The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2010) 52, 7787

doi: 10.1111/j.1440-169X.2009.01138.x

Review Article

Stem cell system in tissue regeneration in fish

Atsushi Kawakami*
Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501,
Japan

During evolution from single-cell to multi-cellular organisms, organisms developed the needed machinery by
which a vast number of functionally different types of cells could be unified into an individual. To attain this goal,
organisms evolved the developmental strategies that produced different cell types and unified them into complex body architecture. However, a more intriguing feature of multi-cellular organisms is that they can maintain
their bodies throughout long life. For tissue maintenance, stem and or progenitor cells in many tissues and
organs are thought to play an important role; however, we know little about their control and the process of tissue reconstitution. As cells are fragile, all animals have the ability, more or less, to replace damaged or dead
cells; however, there are large variations in such abilities, depending on the type of organs and the species.
Though vertebrates cannot reconstitute their bodies from a small piece as do planarians, some lower vertebrates, unlike mammals, have the ability to regenerate body appendages and many internal organs. If we unveil
the nature of stem cells in striking examples of such regeneration, this information can be applied to mammals
and greatly benefit us. The focus in the present review is on the recent advances in our knowledge about the
regeneration mechanism in fish, including the stem cells that are involved.
Key words: blastema, fin, regeneration, wound epidermis, zebrafish.

Introduction
During the evolution from single-cell to multi-cellular
organisms, animals attained longevity by developing
the machinery by which a vast number of functionally
different types of cells could be unified into an individual organism. To maintain the integrity of the multicellular body, animals needed to have specific sizes
and morphologies, and to maintain their properties by
a tissue maintenance mechanism.
The word regeneration is defined as the reproduction or reconstitution of a lost or injured part or as
a form of asexual reproduction. With such definitions, regeneration encompasses a broad spectrum of
natural phenomena that operate through seemingly
different mechanisms. Regeneration may include phenomena such as physiological regeneration (renewal of
blood and epithelial cells and seasonal replacement of
deer antlers), morphallaxis (regeneration of hydra and
some annelids), hypertrophy (compensatory or regen*Author to whom all correspondence should be addressed.
Email: kawakami.a.aa@m.titech.ac.jp
Received 30 July 2009; revised 1 September 2009;
accepted 1 September 2009.
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Journal compilation 2009 Japanese Society of
Developmental Biologists

erative increase in the mass of internal organs), and

reparative regeneration (cellular regeneration, tissue
regeneration), and epimorphic regeneration mediated
by the formation of a blastema (Fig. 1; Carlson 2007).
However, in any of these tissue-maintenance phenomena, how organisms sense the loss or damage of tissues and control the proper tissue restoration has not
been elucidated yet.
One of the difficulties in revealing the regeneration
mechanism is due to our ignorance about the entity of
positional information. When we look at the animal
kingdom, there are many varieties of sizes even
between close species. For example, the largest modern rodent, the capybara, is over 1 m in body length
and 45 kg in weight (there is an even larger rodent in
the fossil record) in contrast to the smallest one, the
pygmy mouse, whose body length is only 56 cm.
Similarly in primates, the smallest pygmy mouse lemur
is about 6 cm in body length; whereas the largest
gorilla is nearly 2 m tall and weighs over 200 kg. Considering the rapid and continuous turnover of cells
within the body, it is surprising that animals are able
to maintain their specific body sizes and morphologies
during their long lives, which implies that the positional
information exists in adult organisms and it keeps
directing the size and shape according to a genetic
program.

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A. Kawakami

Fig. 1. Major types of regenerative phenomena in multi-cellular

organisms. Physiological regeneration includes seasonal or
hormonal cycles of tissues such as deer antlers and replacement
of blood and epithelial cells. Tissue regeneration is defined as the
replacement of damaged tissues without the mediation of a
blastema, whereas epimorphic regeneration refers to a type of
regeneration mediated by the blastema. Hypertrophy is thought
to be a type of regeneration, in which there is a compensatory
increase in the size of a paired organ such as kidneys and lungs
after its pair has been lost or damaged. The regenerative type
refers to restoration of the mass of damaged internal organs
such as liver and pancreas. Morphallaxis refers to reconstruction
of form after severe damage by remodeling the body.

