Download Regeneration in Vertebrates

Developmental Biology 221, 273–284 (2000)
doi:10.1006/dbio.2000.9667, available online at http://www.idealibrary.com on
REVIEW
Regeneration in Vertebrates
Panagiotis A. Tsonis
Laboratory of Molecular Biology, Department of Biology, University of Dayton,
Dayton, Ohio 45469-2320
One way or another, all species possess the ability to regenerate damaged tissues. The degree of regeneration, however,
varies considerably among tissues within a body and among species, with urodeles being the most spectacular. Such
differences in regenerative capacity are indicative of specific mechanisms that control the different types of regeneration. In
this review the different types of regeneration in vertebrates and their basic characteristics are presented. The major cellular
events, such as dedifferentiation and transdifferentiation, which allow complex organ and body part regeneration, are
discussed and common molecular mechanisms are pinpointed. © 2000 Academic Press
INTRODUCTION
The fascination with regeneration of body parts has
mythological origins in ancient times. Stories of regeneration are prominent in mythologies from many different
cultures. In more recent times and especially in the 18th
century, description of regenerative properties of animals
formulated by Trombley, Reamur, Bonnet, and Spallanzani
set the foundation for the birth of developmental biology
and shaped the thought on reproduction (Dinsmore, 1991).
During the past 25 years the field of limb regeneration
provided the most concrete model for formulating concepts
of pattern formation and positional information (Bryant et
al., 1981). Regeneration, however, is a broad field with
important idiosyncrasies. Not all animals are able to regenerate body parts, and not all tissues within a body can be
equally repaired. Several important questions are in need of
answers. What are the different types of regeneration and
how do they differ? What determines the potential of tissue
regeneration in an animal or among different species? Are
there common features or mechanisms among the different
types of regeneration? It is the goal of this review to address
these questions and to present a useful synthesis for the
students of regeneration.
TYPES OF REGENERATION
Not all tissues are equal in their regenerative potential or
magnitude or in the mechanisms involved (Goss, 1969;
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Stocum, 1995). Having this as a starting point it is necessary
to categorize the different types of regeneration and to
pinpoint differences and similarities in the different types
of regeneration. The reward is twofold. First, by defining the
criteria, the mechanisms involved in the different types of
regeneration can be more clearly defined. As a result, the
exercise can provide insights concerning commonality.
Having established such a relation between the different
types of regeneration, it could create the necessary links
and cross-talk between different disciplines and bring together scientists from different areas, which is most imperative for the success of regeneration research.
Apart from wound healing (or wound repair), which is
mostly closure of a wound by scar tissue, the degree of
tissue renewal or regeneration in vertebrates varies in
different tissues. In fact what is different is the complexity
that is involved in the mechanisms and magnitude of
regeneration. The simplest form of regeneration is the
axonal outgrowth seen in severed nervous system. Regeneration by simple proliferation seen in organs, such as
intestines, liver, or adrenal gland, is somewhat more complex. It involves proliferation of cells that compose the
particular organ. Regeneration of other organs and tissues
on the other hand can be channeled through the proliferation and differentiation of stem cells. More complex types
of regeneration involve the process of dedifferentiation. In
these cases (mostly seen in amphibia) cells at the damaged
site dedifferentiate and then redifferentiate into their type.
Regeneration of CNS (brain, spinal cord), intestine, and
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Panagiotis A. Tsonis
TABLE 1
Examples of Tissue and Organ Regeneration
Organ
Source of cells
Axonal outgrowth
CNS (spinal cord)
CNS (auditory system)
PNS
Species
Rats
Experimentally induced
(⫹FGF, NGF),
neurotrophins, Nogo
inhibition
Inner hair cells
Axonal outgrowth
Rats
Rats
Cell proliferation
Proliferation of liver
cells
Proliferation of
glomerulosa cells
Proliferation?
Dedifferentiation?
Mammals
Mammals
Rats
Stem cells
Mammals
Stem cells
Mammals
Epidermis
Liver
Stem cells
Stem cells
Mammals
Mouse
Muscle
Satellite cells
Amphibia, mammals
Proliferation
Gastrointestinal mucosa
Liver
Adrenal
Small intestine
Stem cells
Intestine (small and
large)
Cartilage/bone
Dedifferentiation
CNS (brain, spinal cord)
Ependymal cells form
blastema and back
ependymal cells
Intestine
Serosal and smooth
muscle produce
blastema by
dedifferentiation
Heart
Dedifferentiation of
cardiomyocytes
Dedifferentiation, transdifferentiation
Pancreas
Acinar or duct cells
Remarks/factors
PNS better than CNS
(affinity of cells?)
