This was also observed at the TEM level in
regenerating RNC of E. fraudatrix  and in regenerating fish CNS where apoptotic cells are seen for a long period of time following the lesion .
A different explanation might be involved in explaining the increase
in apoptosis observed within the regenerating RNC in the second week
following the transection. In this case the apoptosis event might be
similar to what has been shown to occur during embryonic development in
vertebrates, where about half of the neurons that are formed in the
spinal cord undergo apoptosis . Neuronal death in regenerating spinal cord has also been reported in the tail of the fish Sternachus [1,52].
In these fishes, younger segments of the regenerated spinal cord have a
larger number of neurons than older segments, once again suggesting
that the regenerating spinal cord recapitulates the known embryonic
developmental process of overproducing neurons that will eventually be
Cell division has been shown to occur in the regenerating arms of several echinoderms, mainly crinoids and starfish [20-22].
In crinoids, the nerve stump is also an active place of cell
proliferation, although the dividing cells in the early stages appear
to be non-neuronal . In the starfish Asterias rubens,
it was found that cell division in the regenerating nerve began 6–7
days following transection and increased during the next 2 weeks,
gradually decreasing to control levels by day 60 post-injury .
Similarly, our results show cell division to occur within the
ectoneural and hyponeural components of the nerve stump, but even more
within the regenerating RNC. However, it is still unclear what type(s)
of cells are proliferating since support cells, macrophages or some
type of precursor cell could be accountable for the extent of cell
division. This is a crucial issue that will be addressed below.
Nonetheless, in recent studies Mashanov and colleagues  describe neurons and glial cells undergoing division in the regenerating RNC of E. fraudatrix.
The overall pattern of cell division within the regenerating RNC has
some similarities to what has been shown to occur in the adjacent
muscle and connective tissue, where cell division peaks between 6 and
12 days following injury .
These two weeks of extensive cell proliferation are undoubtly
responsible for the finding that, at 30 dpi, animals have the same
number of cells in the regenerated and in the uninjured RNCs.
Formation of new neurons has been shown to be an important process
in animals where CNS regeneration is known to occur. In fish, BrdU
labeling together with back-tracing techniques have shown that in the
adult fish, neurons are regenerated from proliferating precursors
following CNS injury . In urodeles, new neurons are known to originate from proliferating ependymal cells .
Whether new neurons take part in the regeneration of the spinal tract
observed in the chick embryo and the neonateal opposum is not known .
However, recent results suggest that stages where the ability to
regenerate is still present, coincide with those stages where there is
ongoing formation of new neurons .
On the other hand it is important to emphasize that some vertebrates
can undergo functional regeneration with little or no contribution of
neurogenesis, or that new neurons can originate from undifferentiated
In view of these findings, our results showing that neurons are
present within the regenerated RNC and that some originate from
dividing precursors provide for interesting comparisons to other
systems. At the same time we must reconcile the finding that the
densities of GFS and NURR1 neurons are lower in the regenerated RNC.
One possible explanation is that the appearance of neurons in the
regenerated RNC is a late event and that at 62 dpi some neuronal
precursors have not fully differentiated. This would be consistent with
experiments in starfish, where the first neurons observed in the
regenerated segment following transection of the RNC were not observed
until 28 days following injury . These results might appear to contradict the observations by Mashanov and colleagues  in E. fraudatrix,
who observed some neuronal division in the early stages of RNC
regeneration, however, this might be explained when we take into
account that early division was observed using TEM, while we used
markers for differentiated neurons. Nonetheless, it is important to
emphasize that in both E. fraudatrix and H. glaberrima neurons
appear in the regenerated RNCs and that at least some of these neurons
originate from dividing precursors. Similarly, in crinoid regenerating
arms, some BrdU labeled neurons were recognized using electron
microscopy, suggesting that in this echinoderm, neurons also originate
from dividing precursors .
However, there are striking differences between neuronal populations
since the GFS neurons were not observed to express BrdU, suggesting
that they do not originate from dividing precursors. Once again this is
consistent with studies in the transected starfish RNC where none of
the neurons identified with the S1 neuropeptide (the starfish
equivalent of GFS) showed BrdU labeling, even when enough time was
allowed following the BrdU treatment for differentiaton to occur .