If we could figure out the entity of such information

and elucidate how the regeneration mechanism surveys the tissue integrity, it will impact many fields of
biology including regenerative medicine. Though trials
that aim to reconstruct tissues by using the induced
and or natural stem cells are already ongoing, a
mechanistic understanding of the regeneration process
is necessary for making organs with specified size,
morphology, and function. Particularly, elucidation of
the mechanism of epimorphic regeneration, a natural
process that completely restores the size, morphology,
and function, will greatly benefit regenerative medicine
and make it feasible. This review focuses on the stem
and or progenitor cell system of tissue regeneration,
especially the epimorphic regeneration, in fish, and
overviews the recent progress in our understanding.

Structure of the tail fin in fish

Technically, epimorphic regeneration has been defined
as a form of regeneration that is initiated by the formation of a blastema, which is a mass of proliferating
undifferentiated progenitor cells, followed by their differentiation to attain restoration of the tissue. This phenomenon has attracted the attention of biologists
since the first description of limb regeneration of crayfish in the early 18th century by Rene-Antoine Ferchault de Reaumur (1712). Since that era, the fish fin has
also been used for regeneration research (Broussonet
1786), which has now spanned more than 200 years.

The fish tail fin is an easily accessible tissue for

examining the regeneration process due to its simple
and radial symmetrical structure, rapid and robust
regeneration, and accessory function for animal survival. Actually, fish are the champions of regeneration
among vertebrates, since they possess a striking
potential to regenerate not only fins, but also scales,
retina, spinal cord, and many internal organs including
the heart and pancreas. Regarding the tissue origin,
the fish tail fin is an organ analogous to the tail of
terrestrial vertebrates; but unlike the tail, it does not
contain the extension of the notochord and muscles.
Common to all types of fish fins, the main constituents
of the tail fin are the fin rays, which are composed of
an array of fin-ray bones that have a shape like a
longitudinally split piece of bamboo (Fig. 2). These
bones are radially arranged to form a fan-like frame
and are covered with an epithelium that has a basal
cell layer at its bottom. Within the concave fin-ray
bones, the hollow spaces contain the actinotrichia,
which is the bundles of collagen fibers, arteries and
nerve axons that run down to the caudal end. The rest
of the space within the fin is filled with non-characteristic mesenchymal or connective tissues.

Epimorphic regeneration in fish species

A number of classical studies have afforded an extensive description of the process of regeneration.
According to those studies and our own observations,
the process goes through the following steps:
(i) wound closure; (ii) epithelial wound healing and formation of a wound epidermis; (iii) the formation of a
blastema at the distal end of the mesenchyme; and (iv)
proliferation of blastema cells, their differentiation, and
tissue reconstruction (Fig. 3).
After the amputation, the opening of the epidermis is
in many cases quickly sealed by prompt contraction of
epithelial tissue surrounding the amputation site, which
is known to be mediated by the formation of the
F-actin purse string around the wound and its rapid
contraction (Martin & Lewis 1992). Such a rapid reaction
of wounded tissue is commonly observed in flies and
vertebrates (Martin et al. 1994; Wood et al. 2002), and
it appears to be a mechanism conserved through
evolution as a first tissue reaction against tissue trauma.
Though the process of early wound closure is accompanied by neither cell proliferation nor migration, in the
following step when the actin purse string disappears,
the epithelial cells around the stump actively migrate to
form the wound epidermis, a specialized epithelial thickening formed over the stump, which has a molecular
identity different from the surrounding epidermal regions
(Poss et al. 2000b; Kawakami et al. 2004).

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Stem cell system in fish regeneration

(a)

(b)

79

(c)

Fig. 2. Structure of adult fish fin. (a) Appearance of a caudal fin. (b) Magnification of boxed area in (a). The segmented fin-ray bones
(arrowheads) are surrounded by mesenchymal cells and covered with thin epithelial cells. Irregularly distributed black spots are the
chromatophores. (c) Schematic illustration of fin rays. A segment of fin-ray bone is composed of two hemi-rays. The pink line depicts
the blood vessel.