Rats
Cheng et al., 1996;
Oudege and Hagg,
1999; Grandpre et
al., 2000; Chen et
al., 2000
Ito et al., 1998
Pestronk et al., 1990;
Daston and Ratner,
1991
TGF-␣ EGF
Jones et al., 1999
Michalopoulos and
DeFrances, 1997
Engeland et al., 1996
Experimentally induced
(EGF, HGF)
Kim et al., 1999
Inhibition of
hepatocyte division,
differentiation to oval
cells
Amphibia, fishes
References
Booth et al., 1999;
Sattar et al., 1999
Prockop, 1997;
Pittenger et al., 1999
Cotsarelis et al., 1999
Petersen et al., 1999
Cameron et al., 1986;
Hansen-Smith and
Carlson, 1979
EGF, PDGF, TGF-␤
O’Hare et al., 1992
Amphibia
O’Steen and Walker,
1962
Amphibia
Oberpriller and
Oberpriller, 1991
Mammals
Dedifferentiation, transdifferentiation, pattern formation
Eye (retina, lens)
Pigment epithelium;
Amphibia
cornea
Limb
Blastema
Amphibia
Limb
Blastema
Mouse
Transdifferentiation
(Tgf-␤, Hox genes)
Bouwens, 1998; Menke
et al., 1999; Sharma
et al., 1999
FGF
Tsonis, 1999; Tsonis,
2000
Tsonis, 1996
Reginelli et al., 1995
Nerves, FGF
Msx-1
Note. When “Mammals” appears, studies may include more than one species and even species lower than mammals.
heart can be achieved by this mechanism of dedifferentiation. A more complex type of regeneration involves dedifferentiation and transdifferentiation and can be seen during
pancreas regeneration. In this type, acinar and/or duct cells
dedifferentiate and then transdifferentiate into insulinexpressing beta cells, thus reconstructing the lost part of
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Regeneration in Vertebrates
the organ. The most complex type of regeneration, however, is seen in limb and eye regeneration (especially in
amphibia) in which cells at the damaged or amputated site
dedifferentiate and transdifferentiate, but they are also able
to build back an exact replica of the lost part. This type
involves the mechanisms of normal pattern formation as
well. Historically, regeneration of amphibian limbs, and
also regeneration of appendages or whole body parts in
invertebrates, has been termed as epimorphic, while the
rest of regeneration has been called tissue regeneration.
Such a distinction, however, has not always been clear-cut
and should be used as such only for the sake of convenience.
Examples of regeneration grouped according to the increasing complexity are outlined in Table 1. In this table remarks
on factors and mechanisms are also presented. It is obvious
from Table 1 that overlapping mechanisms can control the
different types of regeneration. For example, in both amphibian heart and limb regeneration muscle dedifferentiation is common, but limb regeneration is more complex
because it involves the additional steps of transdifferentiation and pattern formation. These ideas could be very
helpful in identifying common criteria and mechanisms in
the different types of regeneration.
As mentioned above, regeneration of amphibian lens,
retina, and limb is the most complex and, therefore, the
most spectacular of all. Such regeneration is possible by the
recruitment of many different mechanisms involved in the
process of dedifferentiation, transdifferentiation, and faithful pattern formation of the regenerated structure. Dedifferentiation is the most basic element of such form of regeneration. It entails terminally differentiated cells reentering
the cell cycle and losing the typical characteristics of their
origin. The only parallelism is when a normal cell is
transformed to a cancer cell. Transdifferentiation allows a
cell to change its identity and become a different cell.
Regenerating an exact copy of a pattern that was lost
involves all the marvels of embryogenesis where formation
of tissues, organs, and whole body structures are controlled
by a unique weaving of genetic programs. Having introduced the basic types of regeneration and outlined examples
and mechanisms, the concepts of dedifferentiation, transdifferentiation, and pattern formation will be further explored in this review. For this I will concentrate on the
paradigms of amphibian retina, lens, and limb regeneration,
which best represent these concepts. Furthermore, as it will
become apparent below, these regenerative processes might
be controlled by similar mechanisms, and this might help
elucidate the crucial molecular pathways that have en-
dowed amphibia with such outstanding regenerative abilities.
