Blackwell Publishing AsiaReview Brain regeneration in ...

Develop. Growth Differ. (2007) 49, 121–129 doi: 10.1111/j.1440-169x.2007.00914.x

ReviewBlackwell Publishing Asia

Brain regeneration in anuran amphibians

Tetsuya Endo,* Jun Yoshino, Koji Kado and Shin Tochinai

Department of Natural History Sciences, Faculty of Science, Hokkaido University, N10W8, Kita-ku, Sapporo 060-0810,
Japan

Urodele amphibians are highly regenerative animals. After partial removal of the brain in urodeles, ependymal
cells around the wound surface proliferate, differentiate into neurons and glias and finally regenerate the lost
tissue. In contrast to urodeles, this type of brain regeneration is restricted only to the larval stages in anuran
amphibians (frogs). In adult frogs, whereas ependymal cells proliferate in response to brain injury, they cannot
migrate and close the wound surface, resulting in the failure of regeneration. Therefore frogs, in particular
Xenopus, provide us with at least two modes to study brain regeneration. One is to study normal regeneration
by using regenerative larvae. In this type of study, the requirement of reconnection between a regenerating
brain and sensory neurons was demonstrated. Functional restoration of a regenerated telencephalon was also
easily evaluated because Xenopus shows simple responses to the stimulus of a food odor. The other mode
is to compare regenerative larvae and non-regenerative adults. By using this mode, it is suggested that there
are regeneration-competent cells even in the non-regenerative adult brain, and that immobility of those cells
might cause the failure of regeneration. Here we review studies that have led to these conclusions.

Key words: amphibian, anuran, brain, regeneration, Xenopus.

Introduction ability is largely confined to the regrowth of neural
processes and the formation of new synapses (see
Amphibians are known to be highly regenerative Kirsche 1983). The type of massive regeneration
animals. They can regenerate a limb, tail including after partial brain removal that is common among fish
a spinal cord, jaw, liver and even brain. After a partial and amphibians is not observed in mammals. There-
amputation, the remaining part of those organs fore fish and amphibians are unique among verte-
reconstitutes the lost part completely. Mammals, brates in having the ability to completely regenerate
including humans, also have regenerative ability to a brain. Whereas urodele amphibians (newts and
some extent, but in general this ability is considered salamanders) retain their regenerative ability in adult
to be very limited. life, this is not the case in anuran amphibians (frogs)
that have high regenerative abilities as larvae, but
Conversely, recent progress in regenerative medi- lose them as adults. The ontogenic decline of regen-
cine has demonstrated regenerative responses in the erative ability occurs not only in the brain but also
mammalian central nervous system (CNS), including the limb (Dent 1962). This phenomenon has fasci-
the discovery of neural stem cells (NSCs) (Doetsch nated researchers for a long time, because it enables
et al. 1999; Johansson et al. 1999). It has been shown us to study regeneration comparing a regenerative
that neurons are replaced even in the adult CNS and non-regenerative phase in one organism. In recent
using the NSC system (Gage 2002). This implies the years, molecular biological studies have revealed
presence of regenerative abilities in the adult mam- much about the mechanisms of larval and adult
malian CNS in response to an injury, although this regeneration, particularly in the limb (reviewed by
Gardiner et al. 2002; Suzuki et al. 2006). These recent
*Author to whom all correspondence should be addressed. studies have built on classical studies that were made
Email: [email protected] before the 1980s by interpreting the old phenome-
Received 30 November 2006; revised 15 December 2006; nology in a more contemporary ‘molecular language’.
accepted 15 December 2006. Such studies will eventually lead us to understand
© 2007 The Authors the deeply hidden mechanisms of regeneration that
Journal compilation © 2007 Japanese Society of researchers could never see in the past.
Developmental Biologists