(a)

(b)

Fig. 3. Epimorphic regeneration in adult fish. Process of fin regeneration (a) and actual tissue appearances (b). After the amputation of
an adult caudal fin, the amputated plane is covered with new epithelial cells by 3 h. However, a tight epithelial sheet is not formed at
3 h. The wound epidermis is formed by 1 day. In the following stages, the blastema (red ovals) is formed, and its active cell proliferation
and growth recover the original fin morphology in approximately 10 days. Irregularly distributed black and golden spots are the
chromatophores.

It has been known for more than 100 years that

the presence of the wound epidermis is an absolute
requirement for the initiation of regeneration. In a
well-designed study, Goss (1956) amputated the
forelimbs of newts and inserted the distal epidermisfree ends into the body cavity; a procedure that
keeps the apical surface epidermis free. These limbs
did not regenerate. Thus, the apical wound epidermis plays a necessary role in regeneration; however,
it remains unclear how the wound epidermis affects
regeneration.

In the successive stages, the blastema proliferation

provides an adequate number of cells to fulfill restoration of the lost tissue part; and further cell differentiation and tissue remodeling reconstitutes the original
tissue architecture.

Origin of the blastema Are blastema cells

stem cells?
It is widely accepted that the presence of the wound
epidermis and blastema is absolutely required for the

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A. Kawakami

initiation of regeneration and that the formation of the

blastema and its active cell proliferation are the crucial
and necessary driving forces for the progression of
regeneration. Although the blastema cells seem to
have the characteristics of undifferentiated cells, are
blastema cells really stem cells? According to the definition, stem cells are cells that have the capacity to
self-renew for an extended period of time and the ability to differentiate into a mature cell. In a broad sense,
the blastema in fish regeneration nearly fulfills the criteria of stem cells, although little is known about the
exact origin and fate of the blastema.
In terms of the origin of the blastema, there are two
opposing hypotheses. One is that the blastema is a
collection of activated cells derived from a population
of dormant and widely distributed stem cells. The tissue regeneration by such a stem cell system can be
seen in the regeneration of planarians, annelids and
other invertebrates (see other reviews in this issue);
however, an apparent example of stem cells in vertebrate tissue regeneration has not been proved. If this
is true, the blastema cell is precisely a stem cell or a
progeny derived from a stem cell.
Alternatively, another prevailing idea is that the blastema originates from cells around the wound site
by de-differentiation. Cell lineage studies in urodele
amphibians have suggested that this actually occurs in
amputated tissues (Tanaka & Brockes 1998; Echeverri
et al. 2001; Echeverri & Tanaka 2002). In such a case,
the blastema cells should be called precursor cells as
a transient cell population that is exhausted at the end
of regeneration. As well as in urodeles, it has been
observed that the mesenchymal cells in the amputated
fish fin undergo disorganization and migration toward
the amputation plane, where the blastema is formed
(Poleo et al. 2001). In addition, the blastema cells
express msx molecules, a family of transcription factors that may have a role for maintaining cells in an
undifferentiated state (Akimenko et al. 1995; Odelberg
et al. 2000). From these and other observations, it has
been thought that the de-differentiation is a more
favorable hypothesis, although a direct demonstration
of the origin of the blastema in fish has not been made
yet. Thus, we cannot completely rule out either of
these possibilities before carrying out a detailed tracing
of cell lineage. In the remainder of this review, I will
tentatively use the term stem cells to refer to the
blastema cells responsible for fish tissue regeneration.