RETINA REGENERATION
During eye development, the optic vesicle that originated
from the anterior neural tube can give rise to either neural
retina, a multilayered structure containing photoreceptors
and neurons, or pigmented epithelium consisting of a single
layer of nonneuronal, pigmented cuboidal cells. The anterior part of the optic vesicle that is in closer contact with
the ectoderm becomes neural retina. Such an induction is
likely to be controlled by fibroblast growth factor (FGF),
which is found in abundance in the ectoderm and which has
been shown to induce the neural phenotype in cultures of
optic vesicle explants (Reh and Pittack, 1995). Retina regeneration takes place in a variety of animals, such as fishes,
birds, amphibia, and mammals (Mitashov, 1996). The
source of retina cells in fishes, frogs, and mammals seems
to be precursor cells in the peripheral growth zone of the
dorsal and ventral iris, the same cells that support normal
growth throughout life. The most spectacular mode of
retina regeneration, however, can be observed in chicks and
amphibia, and it occurs by the transdifferentiation of the
retinal pigment epithelial cells to all types of neural retina
cells. In embryonic chicks (stage 22–24) such a transdifferentiation process can occur only if some neural retina is left
behind. If all neural retina is removed, regeneration occurs
only by treatment with ␤-FGF (Park and Hollenberg, 1989).
FGF, therefore, must be the most important factor in the
induction of transdifferentiation and neural retina regeneration. The stages of these events are as follows. After
dedifferentiation, the depigmented cells start dividing. One
cell commits itself to the restoration of pigment epithelium
and the other transdifferentiates to neural cells. First the
photoreceptor cells are produced, then the cells of the
amacrine layer and finally the ganglion cells (Fig. 1). In vitro
systems for retina regeneration in which pigment epithelium explants can transdifferentiate to neural retina have
been established. Using such in vitro system the effect of
FGFs in inducing transdifferentiation has been verified (Reh
and Pittack, 1995). Little is known about gene regulation
during these events. Obviously FGF must down-regulate
RPE cell-specific gene expression. Two transcriptional factors are known to control pigment epithelium identity. One
is the product of Mitf (microphthalmia) and the other the
product of pax-6 gene. Mutations in Mitf in chick embryos
FIG. 1. The process of dedifferentiation during retina and lens regeneration in the adult newt. (a) A section through an adult newt retina
showing the three layers of the neural retina, photoreceptors (p), amacrine (am), and ganglion cells (g). The dark pigment epithelium (arrows)
lies over the photoreceptor layer. (b) The eye after retinectomy. The neural retina has been completely removed and the pigment epithelium
(arrows) is intact. (c) Dedifferentiating pigment epithelium and transdifferentiation into neural retina cells (r) 14 days after retinectomy.
Arrows denote pigment epithelium cells. (d) Transdifferentiation of the pigment epithelium from the dorsal iris (di) and formation of the
regenerating lens vesicle (lv).
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276
Panagiotis A. Tsonis
FIG. 2. The process of muscle dedifferentiation during limb regeneration in the adult newt. (a) The top of the picture is near the amputation
level. The top half shows the dedifferentiation process in the muscle. The fibers have “melted down” and the characteristic striated nature of the
muscle has been lost. The nuclei from the multinucleated muscle fibers are released to form mononucleated blastema cells and some are shown
dividing (arrow). The loss of the muscle-specific characteristics is shown in b and c. (b) Scanning electron micrograph from a dedifferentiating
muscle cell (such as the ones on the top of a) showing the loss of the muscle cell characteristics. (c) Scanning electron micrograph through a
muscle cell from a normal muscle cell (such as the ones on the bottom of a) showing the normal structure of the cytoplasm with the hexagon-like
shape of the myofibrils. The arrows show the sarcoplasmic reticulum. EM, extracellular matrix; n, nucleus.
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277
Regeneration in Vertebrates
FIG. 3. The limb–lens connection. Lens formation in the regenerating limb. (a) A perfectly formed lens after dissociated pigment epithelial
cells from the dorsal iris were implanted into the blastema. The lens possesses a normal appearance with normal posterior–anterior polarity
(direction of the arrow), with the anterior (front) part facing the wound epithelium. In (c) the lens is magnified to show its normal
appearance. The lens epithelium in the anterior (A) shows the characteristic cuboidal shape, while in the posterior (P) the cells become
elongated and differentiate to lens fibers (lf). The AP polarity of lens does not coincide with the AP axis of the limb. (b) Failure to form a
lens after transplantation of dissociated pigment epithelium cells from the ventral iris. The cells have not dedifferentiated and have
remained pigmented. (d) Magnification of the pigment aggregate shown in b. (Reproduced by permission from Ito et al., 1999.)
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278
Panagiotis A. Tsonis
result in loss of pigment epithelium and its transdifferentiation into neural retina (Mochii et al., 1997). These
mutant animals have in fact developed with two neural
retinas. Mutations in the pax-6 gene result in several
abnormalities in the eye tissues, including in the pigment
epithelium (Ton et al., 1991). FGF in fact down-regulates
both genes. Although expression studies of pax-6 and Mitf
during retina regeneration have not been performed, their
down-regulation by FGF strongly supports their role in
retina regeneration.