122 T. Endo et al.

Although there are many classical studies of anuran (Fig. 1b–d). In mature Xenopus adults, no regener-
brain regeneration, understanding of this phenome- ation occurs and structural defects persist (Srebro
non at the molecular level is still poor. The purpose 1965). The response to injury at the intermediate
of this review is to summarize the classical literature developmental stages is unclear, given the conflicting
on brain regeneration in amphibians, particularly in results reported for the regenerative response in
anurans, in order to provide a foundation for the next froglets (young adults). One is that no regeneration
generation of studies using molecular biological occurs even in 10-day-old froglets, as observed in
techniques. We will focus on the telencephalon and mature adults (Fig. 1e–g) (Yoshino & Tochinai 2004);
the optic tectum of the mesencephalon, because whereas the other is that regeneration takes 3 months
other encephalic regions are involved in maintaining in froglets that undergo cephalotomy immediately
the viability of the animal, such as respiratory and after metamorphosis (Jordan 1958). At this point, it
cardiovascular function. Therefore it is possible to is not possible to resolve these apparently contra-
resect a part of the telencephalon or optic tectum dictory findings because it is unclear if the experi-
with low mortality, thus allowing for regeneration to mental conditions were the same between them. At
occur. Also, they are large and swollen, so they are least, postlarval regeneration takes a long time and
relatively easy to resect. Finally, because the telen- results in a deformed structure based on Jordan’s
cephalon and optic tectum are the centers of olfac- experiment, and postmetamorphic Xenopus eventu-
tion and vision, respectively, functional recovery can ally lose the ability to regenerate the telencephalon.
be assessed behaviorally.
Regeneration of the telencephalon requires recon-
Regeneration of the telencephalon nection with the olfactory nerves in order to reform
the olfactory bulb. Some classic studies, which were
Although successful brain regeneration occurs in mainly based on histology, suggested that numerous
adult urodeles, most studies of telencephalon regen- cells migrate from the olfactory organ to the brain
eration have been carried out in larvae and juveniles along the olfactory nerves and participate in brain
(see Kirsche 1983). The paucity of studies on telen- regeneration as neuroblasts (Srebro 1957; Jordan
cephalon regeneration in adult urodeles may be a 1958; Kosciuszko 1958; Kirsche & Kirsche 1964). In
consequence of the extremely long time it would take this way, reconnection between the telencephalon
in adult-sized brains, and also because of a meth- and olfactory nerves was thought to be a prerequisite
odological problem, both of which would make analy- for regeneration; however, this report regarding the
ses difficult. In many classic studies, for example, source of cells in regeneration has not been confirmed
a whole telencephalon was extirpated to see if it using appropriate cell lineage markers. Conversely,
could be regenerated from the stump on the dien- considerable regeneration of the telencephalon is
cephalon. However, because NSCs from different observed even before the reconnection (Yoshino &
encephalic regions possess each regional character Tochinai 2004). This regenerated part is the anterior
in mammals (Klein et al. 2005), it seems likely that part of the cerebrum, which has differentiated gray
each encephalic region can regenerate only itself, matter and white matter; however, the olfactory bulb
even in the highly regenerative amphibians. For is missing from the regenerate (Yoshino & Tochinai
example, removal of the optic tectum, a dorsal part 2006).
of the mesencephalon, results in successful regen-
eration, as we will describe in the next section The phenomenology of forebrain regeneration cor-
(Minelli & Del Grande 1974; Ohsawa et al. 2003). responds to what is seen in the development of the
Therefore partial removal of telencephalon in adult olfactory bulb. In Xenopus embryos and tadpoles,
urodeles should be tested to see if complete regen- when the connection of the olfactory nerves to the
eration occurs from the cells of the remaining portion forebrain is disturbed, the olfactory bulb fails to
of the telencephalon. develop (Graziadei & Monti-Graziadei 1992). In con-
trast, transplantation of the olfactory placode near
In contrast to urodeles, anuran amphibians can the diencephalon of the tadpole is enough to induce
only regenerate the telencephalon as larvae and lose well-defined structures such as glomeruli that are a
the ability as adults. Young Xenopus tadpoles, such part of the olfactory bulb (Stout & Graziadei 1980;
as stage 47–53 larvae, have quite high regenerative Magrassi & Graziadei 1985). In mammals, innerva-
ability and complete regeneration is observed after tion of the olfactory nerves into the forebrain controls
ablation of a massive part of the telencephalon (Srebro cell cycle kinetics of anterior telencephalic progenitor
1957; Yoshino & Tochinai 2004). Within a month after cells to induce formation of the olfactory bulb (Gong
tissue removal, an almost normal structure is reformed & Shipley 1995). When neurogenesis in the olfactory
epithelium is inhibited by disturbing fibroblast growth

© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists

Brain regeneration in anuran amphibians 123

Fig. 1. Telencephalon (cerebrum
and olfactory bulb) regeneration
in Xenopus larvae and froglets.
(a) Illustration of the brain in
Xenopus. The anterior half of the
telencephalon was removed. The
broken line indicates the ampu-
tation site. cer; cerebrum, ob;
olfactory bulb. (b–g) Horizontal
sections of the telencephalon in
larvae (b–d) and froglets (e–g).
(b, e) Intact; (c, f) immediately after
amputation; (d, f) 30 days after
amputation. Scale bars, 300 µm.

factor (FGF) signaling, the olfactory bulb is not formed 20 (Shumway 1940), which is comparable to about
(Hebert et al. 2003; Kawauchi et al. 2005). In regen- stage 37/38 in Xenopus (Nieuwkoop & Faber 1956),
eration of the Xenopus telencephalon, innervation of show considerable regeneration after a short period
the olfactory nerves into the regenerating telen- following the removal of an optic lobe unilaterally. In
cephalon may play a crucial role in the regeneration contrast, none of the cases operated between stages
of the olfactory bulb through the same mechanisms 21 (Shumway’s normal stage, 1940) and XV (Taylor
involved in the development of this organ. & Kollros’s normal table 1946), which are comparable
to Xenopus stages 40–57, show a complete regen-
Regeneration of the optic tectum erative response (Terry 1956). Similar results were
obtained in Xenopus, where a gradual loss of regen-
It is repeatedly demonstrated that regeneration of the erative ability was observed (Fig. 2) (Filoni & Gibertini
optic tectum is possible in adult urodeles (Minelli & 1969). Tadpoles before stage 50 can repair the lesion
Del Grande 1974; Ohsawa et al. 2003). Ohsawa et al. in the operated ventricle with undifferentiated cells
removed 70% of the tissue of the left optic tectum quickly and can reconstitute the optic lobe completely
from adult newts and observed optic nerve projec- with regards to the number of layers of the tectum
tions onto the regenerated structure by anterograde and its thickness after removal. After stage 51, irreg-
labeling with horseradish peroxidase from the right ularities in the thickness and alignment of the layers
eye. The left optic tectum regenerated up to 80% of increase progressively. The stratification disappears
the volume of the contralateral intact tectum and from the regenerates that are operated at stage 55.
regeneration was accompanied by de novo differen- At stage 59, the ventricle never completely heals
tiation of nerve cells from ependymal cells (Ohsawa even 90 days after operation and has no stratified
et al. 2003; M. Okamoto, pers. comm., 2006). organization (Filoni & Gibertini 1969). After meta-
morphosis, the wound surface is never closed and
Anuran amphibians have the ability to regenerate its histological appearance has not changed around
the optic tectum, as well as the telencephalon, at the surface even a month after the operation
early larval stages and there is also a decline in (Fig. 2f,g). However an increase in the BrdU labeling
regenerative ability in a stage-dependent manner. In index is observed 9 days after the tectotomy, suggesting
Rana pipiens, embryos and early larvae until stage

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Journal compilation © 2007 Japanese Society of Developmental Biologists

124 T. Endo et al.

Fig. 2. Optic tectum regeneration
in Xenopus larvae and froglets.
(a) Illustration of the brain in
Xenopus. mes; mesencephalon.
(b) A 3-D image of the anterior
half of the mesencephalon as seen
from the posterior. The right optic
tectum was removed from the
whole mesencephalon. (c–g) Cross-
sections of the mesencephalon in
larvae (c–e) and froglets (f, g).
Immediately (c, f), 4 days (d) and
30 days (e, g) after tissue removal.
Scale bar, 300 µm.