clarified. A cell lineage analysis was recently carried out

during limb regeneration in the axolotl, and it surprisingly showed that the de-differentiated cells derived
from various types of cells such as dermis, muscle, epidermis, and Schwann cells produced the same respective type of cells after regeneration (Kragl et al. 2009).
Though it has been postulated that the blastema is a
sort of stem cell that can produce several cell types,
the result in the axolotl suggests that the blastema itself
is a group of heterogeneous cells that are weakly
de-differentiated and re-enter the cell cycle.
In line with such a heterogeneity of regenerating axolotl tissue, a number of observations in fish have
shown that the expression of sonic hedgehog (shh)
and bone morphogenetic protein 2b (bmp2b) is localized in lateral domains of the basal layer of the wound
epidermis (Laforest et al. 1998) and that lef1 has a
similar overlapping expression in the basal wound epidermis (Poss et al. 2000a). Further, a study by Nechiporuk and Keating (2002) demonstrated that the
distal most part of the blastema contains a group of
slow cycling cells that are apparently segregated from
the proximal blastema cells. Moreover, we recently
demonstrated from the expression analysis of a number of regeneration-induced genes that the wound epidermis and blastema are composed of several cellular
compartments with distinct molecular identities (Yoshinari et al. 2009; Fig. 4). According to our results, the
wound epidermis contains at least four different compartments including the apical cells and several types
of basal epidermal cells. The blastema is also divided
into at least three types, the distal, proximal, and lateral populations, which appear to be the early osteoblasts (Yoshinari et al. 2009; Kawakami in press).
Thus, the emerging data suggest that the regenerating
tissue is a much more complex tissue than previously
thought.
However, there is no concrete study on fish that
addresses the fate of each blastema cell. So, the fate
mapping study will be one of the important ones to carry
out. Though the cell lineage tracing in the axolotl was
carried out with transplanted cells (Kragl et al. 2009), it
can ideally be carried out by using genetic labeling.
Recently, it was reported that inducible or tissue-specific in vivo recombination using the Cre-ER and lox system was successfully used for a long-term marking of
specific cells in zebrafish (Hans et al. 2009). By using
such genetic labeling, we will be able to trace the origin
and fate of the cells participating in regeneration.

Heterogeneity of cells in regenerating

tissues

Stem cells in heart regeneration

As well as the origin of cells for regeneration, the differentiation potential of the blastema has also not been

Despite the histologically distinguishable morphology

of the blastema in limb and fin regeneration, the

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81

Intermediate layer of wound epidermis

Proximal blastema

Distal blastema

mmp9+ cells

Apical wound epidermis

Differentiating osteoblast
Basal epidermis

Proximal basal wound epidermis

Early osteoblast

Fin ray bone

(Lepidotrichia)

Fig. 4. Cellular compartments in the blastema of a regenerating adult fin. The illustration schematically depicts a regenerating fin ray
(approximately 2 days post amputation [dpa]). Gene expression analysis (Yoshinari et al. 2009) revealed that the regenerating tissue
contains many cell types with different molecular identities. Respective cellular compartments are expressed with different colors.

blastema in other tissues has been poorly characterized. However, regeneration of other tissues in fish is
thought to be mediated by the blastema or blastemalike cells. One such tissue is the heart, as the partially
resected organ in fish can be completely restored
without scar formation (Poss et al. 2002; Poss 2007).
In mammals, however, the lesions in heart muscle
caused by an infarction are never recovered to normal,
but form fibrous scar tissues that may cause severe
contractile dysfunction and lead to heart failure.
During the course of regeneration of the zebrafish
heart, cardiac progenitor cells are successfully driven
to regenerate by interaction with the epicardium,
the thin epithelial layer enveloping the chambers. The
epicardial cells close to the site of injury invade the
regenerating tissue through a process reminiscent of
the epithelial-to-mesenchymal transition (EMT) that
occurs in the developing heart, and provide new vasculature to the regenerating muscle (Lepilina et al.
2006). In this process, epicardial and myocardial
cross-talk is mediated by FGF signaling, and inhibiting
the FGF receptor blocks cardiac regeneration (Lepilina
et al. 2006). Not only in regeneration but also during
homeostatic cardiac growth the epicardium regulates
the addition of new myocardial and epicardial cells
(Wills et al. 2008). Although dedifferentiation of preexisting cardiomyocytes has also been postulated for
the zebrafish heart, this possibility has not been supported by experimental evidence (Lepilina et al. 2006).
Thus, it remains unclear whether newly formed cardiomyocytes originate from epicardium-derived cells or
from de-differentiated progenitor cells after epicardium-mediated activation. However, in any case, a