LENS REGENERATION
During development, the lens is induced by contact
between the ectoderm and the optic vesicle. Likewise in
retina development, FGFs seem to control such an induction (de Iong and McAvoy, 1993). Also, FGFs determine the
polarity of the lens. It has been shown that FGF-1 is present
as a gradient in the eyeball, with higher concentration
needed for fiber differentiation in the posterior chamber and
lower concentration in the anterior, where the lens epithelial cells are (Caruelle et al., 1989). Other important genes
for such an embryonic induction are pax-6 and six-3 (Oliver
et al., 1996; Altman et al., 1997). When the lens is removed,
however, regeneration occurs by the dedifferentiation of the
pigment epithelial (PE) cells of the dorsal iris (Fig. 1). These
cells proliferate and produce a lens vesicle and later differentiate to form lens fibers and finally, 20 –25 days later, a
normally polarized lens (see Tsonis, 1999, 2000, for review).
As in retina regeneration, lens regeneration is restricted
mostly to some urodeles. Some fishes can also regenerate
their lens, while in chickens it is possible during a very
narrow window during development. In vivo lens regeneration is possible only from the dorsal iris (never from the
ventral). Nevertheless, pigment epithelial cells from anywhere in the eyecup are capable of transdifferentiating to
lens in culture, and this ability has no species restrictions
(Kodama and Eguchi, 1995). Even PE cells from aged humans have this capability. This suggests that in vivo, but
not in vitro, there is spatial restriction to a specific position.
Such a restriction implies that there must be specific
regulation in the dorsal versus ventral iris. This is an
excellent system in which to identify the components of
the system that lead to inhibition except at the dorsal iris.
Despite the difference between the induction during
development and regeneration, it seems that similar regulatory events occur in both. Some of the factors that have
been studied in detail are the FGFs and their receptors.
During dedifferentiation of the PE from the dorsal iris,
expression of FGF-1, FGFR-1, FGFR-2, and FGFR-3 is
prominent in the dedifferentiating cells and in the subsequent regenerating lens vesicle and differentiating lens
fibers (Del Rio-Tsonis et al., 1997). However, only FGFR-1
product seems to be present specifically in the dorsal iris
during dedifferentiation. Its role in regulating lens regeneration has also been strengthened in experiments in which an
inhibitor of FGFR-1 inhibited lens regeneration and lens
fiber differentiation (Del Rio-Tsonis et al., 1998). In addition, as in cases of transgenic mice, exogenous FGFs were
capable of inducing similar abnormalities in the regenerating lens (Del Rio-Tsonis et al., 1997). These abnormalities
included vacuolated lens, double lens formation, and lenses
with abnormal polarity. The FGF story can tell us that this
particular signaling pathway is important for both retina
and lens development and regeneration and thus conserved
in both. In older experiments it has been shown that
proteoglycans are lost from the dorsal iris during the processes of regeneration (Zalik and Scott, 1973). It is possible
that this event is linked to FGFs since FGFs bind proteoglycans and affect availability of FGFs to activate the
function of receptors. Such a line of research is very
important for this field because it might provide clues about
the key cell surface changes that govern the dedifferentiation process and thus lens regeneration.
Spatial regulation along the dorsal–ventral axis may imply that genes such as Hox genes are involved. It has been
well established that these genes are major players in cell
fate determination, organogenesis, and pattern formation. It
is, therefore, likely that Hox genes regulate the events of
lens regeneration. Several Hox genes that are expressed in
the newt eye have been studied, but two homeoboxcontaining genes have been pinpointed as key regulators so
far. One is pax-6, a known lens-inducing gene. Pax-6 has
been found to be expressed in the adult newt eye and during
regeneration of the lens. However, its expression was not
evident in the axolotl, a urodele unable to regenerate the
lens (Del Rio-Tsonis et al., 1995). Prox-1, another Hox gene
which seems to be important for lens development
(Tomarev et al., 1996), has been found to be specifically
expressed and regulated in the pigment epithelium of the
adult newt dorsal iris and in the dorsal iris during lens
regeneration (Del Rio-Tsonis et al., 1999). Such expression
patterns suggest a role for these two genes in regenerationcompetent PE cells.