that tectum cells might be able to respond to injury of importance in the regenerating tectum. When a
even in froglets (Kado & Tochinai; unpubl. data, 2006). single eye is removed concomitantly with ablation of
a contralateral tectum in order to eliminate the effect
Complete stratification of the tectum during regen- of the optic nerves during tectum regeneration, a
eration seems to require neural connection into the hypoplastic tectum is regenerated, suggesting that
regenerating tectum in a manner that is comparable the final migration of tectal cells to the definitive posi-
to the requirement of the olfactory nerve connection tion in the regenerate is dependent on the invasion
in telencephalon regeneration. As described above, of the optic nerves into the regenerating tectum (see
the highly stratified structure is a distinctive feature Levine 1984).
in the optic tectum and is a good measurement of
how much of the tectum has been regenerated. After Functional assay for brain regeneration
the operated ventricle is closed with undifferentiated
cells, laminar formation begins as a consequence of Typically, studies on amphibian tissue/organ regen-
the migration of the cells from the inner layers toward eration have focused only on morphology and rarely
the outer layers (Filoni & Gibertini 1969). There are on restoration of function. The assessment of function
nine layers in the Xenopus optic tectum (Lazar 1973; in tissues/organs that are moved by the organism,
Lazar 1984). The outer layers nine, eight and seven such as a limb or tail, is less challenging than the
are always the first ones to appear in regeneration assessment of brain function. In the former case, it
(Filoni & Gibertini 1969), which is comparable to the wound be obvious if regenerates are functional, if
sequence of formation during tectum development operated animals actually recovered to walk or swim
(Kollros 1953). If the tectum is permanently denervated using the regenerate. For brain regeneration, how-
during development by removing a contralateral eye, ever, the answer is not as simple, because the brain
the total number of synapses decreases rapidly to is an information-processing organ that consists of
about 40% of the original number, and the thickness complex neural networks. Morphological evaluation
of the superficial layers eight and nine is reduced to is not enough to determine if the neural networks are
80% of the control tectum (Ostberg & Norden 1979). functionally recovered and therefore a functional assay
It is likely that innervation of the optic nerves is also

© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists

Brain regeneration in anuran amphibians 125

is required. For this reason, evaluation tests have been be sufficient to restore the missing structures. Among
developed for studies on mammalian spinal cord these animals, half responded to the odor stimulus
regeneration, including the Basso-Beattie-Bresnahan within 2 min, flicking their forelimbs to the mouth.
(BBB) method (Basso et al. 1995). In this test, func- When the animals that did not show the sweeping
tional recovery from a spinal cord injury can be scored action were dissected, it turned out that all of them
by observing behavioral outcomes. had regenerated only the cerebrum but not the olfac-
tory bulb, which resulted from the failure of olfactory
It is possible to assess functional regeneration of reinnervation. Thus, all animals at this stage can
the telencephalon by examining if frogs can sense regenerate the telencephalon and if the olfactory
odors, because the telencephalon in frogs principally nerves reconnect to the brain, olfactory function is
processes olfactory information (Scalia 1976). Water- also restored.
soluble odorants are primarily detected by olfactory
receptor neurons in the nasal cavity. Olfactory recep- In contrast to studies of regeneration of the telen-
tor neurons innervate the olfactory bulb through the cephalon, behavioral experiments have not yet been
olfactory nerve layer and make primary synapses reported for regeneration of the optic tectum. There
onto mitral and tufted cells in the glomerular layer, are some behavioral studies about vision in Xenopus
where they are the only output neurons in the olfac- (see Elepfandt 1996) that might be useful for evalu-
tory bulb (Wilson & Mainen 2006), and project axons ating functional recovery of the regenerated optic
onto the main olfactory cortex in the cerebrum through tectum. However, it must be noted that Xenopus
the lateral olfactory tract (Schwob & Price 1984). young tadpoles have the pineal eye as a third
Consequently, if the anterior half of the telencephalon photoreceptor organ (Roberts 1978; Jamieson &
is removed and it is not functionally regenerated, Roberts 1999; Jamieson & Roberts 2000), and
frogs cannot recognize a smell at all. pineal-dependent responses during swimming are
observed up to stage 44 (Jamieson & Roberts 2000).
The forelimb sweeping behavior in response to food Thus researchers need to pay attention to the role
odors provides the opportunity to evaluate functional of this visual organ when designing experiments
regeneration of the telencephalon. The detection of using such young tadpoles.
food by olfaction is thought to initiate the appetitive
stage of the normal feeding behavior in Xenopus Origin of the cells
(Hutchison 1964). During this stage, the animal
becomes active and begins to swim randomly, flick- In regeneration of both the telencephalon and optic
ing its forelimbs to its snout. Once they touch a food tectum in amphibians, it is presumed that the cells
object by the forelimbs or the snout during this stage, participating in the regenerative response originated
the animal attempts to ingest the food (Hutchison from the periventricular zone (the ventricular zone
1964). Therefore, flicking forelimbs, also referred to (VZ) and subventrucular zone (SVZ) at the embry-
as a ‘sweeping’ action by Avila and Frye (1977), is onic/larval stages and the ependymal layer (EL) and
a behavioral response by which to evaluate olfactory subependymal layer (SEL) in the adult. The terminology
function of a regenerated telencephalon. for these layers is based on The Boulder Committee
1970). The VZ is located adjacent to the ventricular
The response behavior to food odors has been lumen and differentiates perinatally into a cuboidal
used as a functional assay to demonstrate that a epithelial cell layer, the EL (Takahashi et al. 1996).
regenerated telencephalon that was injured at stage The SVZ originates from the VZ embryonically and
53 recovered the ability to process olfactory informa- is considered to be the SEL in the adult that contains
tion (Yoshino & Tochinai 2006). Trout food pellets were several layers of tightly packed SVZ-like cells
dissolved in distilled water, filtered and centrifuged between the EL and the mature nervous tissue (Taka-
to obtain a colorless supernatant. The supernatant, as hashi et al. 1996; see Peretto et al. 1999). The term
an olfactory stimulus, was applied to intact (positive SVZ seems to be preferred recently to SEL even for
control), olfactory nerve-cut (negative control) and the adult tissue. In adult mammals, it is suggested
telencephalon-regenerated frogs (experiment) to test that the SEL retains multipotent neural stem cells
if it triggered a feeding response as evidenced by (Doetsch et al. 1999; see also Alvarez-Buylla et al.
the sweeping action. As expected, none of the frogs 2002). Although ependymal cells in mammalians have
with non-regenerated, severed olfactory nerves showed also been suggested to function as neural stem cells
the sweeping action, while 100% of intact frogs showed for the SEL (Johansson et al. 1999), several other studies
the sweeping action within a minute. The experimental do not support this interpretation (Chiasson et al.
animals had the anterior half of the telencephalon 1999; Doetsch et al. 1999; see also Alvarez-Buylla
removed at stage 53 and had been allowed to
undergo regeneration for a period of time that would