group of cells that undergo active mitosis and produce

the mature cardiomyocytes, which can be called blastema-like cells, is formed around the site of injury.
In mammals, it has long been believed that the cardiac muscles never renew in their postnatal lives (Leu
et al. 2001). However, a recent study has shown that
the adult heart achieves a modest, but nonetheless
convincing, self-renewal after injury that was attributed
to a pool of resident stem cells (Hsieh et al. 2007). In
addition to this, it has been suggested that the epicardium also has a crucial stimulatory role essential for
proper development and regeneration, as in the zebrafish. One of the factors that influences this instructive
role is thymosine b4 (Tb4), a G-actin monomer-binding
protein implicated in cytoskeleton reorganization. Tb4
secreted from the developing myocardium stimulates
the proliferation, differentiation, and inward migration of
epicardial cells (Smart et al. 2007). Another potential
source of cardiac stem and progenitor cells may be
the vasculature. Vessel-associated progenitor cells,
called mesoangioblasts, have recently been identified
in the juvenile mouse heart (Galvez et al. 2008).
Although it is controversial as to which stem cell
source is responsible for the natural process of selfrenewal in the heart (Ausoni & Sartore 2009), it is
intriguing to compare the heart stem cell systems in
different species with different regeneration potentials.

Stem cell system in scale regeneration

The fish scale is also known as one of the regeneration-competent tissues (Sire 1989). Though the term
scale often covers a variety of different structures,

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A. Kawakami

the elasmoid scale is the commonest type of scale

within teleost species (Sire & Akimenko 2004). In
medaka and zebrafish, the body is covered with several hundreds of large elasmoid scales, arranged in
longitudinal and vertical rows, forming a regular pattern. The elasmoid scale, like the other elements of the
dermal skeleton including the membrane bones, forms
in the dermis without the presence of a cartilaginous
initium (Zylberberg & Nicolas 1982).
Though hair and fish scales are distributed over the
body surface in an orderly pattern, it has been thought
that they are morphologically and evolutionally different
(Sharpe 2001). However, recent studies have shown
that the formation of these skin appendages is
governed by the same signaling pathway including a
transmembrane protein, ectodysplasin or EDA, and a
TNF-like receptor, EDAR, that interacts with EDA
(Kondo et al. 2001), indicating the existence of a common developmental and molecular mechanism for
appendage formation involving epidermaldermal interactions. In addition, the expression of several signaling
molecules such as shh is shared in mammalian hair,
bird feather, and fish scale (Iseki et al. 1996; Nohno
et al. 1995; Sire & Akimenko 2004), supporting a
molecular commonality among these skin appendages.
During the past decade, it has been shown that the
hair bulge, an upper permanent region of the hair follicle below the sebaceous glands, is the residence of
stem cells that can contribute to epidermal repair
when a wound cannot spontaneously repair itself
through the migration of epidermal cells from the
neighboring unwounded epidermis (Ito et al. 2005). On
the other hand, little is known about the stem cells
within the epidermis and or dermis in fish skin. It
would be intriguing to characterize such cells in the
fish skin and compare the underlying mechanism with
that operating in mammals.

Control of cellular supply during

regeneration
Regardless of the type of regeneration, either epimorphic regeneration, simple tissue regeneration or
other types of tissue restoration, it is essential for
organisms to supply the cells that replenish the
damaged parts. Although we are not sure of the
nature of blastema cells, cells that are not terminally differentiated and retain their proliferation activity
are involved in the process and are the source
of new cells that participate in the repair of tissues.
The control of morphogenesis and tissue architecture
is also an important issue in regeneration, but it acts
later on. Thus, in a sense, regeneration can
be defined as a process in which new cells are

supplied in response to tissue loss or incompleteness of positional information.

The strategies of cell supply are different depending
on the tissue types and or extent of the damage.
Small wounds can completely be repaired by cell division of surrounding epithelial cells and fibroblast cells,
whereas larger tissue loss requires the consecutive
occurrence of several distinct processes including epithelial wound healing, induced proliferation of stem
cells such as the blastema, and production of new
cells to form new body parts. Seemingly, re-epithelialization occurs in a similar way in all animals, and
appears to be possibly universal machinery that serves
as a primitive response of organisms to their body
damage (Gurtner et al. 2008). In regeneration-competent species or tissues, the first step for complete
regeneration is the formation or activation of stem cells
such as the blastema. Otherwise, in many mammalian
tissues, damaged tissues form a scar, a fibrous tissue
that contains plenty of collagen matrices by way of the
transient formation of granulation tissue. A leading
hypothesis is that the immune system is involved in
the switch between regeneration and fibrotic healing,
because human fetuses, which heal without scarring,
have immature immune systems (Mescher & Neff
2005). Although the significance of scarring and its
relation to regeneration have not been elucidated, the
mechanism of cell supply, in other words, the activation of the cell cycle and formation of proliferating cells,
is one of the critical issues to be addressed in order to
understand the process of tissue repair either in fish or
in mammals.