The field of lens regeneration has benefited enormously
from in vitro studies. As mentioned above, PE cells placed
in culture undergo dedifferentiation and subsequent transdifferentiation to form lentoids. This has helped to unequivocally prove that PE cells indeed can transdifferentiate
(the process has been observed even from a single cell;
Kodama and Eguchi, 1995). In vitro experiments have
shown the promoting effect of FGF, which is consistent
with the role of FGFs and their receptors during in vivo
regeneration (Hyuga et al., 1993). Finally, the ability of PE
cells from other species to transdifferentiate in vitro opened
new avenues in the field. We now know that PE cells from
other species, including humans, do have the program to
produce lens, and that knowledge could help in future
applications (Kodama and Eguchi, 1995). Culturing has also
been proven indispensable in the study of PE cells. For
example, ventral iris or retina PE cells can transdifferentiate
into lens cells in vitro. Ventral cells or explants from the
ventral iris, cultured in vitro, are able to produce a lens
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Regeneration in Vertebrates
upon transplantation into a lentectomized eye, but this
depends on the duration of the culturing before transplantation. Obviously, some disruption in cell-to-cell communication and cell surface, which occurs when cells are
cultured, is important for the initiation of dedifferentiation.
Furthermore, Ito et al. (1999) have shown that ventral iris
PE cells can in fact deprive the dorsal iris PE cells from
dedifferentiating in vivo. When a number of dorsal iris PE
cells (a number of cells able to produce a lens upon
transplantation) is mixed with fivefold the number of PE
cells from the ventral iris, their potential for lens transdifferentiation is inhibited. It is possible that the ventral PE
cells produce a factor that inhibits their ability for dedifferentiation in vivo. Transplantation experiments have also
revealed that the eye is not necessarily needed for dedifferentiation and transdifferentiation of PE cells into lens cells.
When dissociated PE cells were implanted into the blastema of a regenerating limb a perfect lens can be formed with
even a correct anterior–posterior orientation (see also below) (Ito et al., 1999).
LIMB REGENERATION
While the process of retina or lens regeneration is basically the transdifferentiation of one cell type to others, the
regeneration of amphibian limbs (and tails) is more complex. After amputation, the wound is quickly covered by a
specialized epithelium, the so-called wound epithelium. It
is strongly believed that this epithelium provides the necessary signals for the underlying tissues to dedifferentiate,
proliferate, and form the blastema. All the tissues at the
stump undergo dedifferentiation, including muscle, bone,
and other mesodermal tissues. The dedifferentiation process leads to cells that by proliferation form the blastema
(Fig. 2). After a period of about 2 weeks the blastema
redifferentiates to form an exact replica of the severed part
(Tsonis, 1996). Due to its complexity the process of limb
regeneration is regarded as more spectacular.
Expression in the wound epithelium has been studied
with the hope of identifying the factors that signal the
initiation of the dedifferentiation process. While several
factors have been found unique to the wound epithelium,
FGFs and their receptors are also thought to be paramount
for the signaling that leads to regeneration. In urodeles,
FGF-1 and FGF-2 have been found in the wound epithelium.
Interestingly, FGF-1 and FGF-2 have been implicated in the
nerve dependency of limb regeneration (Mullen et al., 1996;
Zenjari et al., 1997). FGFRs are also expressed in the newt
wound epithelium. The KGFR variant of FGFR-2 is specifically located in the basal layer of the wound epithelium,
while the bek variant is mesenchyme specific (Poulin et al.,
1993). In another study using Xenopus limbs it was found
that FGFR-1 and FGFR-2 are expressed in the wound
epithelium of stumps at stages at which regeneration is
permissive (premetamorphic), but they are absent from the
wound epithelium of stumps that are unable to regenerate
(postmetamorphic). More importantly, however, when
premetamorphic amputated limbs were treated with FGFR
inhibitors, regeneration was impaired, with formation of
spikes reminiscent of the quality of regenerates seen in
postmetamorphic frogs (D’Jamoos et al., 1998).
The mechanism of dedifferentiation, which allows cells
to reenter the cell cycle, must be a key for the ability to
regenerate. Cells, such as muscle cells, lose their characteristic architecture (such as the actin–myosin arrangement);
they become mononucleated and they start to divide (Fig.
2). Cell lines of dedifferentiated muscle cells, produced
from muscle explants, have been useful in studying cellular
and molecular events (Lo et al., 1993; Tsonis et al., 1995).
When these cells were implanted into an amputated limb,
in addition to muscle they were also able to produce
cartilage, thus providing significant evidence for the process
of transdifferentiation in limb regeneration as well (Lo et
al., 1993; Tsonis et al., 1995). Also, a key molecular event
associated with the process of dedifferentiation has been
identified. When these cells are allowed to form myotubes
in vitro and then induced to enter the cell cycle and become
mononucleated (by serum stimulation) the product of the
retinoblastoma (Rb) gene is specifically phosphorylated.