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126 T. Endo et al.

Fig. 3. Schematic illustration of
regenerative differences between
larval and adult brain tissues in
Xenopus. (b) After brain injury,
periventricular cells in the vicinity
of the wound migrate and close
the wound in larvae, but not in
adults. (c) The rate of cell
proliferation increases around
the regeneration site and the
regenerating tissue thickens in
larvae. A temporal increase in cell
proliferation is also observed in
adult brains. (d) Regeneration of
a functional brain is completed
in about a month in larvae.
Conversely, the wound site is
never closed and no regeneration
occurs in adults.

et al. 2002). In amphibians, cells from both the EL obvious that the proliferating cells do not participate
(or VZ) and SEL (or SVZ) proliferate after CNS injury in regeneration. Wound closure seems to be critical
and appear to participate in CNS regeneration (Srebro for successful regeneration because it is one of the
1957; Jordan 1958; Srebro 1959; Filoni & Gibertini 1969; earliest events that are followed by a sequence of
Filoni & Margotta 1971; Minelli et al. 1987; Minelli et al. regeneration processes. We consider that the failure
1990; Zhang et al. 2000; Yoshino & Tochinai 2004; of wound closure is one of the causal events that
for a review see Kirsche 1983; Chernoff et al. 2003). make froglets unable to regenerate the telencephalon
(Fig. 3).
The relationship between the response of periven-
tricular cells and the ontogenetic decline of regener- It may also be the case that regenerative failure of
ative ability has been studied in Xenopus. In the case the optic tectum is a consequence of a failure to
of the regeneration of the telencephalon in larvae close wounds after metamorphosis (Fig. 2g). In young
(regeneration-competent), an early response to tissue regenerative tadpoles, high mitotic activity is observed
removal is closure of the wound surface, which particularly in the VZ of the non-operated optic lobe
occurs after several days (Yoshino & Tochinai 2004) and the area of transition between the removed optic
(cf. Fig. 2d). Presumably the cells that close the lobe and the ipsilateral semicircular torus. It is the
wound are derived from the VZ. The level of cell cells from these two areas that are presumed to
proliferation is high during regeneration; however, migrate to cover the cut surface. In later stages, as
because the animal is still developing at this stage, regenerative ability declines, the presumptive source
the labeling index of an uninjured, control brain is of cells for regeneration becomes confined eventu-
also high. Eight days after ablation, the proliferation ally to the non-operated optic lobe (Filoni & Gibertini
activity in the VZ is at its maximum, which corre- 1969). There is a temporal increase in cell prolifera-
sponds to the stage in regeneration when the reform- tion in froglets in response to tectotomy; however,
ing brain tissues thicken. The proliferating cells are BrdU is incorporated only in ependymal cells in the
positive for the NSC marker, Musashi-1, suggesting vicinity of the wound edge. In contrast, during regen-
that they are NSCs. Although postmetamorphic froglets eration at larval stages, BrdU-positive cells are widely
do not regenerate the telencephalon, the frequency distributed, including within the SVZ of the intact side
of proliferating cells is increased in response to injury. of the mesencephaolon (Kado & Tochinai; unpubl.
However, wound closure does not occur in froglets data, 2006). This difference might be potentially of
(Fig. 1g) (Yoshino & Tochinai 2004) and thus it is interest, as we describe below.