Molecules and signals that are involved in

regeneration
Then, how does the epithelial wound healing proceed
to regeneration in fish? Further, why does such a
regeneration system not operate in large wounds of
mammalian tissues? To approach these issues, we
need to know the molecular basis of regenerationcompetent systems and compare it with that of incompetent ones. During the last decade, studies aimed at
the molecular basis of regeneration were conducted
and identified a number of molecules and signaling
pathways involved in regeneration (reviewed by Poss
et al. 2003; Iovine 2007; Nakatani et al. 2007; StoickCooper et al. 2007). A number of signaling pathways
such as Hedgehog (Hh), Wnt, Fibroblast growth factor
(Fgf) and Activin bA signaling as well as many other
molecules have been shown to be involved in regeneration. The details of these respective molecules and
signaling pathways are outside the scope of this
review. In spite of the increase in our knowledge about

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Stem cell system in fish regeneration

the molecular players involved, we still must figure out

how these molecules are interconnected to lead to the
respective processes of regeneration such as its initiation, blastema formation, cell proliferation, and morphogenesis. As the regenerating tissue has a complex
cellular composition, we will need to examine the function of these respective molecules in the cellular compartments as well as the role of each compartment.

Use of larval zebrafish finfold as a model of

tissue regeneration
To facilitate the analysis of regeneration at the molecular level, we previously proposed the use of infant
tissue regeneration (Kawakami et al. 2004). The newly
hatched zebrafish larva at 2 days postfertilization (dpf)
has already completed the formation of most of its
organs and tissues, and thus it serves as a useful system for analyzing the tissue repair process. According
to a previous study, even such infant tissues respond
to tissue trauma to form a wound epithelium with a
molecular identity distinct from that of the surrounding
epithelial cells and to cause apparent activation of the
cell cycle in cells of the adjacent mesenchymal region,
where the induction of msx-family genes is also
observed. These processes are followed by a complete recovery of morphology within 3 days (Fig. 5).
Thus, in light of the criteria of epimorphic regeneration
such as the formation of specialized epithelial covering,

(a)

83

the induction of genes that represent an undifferentiated state, and the activation of cell cycle, the larval
finfold regeneration fulfils these criteria of epimorphic
regeneration.
The larval finfold is mostly composed of mesenchymal
cells covered with a thin epithelial sheet. At this stage,
differentiated cells cannot be detected, except that the
growing actinotrichia, bundles of collagen fibers, begin
to radially elongate. Though the nerve fibers from the
spinal cord have entered and become distributed all
over the finfold, there is no detectable presence of
blood vessels or their arbors by 5 dpf (not shown).
Despite its functional homology with the adult caudal
fin, the larval finfold is a transient tissue that is later
replaced by the developing adult fin. The precursor cells
that form the adult fin rays arise from the ventral side of
posterior somites and or notochord and migrate to form
the actively proliferating fin ray progenitors, the process
of which was shown to be dependent on Hh signaling
(Hadzhiev et al. 2007). These fin rays precursors gradually shift to the dorsal side to form the adult caudal fin as
if spreading a fan (Sakaguchi et al. 2006; Hadzhiev
et al. 2007; Fig. 6). In view of such differences in origins
and structures between the larval finfold and adult fin,
the similarity in the repair processes between these tissues is striking and suggests a commonality of basic
mechanisms that do not depend on the tissue types.
Given the commonality with adult epimorphic
regeneration, the model of larval finfold regeneration is

(c)

(b)

Fig. 5. Regeneration of juvenile tissue. (a) Zebrafish larvae at 2 days post fertilization (dpf). The finfold can be used as a model of
regeneration. (b) Temporal process of larval finfold regeneration. Hematoxylin staining. By 6 h, the wound epithelium (arrowhead)
composed of tightly packed cells is formed. From 12 to 48 h post amputation (hpa), many cells with condensed chromatin, which
seems to represent the actively dividing cells, are observed in areas adjacent to the stump (bracketed areas). (c) Schematic illustration of
larval regeneration.
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A. Kawakami