Myotubes dominantly express the hypophosphorylated
form of Rb, which inhibits the entry into the S phase. But as
dedifferentiation and entering the cell cycle ensues, the
hyperphosphorylated form of Rb (inactive) becomes dominant. This Rb phosphorylation event must be
dedifferentiation-specific (Tanaka et al., 1997). Indeed,
mouse myotubes are not able to enter the cell cycle (or
undergo phosphorylation of Rb) after serum stimulation
(Tam et al., 1995). The serum factor seems to be thrombin,
which alone is able to stimulate newt myotubes to reenter
the cell cycle (Tanaka et al., 1999). Only cells transfected by
viral oncogenes are induced to enter the S phase by sequestering the Rb product (Cardoso et al., 1993).
After the blastema is built it redifferentiates to reconstruct the lost part. This process is obviously very complex
and key developmental genes are expressed to secure that
the exact pattern will be regenerated. In this sense, it is
expected that genes that control pattern formation during
development will also be expressed and control patterning
during regeneration. In the late 1970s it was shown that
retinoic acid is able to respecify the positional memory of
the blastema cells (Niazi and Saxena, 1978). Retinoic acid is
able to proximalize the pattern when a limb is amputated
distally, i.e., at the wrist level. Such a treated limb will
regenerate as if it was amputated at the humerus level (for
details on limb regeneration see Tsonis, 1996). This ability
of retinoic acid suggested that control of limb morphogenesis might occur via genes that are affected by retinoic acid
and provided the first hope for isolation of these genes. The
excitement grew the next decade when it was found that
the receptors for retinoic acid (RARs) are in fact transcriptional factors with very distinct properties (Evans, 1988).
Several RARs have been isolated and found to be expressed
in the newt blastema, among them, the RAR ␣ (both
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280
Panagiotis A. Tsonis
isoforms ␣1 and ␣2), RAR ␤, and RAR ␦ (isoforms ␦1 and ␦2).
With a series of experiments that involved the creation of
chimeric receptors and transfection of these receptors in the
regenerating limb, Brockes and his colleagues were able to
show that different receptors account for the different
actions of retinoic acid. RAR ␣1 mediates the growth
inhibition of blastema cells, one of the earliest effects of
retinoic acid treatment (Schilthuis et al., 1993). RAR ␦1 is
responsible for inducing wound epithelium-specific genes
and RAR ␦2 is the one that mediates the ability of retinoic
acid to proximalize the pattern (Pecorino et al., 1994, 1996).
Similar to these RARs, attention has been paid to HOX
genes as regulators of the morphogenetic events during
limb regeneration (Gardiner and Bryant, 1996). Indeed some
of the Hox genes have shown a proximodistal gradient of
expression which is also regulated by retinoic acid (see
Tsonis, 1996; Beauchemin et al., 1998). Hox genes (especially the 5⬘ genes from the A and D cluster) have been
shown to be regulators of the developing limb pattern in
chicks and mice. Consequently, it was thought that expression studies of Hox genes of the A and D cluster would
provide important insights of how the pattern is formed in
the regenerating limbs. HOXA-9 and A-13 expression in
regenerating axolotl limbs showed some differences compared with expression during development. While in developing limbs A-13 was more distally expressed than A-9,
during regeneration there was no difference. Moreover,
A-13 was down-regulated with retinoic acid treatment
during regeneration (Gardiner et al., 1995). HOXD-11 (but
not D-8 or D-10) seemed to be specific for blastema formation. HOXD-11 appeared first in the early bud blastema at
the posterior-distal part, when only the hand region is
represented in the blastema (Torok et al., 1998). This might
indicate that (unlike developing limb) during regeneration
the distal tip is the first to be specified. Such expression
patterns of HOX genes might explain the mechanisms of
pattern formation during limb regeneration in line with
theoretical models that have been established in the past
(Bryant et al., 1981). Thus, the regenerating limb provides
an invaluable system to study pattern formation and
mechanisms of morphogenesis.
Even though mammals are not endowed with such regenerative abilities, the tips of the toes in mice and even
humans (young children) can be regenerated only when
amputated distally to the last interphalangeal joint. An
interesting association with this digit regeneration and the
expression of the homeobox-containing gene msx-1 has
been reported in mice. The domain of regeneration lies
within the domain of msx-1. Msx-1 is believed to play a role
in the maturation of limb tissues (Reginelli et al., 1995).