In order to fully identify the origin of neurons it will be
important to determine the role of glia in holothurian RNC
regeneration. Particularly in view that in E. fraudatrix Mashanov
and colleagues propose that glial cells might be paving the path for
neuron and fiber migration and even differentiating into neurons .
The role of the glial cells is not the only question that remains to be
studied; several other questions remain to be answered. For example,
does the observed anatomical regeneration provides for complete
functional recovery? In many other model systems the extent of
regeneration has been evidenced by backtracing neuronal connections and
comparing the regenerated versus the uninjured connections. However, in
view that little is known about the echinoderm radial nerve circuitry
and nervous system physiology, it is not possible to determine at
present to what extent there is morphological connectivity. Similarly,
in other animals functional recovery can be documented by behavioral
tests, that, at present, are not available for holothurians.
Comparison with other wound healing/regenerative processes
Transection of the nervous system, whether experimentally induced or
by accidental cause rarely, if ever, exclusively affect the nervous
system. Thus, it is always important to view the regeneration of the
nervous component as a process that occurs parallel to the regeneration
of surrounding tissues. Granted, there are nerve-specific processes,
such as axonal regeneration and synaptic specificity, that only occur
in the nervous system. However, it is interesting that events that
occur in the surrounding muscle and connective tissue previously
published by our group 
parallel those occuring within the nerve cord itself. One of them has
already been addressed above: the pattern of cell division, showing
that in muscle, coelomic epithelium and connective tissue the
proliferation pattern coincides with that observed in the regenerating
Another event that provides for an interesting comparison is the
presence of morula or spheruloytes cells within the RNC. These cells
are rarely observed in uninjured nerves, however, their numbers
increase after injury. This increase in cell numbers parallels what
occurs within the regenerating intestine  and following the transection injury in the adjacent tissues .
These cells have been proposed to play important roles in extracellular
matrix remodeling, phagocytosis and antimicrobial activity, among
others . The presence of these cells within the regenerating nerve is quite intriguing and should be studied in further detail.
The extent of apoptosis observed after nerve transection can also be
compared to what occurs during intestinal regeneration. We have
recently completed a study of apoptosis during intestinal regeneration
showing that high levels of apoptosis take place in the mesothelium 7
days following evisceration (unp. Observation). In both events, nerve
regeneration and intestinal regeneration, apoptosis is rarely observed
in the early days (2–5 days) following the injury or evisceration but
occurs more likely during the second week (7–14 days). Moreover,
apoptosis is more likely to take place within "new" tissues that have
regenerated such as the newly formed intestinal blastema-like structure
and the regenerated RNC, than in adjacent "old" tissues, such as the
remaining nerve stumps or the intestinal mesentery.
For many investigators involved in studies of neurogenesis or
neuronal regeneration, their focus has been strictly on the nervous
system. However, our previous results show that the same events that
occur during wound healing, also occur during intestinal regeneration .
And now we show that some of these events also occur during nervous
system regeneration. Moreover, at the species level, the same species
that can successfully regenerate their nervous systems are known to be
exceptional regenerators of others tissues or organs, not just the
nervous system. Thus, a major implication of our work is that the
capacity to regenerate nervous system might be determined by the same
processes and mechanisms in all tissues and that some animals have
developed or retained these capabilities while in others they are
absent. Although there are obvious exemptions to this statement, such
as liver regeneration in vertebrates, the general rule remains that
animals that have some capacity to regenerate their nervous system are,
in general, good regenerators of many tissues or organs. If this is
correct, then investigators interested in deciphering the mysteries of
nervous system regeneration should also focus on regenerative or wound
healing phenomena in other tissues and organs and on species where
these regenerative processes are prominent.
JSMR carried out most of the experiments and helped in the analyses
of the data and preparation of the manuscript. ARMS performed the
birthdating experiments, analysed the data, and prepared the
corresponding figures. JEGA conceived the study, participated in its
design, data analyses and helped in the manuscript preparation. All
authors read and approved the final manuscript.
The authors would like to thank Dr. V. Mashanov for helpful comments
on the manuscript. Funded by NSF (IBN-0110692), NIH-MBRS (S06GM08102),
NIH-1SC1GM084770-01), and the University of Puerto Rico. JSMR and ARMS
are fellows of the NIH-MARC Program.