(a)

(b)

4 dpf

~7 dpf

(c)

~15 dpf

Fig. 6. Transition from larval fin fold to adult fin. (a) Bromodeoxyuridine (BrdU) labeling of zebrafish larva. A population of
proliferating cells migrating from the ventral side of the notochord
(arrowhead) is the precursor of the adult fin ray. (b,c) Schemes
illustrating the change in migration pattern. Cells forming the
fin-ray precursors migrating in the ventral direction gradually
move to the caudal and then dorsal directions, as the caudalmost few segments of the notochord tilt toward the dorsal side.

amenable to the conventional molecular approaches

established for the analysis of embryogenesis, such as
morpholino antisense oligonucleotide (MO)-mediated
gene knockdown. In particular, further merit is that an
enormous number of larvae can be used for the assay
due to the high fertility of zebrafish. In addition, the
small size of newly hatched larvae in combination with
their aquatic habitat is useful for carrying out a pharmacological test or screening in a multi-plate dish
(Mathew et al. 2007; Yoshinari et al. 2009). Moreover,
a useful but a slightly tricky application of this model is
that we are able to use plenty of mutant resources for
dissecting the regeneration process by a genetic
means, because even the lethal mutations can be
used for the assay as far as they do not affect finfold
formation and survive up to 5 dpf in a good condition
(Yoshinari et al. 2009). Though the larval tissue model
may not have processes of apparent cell differentiation
and complex organ formation, it is certain that it
should have a basic regeneration process such as the
formation of highly proliferating cells and control of cell
supply. We will be able to use this model along with
the adult tissue model to further dissect the molecular
pathway underlying tissue maintenance and regeneration.

Initiation of regeneration and stem cell

proliferation
Whereas the epithelial wound healing appears to be
similar in many species and tissues, why does such a
common reaction proceed to regeneration only in some
species or organs? Still, we cannot completely exclude
the possibility that the wound healing process itself,
including the early immune reaction, differs between

regeneration-competent and -non competent species

or organs (Mescher & Neff 2005); however, no apparent
early difference has been documented so far, including
the immediate induction of Jun family transcription factors (Ishida et al., in preparation), which are suggested
to have a crucial role in wound healing (Yates & Rayner
2002). Then, what is the determinant of regeneration?
Is there a fish-specific regeneration protein? Furthermore, when does regeneration start?
Previously, we reported that the expression of two
members of the junb family genes in zebrafish was
induced during regeneration of the larval finfold and
adult fin. It appears that the expression of these genes
demarcates regeneration from wound healing. The
immediate induction of Jun family genes, c-Jun and
JunB, is soon attenuated in mammalian tissues; however, only the expression of two zebrafish junb genes,
but not that of c-jun, is maintained until the regeneration stage (Ishida et al., in preparation). This maintained expression seems to be the earliest event that
is specific to regeneration.
It has been shown in Drosophila that Jun and its
upstream kinase, the Jun N-terminal kinase (JNK), are
necessary for epithelial cell migration during wound
closure (Jacinto et al. 2000). In mammals, the
functions c-Jun and JunB for proper epithelial wound
healing have also been suggested by studies using
skin-specific conditional gene knockout mice (Grose
2003; Zenz et al. 2008). Generally, fish have two junb
genes, and their expressions are seen in different cellular compartments during regeneration (Yoshinari
et al. 2009). Thus, the original Junb function is thought
to be divided into two independent Junb proteins in
different cell groups. Intriguingly, we recently observed
that two junb genes were immediately induced in the
forming wound epidermis, but as soon as the initiation
of blastema formation the junb-like (junbl) expression
shifted to the blastema. In our recent study, we succeeded in showing that the functions of Junb proteins,
particularly the function of Junbl, are indispensable for
regeneration, Junbl is thought to be the earliest
functional molecule necessary for regeneration (Ishida
et al., in preparation).
To our further surprise, we also found that Junb
family proteins were phosphorylated in response to the
wounds (Ishida et al., in preparation). It has been
shown in mammals that the consensus target
sequence for JNK is not conserved in JunB and that
this sequence in humans and mice is never phosphorylated (Kallunki et al. 1996). However, the fish Junb
proteins partially retain the consensus sites and indeed
are phosphorylated in vivo (Ishida et al., in preparation). However, curiously, in spite of the early role of
JNK in the Drosophila epithelial healing, our analysis in