This mammalian model can become a very important
system in which similarities and differences with the
amphibian counterpart can be studied. Related to these
mammalian models, Goss has proposed that inhibition of
blastema formation by dermal healing (as opposed to wound
epithelium healing) seen in mammalian limb amputations
could account for the loss of regenerative ability in mam-
mals (Goss, 1980). These differences in healing could be
explored in order to identify factors that are differentially
present in the one healing mode versus the other. The
identification of msx-1 in mouse (Reginelli et al., 1995) and
of FGFRs in Xenopus limb regeneration (D’Jamoos et al.,
1998) are good prospects toward this direction.
THE RETINA–LENS–LIMB CONNECTION
Despite the fact that the processes of retina, lens, and
limb regeneration are quite different from each other and
involve different tissues and structures, they all start with
the dedifferentiation process. In this regard, we could hypothesize that what triggers dedifferentiation could be
common in these regenerative processes. From what was
mentioned above, some very interesting similarities are
inescapably obvious. The most striking is the possible role
of FGFs in signaling the initiation of all these events. As
shown in Fig. 3, a perfect lens can be formed after transplantation of PE cells into the limb blastema. Even the
anterior–posterior polarity of the lens is intact, with the
anterior part of the lens (front) facing the wound epithelium
(the reader should not confuse the AP axis of the limb with
the polarity of the lens). This is interesting because FGF has
been implicated in determining this polarity in the eye and
because the wound epithelium is a rich source of FGF. It is
tempting to speculate that FGF signaling does indeed play a
more general role in inducing the dedifferentiation process
in the different regenerative tissues in newt. FGFs have also
been found to support spinal cord regeneration and to
initiate liver development from the gut endoderm (Chen et
al., 1996; Jung et al., 1999). Such similarities and common
themes in regeneration can be studied further as more
mechanisms of dedifferentiation are revealed (e.g., Rb phosphorylation). Identification of a common mechanism for
dedifferentiation could unify concepts and prove paramount in expanding the study of regeneration in other
animals as well.
STEM CELLS IN REGENERATION
Stem cells seem to be a prominent feature in regeneration
of many organs and tissues such as intestine, cartilage,
bone, epidermis, liver, and muscle. On the other hand,
dedifferentiation of cells of local origin has been proven
beyond any doubt in newt retina, lens, and limb regeneration. The search for cells other than local as a source of
epimorphic regeneration has not been fruitful. However,
some participation of stem cells, especially in limb regeneration, should not be ruled out. Mesenchymal stem cells
(MSCs) from bone marrow have been shown to differentiate
to bone, cartilage, adipocytes, or muscle and are thought to
contribute to repair of these tissues (Prockop, 1997; Pittersen et al., 1999). But can such MSCs participate in the
restoration of an amphibian limb? While the local origin of
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281
Regeneration in Vertebrates
TABLE 2
Regeneration of Body Parts and Origin of the Blastema in the
Animal Kingdom
Species
Part of the body
Stentor (protozoa)
Hydroids
Flatworms
Annelids (worms)
Echinoderms
Arthropoda
Whole
Whole
Whole
Whole
Whole
Extremities
Fishes
Amphibia
Fins, retina, lens
Limbs, retina,
lens, jaw, tail
Fingertip
Mammals
liver regeneration). Local dedifferentiation and production
of a blastema could in fact preserve such memory and
secure the faithful regeneration of a particular pattern.
Mode of regeneration
Recapitulates cell division
No blastema
Reserve cells
Blastema
Blastema
Related to growth
(molting); blastema?
Blastema
Blastema
Blastema
Note. Some hydroids and flatworms can regenerate by morphallaxis, in which the remaining tissues reorganize to replace the
missing part with little dedifferentiation and proliferation. The rest
(except in protozoa) can be considered epimorphic regeneration. For
details see Goss (1969).
blastema renders this unnecessary, such a case should not
be impossible, especially when it is known that bone
marrow contains MSCs that differentiate to cells that
comprise the limb tissues. Recently it was shown that bone
marrow stem cells were able to produce oval cells that
contributed to liver regeneration when proliferation of
hepatocytes was inhibited (Petersen et al., 1999). It seems
that if a system is driven toward a particular direction, stem
cells might take over. Due to lack of bone marrow markers
and techniques, such experiments are lacking in the newt
limb regeneration field. Since such experiments are feasible
now and could be very informative, they should be pursued.
Several issues could be addressed from studies with newt
bone marrow MSCs. It will be very informative and productive to define similarities or differences between blastema
cells and MSCs. Not much is known about specific gene
expression in MSCs; however, some striking similarities do
exist. Both MSCs and blastema cells are fibroblastic-like
and they synthesize similar extracellular matrix rich in
collagens and fibronectin. In a strict terminological sense a
blastema cell that is differentiating to a cartilage cell should
be similar to a MSC differentiating to cartilage. Consequently, understanding the biology of the blastema cells
should be complementary in the study of stem cells and
vice versa. Obviously, different strategies of regeneration
have made use of either stem cells or locally produced
dedifferentiated cells. Why one strategy is preferred over the
other depending on the tissue or the mode of regeneration is
not clear, but it will certainly be very important to answer
this question. For example, an intriguing difference could
be the positional memory of the cells. In cases of epimorphic regeneration the missing structure should be replicated with the exact pattern (something that requires positional information, but is not necessary, for example, in
ORIGIN OF THE BLASTEMA IN
VERTEBRATES
In Table 2 the occurrence of body part regeneration in the
animal kingdom is presented with respect to the origin of
the blastema. Admittedly, invertebrates possess much
more spectacular regenerative abilities and can readily
regenerate whole body parts. In contrast, in vertebrates
regenerative abilities have been scaled down and limited to
appendages and eyes. Also, it is evident from Table 2 that as
we climb from simple to more complex organisms, dedifferentiation or blastema formation is the only means for
epimorphic regeneration and, of course, best represented in
some fishes and amphibia. But even among urodeles, regenerative abilities are not, however, widespread nor do they
follow a clear cladistic pattern. In closely related urodeles
the capability of lens or limb regeneration is not universal
and does not follow a cladistic pattern (Fig. 4). Is, therefore,
the occurrence of blastema an evolutionary “remnant,” or
atavistic feature, or a tangible physiological and developmental event that depends on specific molecular mechanisms?
In order to answer the question of the origin of the
blastema we should very carefully consider the molecular
and cellular mechanisms that characterize its formation.
The most seminal feature of the blastema is dedifferentia-
FIG. 4. Relationship of salamander families as inferred using
maximum parsimony analysis of aligned ribosomal RNA sequences (after Larson, 1991). Yes indicates presence and No absence of limb regeneration in adult salamanders (when known). No
obvious cladistic pattern correlating with the capability of limb
regeneration can be noted.
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282
Panagiotis A. Tsonis
tion and the loss of tissue characteristics. In order for this to
happen, terminally differentiated cells (such as muscle)
should reenter the cell cycle. As mentioned above a very
interesting molecular signature of the amphibian limb
blastema is the phosphorylation patterns of the tumor
suppressor Rb, which result in its inhibition. In mammals,
this is seen only in cells allowed to enter the cell cycle after
transfection with viral oncogenes (Cardoso et al., 1993).
Another interesting and intriguing similarity between
blastema and cancer cells is the identification of immortalizing sequences in a newt blastema cDNA expression
library. Immortalizing sequences are usually oncogenes,
genes that are paramount in the induction of cancer (Powell
et al., 1998). Indeed, cancer cells do look like blastema cells;
they are dedifferentiated and pluripotent. While we should
exercise caution in equating blastema and cancer cells their
similarities can be very informative and instructive. In the
past it has been speculated that cancer and regeneration are
inversely associated. In other words, an animal with regenerative capabilities is refractory to spontaneous or experimental cancer. This is especially true for the regenerationcompetent tissues of amphibia. It has been well
documented that spontaneous tumors are difficult to find in
amphibia (Tsonis and Del Rio-Tsonis, 1988). Also, carcinogen application in the regeneration-competent tissues of
amphibia (such as limbs and eye) would result in normal
morphogenesis and differentiation rather than tumor formation (Eguchi and Watanabe, 1973; Tsonis, 1983). These
facts could suggest that cancer originated at the expense of
regeneration. Perhaps as epimorphic regeneration ceased to
exist cancer originated. The trade could be significant for
the development of more advanced immune system and
immunosurveillance mechanisms (Prehn, 1971). Reexpression of embryonic antigens in blastema requires a weak
immune system, which in turn is a high cost to pay.
Perhaps regeneration is not the result of a “trait” but a
well-orchestrated developmental event governed by genes
that control normal growth and differentiation.
The synthesis presented in this review does not separate
the different aspects of regeneration research, but rather
leads to finding links that would increase collaboration of
the different subfields of regeneration research and eventually will lead to a better understanding and to applications
in medicine. It is my conviction that regeneration research
will occupy a bright spot in the pantheon of biomedical
achievements of the 21st century.
ACKNOWLEDGMENTS
This work was supported by NIH Grant EY10540. I thank Dr. M.
Okamoto for providing Fig. 3. Due to space limitations it was not
possible to include all references for such a broad topic. I apologize
to authors whose work was not cited. I am also grateful to two
anonymous reviewers for their helpful comments.
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Received for publication September 22, 1999
Revised February 12, 2000
Accepted February 12, 2000
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