© 2007 The Authors
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Brain regeneration in anuran amphibians 127

Cell proliferation in response to the ablation of brain from regions of the telencephalon was taken out from
tissues might be mediated by a diffusible factor(s). larvae or froglets, dissociated into single cells and
The increase in the cell proliferation rate is observed grafted as a dense cell suspension into the cavity
not only in the vicinity of the removed portion but that had been made in froglets after partial brain
also in other parts of the brain (Del Grande et al. removal. Surprisingly, grafted cells closed the wound
1990; Minelli et al. 1990; Franceschini et al. 1992; surface of the telencephalon and formed a brain-like
Filoni et al. 1995). These findings are suggestive of structure. Although it was deformed, it connected
the existence of a diffusible factor(s) that is released with the olfactory nerves and roughly separated into
into the cerebrospinal fluid and/or blood where it is three layers, including an EL lined smoothly from the
mitogenic for its target tissues (Yoshino & Tochinai host ependymal cells. These results suggest that
2004). It has been demonstrated that the fluid even froglet brain cells, that might be NSCs, have
secreted into the wounded brain cavity in rats has the ability to not only proliferate, but can participate
neurotrophic activity (Nieto-Sampedro et al. 1982). In in the regeneration of well-patterned brain structures.
mammals, there are cytokines that are upregulated
around the wounded area following brain injury, such Although a functional analysis has yet to be per-
as FGF2 and transforming growth factor (TGF)-β (see formed for brain-reconstituted animals, these results
Ghirnikar et al. 1998). Although we have limited infor- provide a clue as to the relationship between the
mation on the molecular mechanism of CNS regen- decline of the regenerative ability and metamorphosis.
eration in amphibians, it has been shown that FGF2 In contrast to the classic and simple idea that the
induces proliferation of neural progenitor cells in number of undifferentiated and regeneration-competent
urodele tail regeneration (Zhang et al. 2000) and cells in the brain decreases during metamorphosis,
that epidermal growth factor (EGF) enhances cell it appears that regeneration-competent cells persist in
migration and proliferation from ependymal explants the adult brain. Our current view is that regeneration-
in vitro (O’Hara & Chernoff 1994). Therefore these competent brain cells exist in both the larval and
factors might be released into the ventricles and adult brains, but that with progressive development
induce NSCs to proliferate during brain regeneration and metamorphosis, environmental conditions even-
in amphibians. tually make the cells unable to initiate brain regen-
eration. Filoni et al. (1979) examined the effect of
Attempts to induce brain regeneration inhibition of metamorphosis by 4 (6)-Propyl-2-thiouracil
(PTU) treatment on regeneration of the optic tectum
Because anuran amphibians progressively lose regen- and suggested the main cause of the decreasing
erative abilities in various tissues and organs at later regenerative ability is the progressive differentiation
developmental stages, researchers have attempted of the optic tectum by the time of surgery. While a
to rescue the regenerative ability in older tadpoles certain concentration of thyroid hormone (TH) treat-
or metamorphosed frogs. ment on early stage Xenopus larvae can increase
the mitotic activity in the regenerating optic lobe, it
As regenerative decline appears to be intrinsic to also accelerates histological differentiation (Filoni
the cells that participate in regeneration (Sessions & et al. 1974). During TH-induced metamorphosis, the
Bryant 1988), it is likely that regenerative abilities brain cells differentiate and constitute complex struc-
could be restored by treatments that cause these tures together with the extracellular matrix (ECM).
cells to revert to a more pluripotent state, or by graft- Regeneration-competent cells in late-stage tadpoles
ing tissue or cells from young regenerative animals and adults might be unable to participate in brain
into adult non-regenerative hosts. Srebro (1965) tried regeneration in this mature environment.
to implant the brains from Xenopus tadpoles at
various stages in place of the removed telencephalon; Conclusions
however, in most cases the implants did not persist
at the host site, suggesting that they were rejected Given the information about the difference between
because of the immune response. Yoshino and Tochinai Xenopus larvae and adults during brain regeneration,
(2004) used the Xenopus J strain (JJ) in their experi- we consider it noteworthy that the ability of ependy-
ments to eliminate immunological rejection. No mal cells to become mobilized in response to injury
immunological rejection occurs in JJ against JB is correlated with regenerative ability (summarized
(hybrids between J females and X. borealis males) in Fig. 3). Because there are proliferative and NSC
and they can be distinguished from each other by marker-positive cells in the EL of the adult brain,
differential quinacrin staining (Thiebaud 1983; Koibuchi immobility of regeneration-competent cells, rather
& Tochinai 1999). In this experiment, tissue removed than the lack of such cells, might cause failure of