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Journal compilation 2009 Japanese Society of Developmental Biologists

Stem cell system in fish regeneration

85

zebrafish suggested that JNK signaling was not necessary for the epithelial wound healing, but was required
for regeneration some time after wounding (Ishida
et al., in preparation). From these observations, we
propose that Junb family molecules and their JNKdependent phosphorylation are an important determinant that specifies the initiation of the regeneration
process (Fig. 7). Our data imply that the initiation of
regeneration is determined some time after injury,
where the wound healing itself has a minor role for
determination, although it is still possible that the early
phosphorylated Junb proteins might have some effect
on the wound healing. In any case, it appears that
regeneration proceeds in a stepwise way.

(a)

Maintenance of blastema cells

Wild-type

The induced blastema increases in size by cell division

and at the same time its cells differentiate to produce
a number of mature cells. Thus, the blastema stem
cells possibly keep self-renewing at least during regeneration. Our recent study has suggested that an additional factor is required for the maintenance of
blastema cells. We have identified a zebrafish mutation
in which the larval blastema cells once formed
undergo apoptosis instead of active cell proliferation
(Fig. 8). The molecule responsible for this mutation has
not been identified, but it is thought to be a key
molecule for blastema maintenance. Another zebrafish
mutant, pinball eye (piy), has the phenotype of retinal apoptosis (Yamaguchi et al. 2008), in which a

Fig. 8. A zebrafish mutant that cannot maintain the blastema.

(a) Mutant phenotype during regeneration (left) and abnormal
apoptosis in the blastema (right). Arrowhead indicates a
characteristic abnormal tissue appearance, which is probably
due to the blastema apoptosis; and the bracket indicates the
terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL)-positive apoptotic cells. (b) Wild-type sibling.

(a)

(b)

3 dpa

TUNEL 1 dpa

Mutant

(b)

missense mutation occurred in the small subunit of

DNA primase (Prim1), a molecule essential for DNA
replication; however, this mutation does not affect cell
proliferation but rather induces neuronal apoptosis.
The cellular maintenance mechanism may be different
between the stem cells in regeneration and that of
other precursor cells during retinal development, which
is intriguing considering the mechanism for stem cell
maintenance in regeneration.

Perspectives

Fig. 7. Function of Junb family molecules in fish regeneration

and comparison with that of mammalian c-JUN and JUNB.
(a) Function of c-JUN and JUNB in mammals. The active
phosphorylated form of c-JUN has a strong positive role in cellcycle progression, whereas JUNB lacks the phosphorylation site
and acts antagonistically toward c-JUN. (b) Function of fish Junb
proteins. The fish Junb and Junbl retain a phosphorylation site
and are activated by the wound. These proteins are expressed in
different cells and have distinct functions in the blastema and
wound epidermis.

During the 1980s, little was known about the molecular background of regeneration, although there were
some trials for identifying molecular players (Tabin
1989). Nowadays, many advances have been made in
our knowledge about the molecules involved in regeneration as well as the nature of the blastema and its
induction. However, many questions still remain to be
answered. The following are some of them:
What is the first trigger for wound healing?
What regulates the regeneration-specific gene expression?
What is the origin and fate of cells participating in
regeneration?

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Journal compilation 2009 Japanese Society of Developmental Biologists

86

A. Kawakami

What are the roles of the cells in the respective cellular

compartments?
What regulates the JNK activity?
How is the blastema maintained during regeneration?
How is the positional information integrated into
regeneration?
By elucidating the answers to these questions along
with clarification of the molecules involved and their
networks at the cellular level, we should be able to
obtain a complete picture of the epigenetic landscape
of tissue maintenance.

Acknowledgments
I would like to thank lab members T. Nakajima and
N. Yoshinari for their help in preparing the illustrations,
providing pictures, and valuable discussions.

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