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Journal compilation © 2007 Japanese Society of Developmental Biologists

128 T. Endo et al.

the wound surface to close. Compared with the thick ependymal cells demonstrate proliferative potential, but only
periventricular zone in larvae, the adult periventricular subependymal cells have neural stem cell characteristics.
zone is very thin. This morphological difference is a J. Neurosci. 19, 4462– 4471.
consequence of the differentiation of progenitor cells Del Grande, P., Franceschini, V., Minelli, G. & Ciani, F. 1990.
into nerve cells to form the neural network in the adult Mitotic activity of the telencephalic matrix areas following
brain, leaving a small population of undifferentiated optic tectum or pallial cortex lesion in newt. Z. Mikrosk. Anat.
cells localized only in the EL. Because of this com- Forsch. 104, 617– 624.
plex neural network, regeneration-competent cells in Dent, J. N. 1962. Limb regeneration in larvae and metamor-
the EL might be physically constrained to this region phosing individuals of the South African clawed toad. J.
and thus unable to migrate to the wound surface. Morphol. 110, 61–77.
In urodele amphibians, ependymal cells produce Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M. &
matrix metalloproteinases (MMPs) during spinal cord Alvarez-Buylla, A. 1999. Subventricular zone astrocytes are
regeneration (Chernoff et al. 2000) that would liberate neural stem cells in the adult mammalian brain. Cell 97,
cells from the matrix and thus allow the ependymal 703 –716.
cells to migrate. It will be important to examine MMP Elepfandt, A. 1996. Sensory perception and the lateral line
activity during brain regeneration in Xenopus larvae system in the clawed frog, Xenopus. In The Biology of
and adults and to test if regeneration can be initiated Xenopus (eds R. C. Tinsley & H. R. Kobel), pp. 97–120.
by application of MMP into the non-regenerative Oxford University Press, Oxford.
adult brain in Xenopus. Filoni, S., Bernardini, S. & Cannata, S. M. 1995. Differences in
the decrease in regenerative capacity of various brain
Acknowledgements regions of Xenopus laevis are related to differences in the
undifferentiated cell populations. J. Hirnforsch 36, 523 –529.
We are grateful to Dr David Gardiner for his critical Filoni, S. & Gibertini, G. 1969. A study of the regenerative
reading of the manuscript and for Dr Mitsumasa capacity of the central nervous system of anuran amphibia
Okamoto’s helpful information. We also thank the in relation to their stage of development. I. Observations on
members of the Tochinai laboratory for their helpful the regeneration of the optic lobe of Xenopus laevis (Daudin)
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© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists