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Strategies for Retinal Tissue Repair and Regeneration in Vertebrates: From Fish to Human,
2007: 77-96 ISBN: 978-81-308-0200-8 Editor: Chikafumi Chiba
5
A comparative study of
amphibian retinal regeneration
by tissue culture technology
Masasuke Araki
Developmental Neurobiology Laboratory, Department of Biological Sciences
Nara Women’s University, Nara 630-8506, Japan
Summary
Amphibian retinal regeneration has been intensely
studied, using the urodele as a model organism.
Transdifferentiation of retinal pigment epithelium
(RPE) into retinal stem cells play a crucial role in
regeneration. Recently, it was found that anurans
(Xenopus laevis) in the adult stage also can
regenerate the retina similarly to the newt. This
provides a new tool of a model for the molecular
mechanisms involved in retinal regeneration. A
precise analysis of the initial cell dynamics in vivo
and under culture conditions indicates that the
interaction of RPE cells with extracellular cues, such
Correspondence/Reprint request: Dr. Masasuke Araki, Developmental Neurobiology Laboratory, Department of
Biological Sciences, Nara Women’s University, Nara 630-8506, Japan. E-mail: masaaraki@cc.nara-wu.ac.jp
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Masasuke Araki
as the choroid tissue and basement membranes, has an important role in the
transdifferentiation of RPE. Further development of culture methods now gives
a system in which the RPE monolayer can regenerate into a well-organized
structure consisting of all layers of the retina, providing an excellent culture
model for retinal regeneration.
Introduction
Urodelian amphibians possess a prominent capacity to regenerate organs
when they are partially damaged or are lost. Anurans also have this ability but
only in their embryonic stages. The ocular tissues such as lens, cornea, and retina
have been experimental materials most intensively examined in regeneration
studies since the initial observations by biologists in the 19 century (Philipeaux,
1880; Colucci, 1891; Wolff, 1895). In recent years many different animal species
from fish to mammals have been studied to search for their regeneration
potential. Numerous review articles have repeatedly described details of the
phenomena (Eguchi, 1979; Stroeva and Mitashov, 1983; Okada, 1991;
Hitchcock and Raymond, 1992; Park and Hollenberg, 1993; Mitashov, 1997;
Reh and Levine, 1998; Raymond and Hitchcock, 1997) and in recent years more
information has accumulated on the molecular bases of retinal regeneration (Del
Rio-Tsonis and Tsonis, 2003; Hitchcock et al., 2004; Araki, 2007a).
The vertebrate eyes are planed on a common developmental progression and
built also in a common structural arrangement. Similar molecular mechanisms in
part also underlie the development and regeneration of the vertebrate retina
(Araki, 2007b). Vertebrate retinas basically have the same physiological
functions, and researchers have been utilizing a variety of animal species for
investigation. Retinal regeneration, however, seems to have diverse aspects
depending on species; in some animals like urodeles a complete retina
regenerates after surgical removal of the whole retina, but most other animals are
unable to undergo such a complete retinal regeneration except during embryonic
stages in certain species. In some species like fish or avians, the retina can be
repaired by intraretinal stem cells after it is partially injured. More recently,
evidence has been found for the existence of retinal stem cells in mammals,
including humans. Various culture technologies are excellent approaches for the
regeneration studies, because we are able to compare results from different
animal models under the artificially organized conditions. Such comparative
studies will bring us a basic understanding on the cellular and molecular
mechanisms involved in vertebrate retina regeneration. The purpose of this
review is to provide a comparative analysis of retina regeneration in urodele and
anuran under the culture condition as well as in vivo circumstances so as to
obtain a common view on vertebrate retina regeneration. We particularly want to
focus on transdifferentiation of retinal pigment epithelium (RPE).
In vitro analysis of amphibian retinal regeneration
79
Regeneration of vertebrate retina
Retinal regeneration can take place to a certain extent in some vertebrates,
depending on animal species and in a perfect manner only in the urodele
amphibian. These include fish, amphibians, birds and mammals, although
regeneration is restricted to certain stages of development in most animals (Del
Rio-Tsonis and Tsonis, 2003; Reh and Fischer, 2001; Okada, 2004). Partial
retina regeneration has been described in many adult animal species, such as fish,
amphibians and birds; cells sequestered in the retinal tissue including rod
precursors or Müller glial cells are the sources of regenerating retina in addition
to retinal stem cells or precursor cells in the ciliary marginal zone (CMZ) (Reh
and Levine, 1998; Fischer and Reh, 2002). In teleost fish such as goldfish, for
instance, retinal regeneration occurs either after surgical removal of a portion of
the central part of the retina or pharmacological lesioning by ouabain that
destroys the neural retina but leaves rod cells intact (Hitchcock and Raymond,
1992). The rod precursors, in addition to the stem-like cells located within the
retina (in the inner nuclear layer), appear to be the origin of retinal regeneration.
Urodelian amphibians like the newt are unique animals that possess the
ability even in the adulthood to regenerate the whole retina after its complete
removal. No other animals had been shown to have this ability until recently
when Yoshii et al. (2007) reported that anuran amphibian, Xenopus laevis, have a
similar ability at the post-metamorphosis stage. Previously, it was reported that
retinal regeneration occurs only in embryonic stages of anuran amphibians and in
embryonic avian embryos after whole retinal removal. In the latter case,
however, exogenous administration of cell growth factor (FGF2) is necessary
(Park and Hollenberg, 1989; 1993). In amphibian retina regeneration at the
mature stages, the cellular sources are the RPE cells that subsequently undergo
differentiate into retinal progenitor cells. Stem cells in the CMZ that contribute to
the ongoing growth of the retina also participate in retinal regeneration of these
animals, particularly in anurans (Hitchcock et al., 2004).
Mammals also possess cells that can proliferate and differentiate into
various types of retinal cells under certain conditions of cell cultures; these are
iris epithelial cells or ciliary pigmented epithelial cells. Such findings open the
possibility of clinically oriented autologous grafts for retinal regeneration
(Ahmad et al., 1999; Haruta et al., 2001), although it appears impossible, at the
moment, to restore the large part of damaged retinal tissue in mature mammals.
Details on this topic of mammalian retina regeneration will be mentioned in
another chapter of this book.
It is apparent that retinal regeneration takes place by means of several
different mechanisms and from cell sources depending on animal species.
Retina can be replaced or repaired by RPE cells via a transdifferentiation
process or by stem-like cells in the CMZ and/or within the retina. Different
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Masasuke Araki
animals use different strategies, and regeneration also depends on the age of
the animal, although potent cells are retained in a wide variety of animals. In
the following part of this article, we will focus the transdifferentiation process
of RPE cells that are the main source of retinal regeneration in amphibians
such as the newt, Cynops pyrrhogaster and African toad frog, Xenopus laevis.
Retina regeneration in these animals can take place through transdifferentiation
of RPE cells but seemingly involve different cellular steps in various species.
We will first discuss the regeneration processes of amphibian retinas briefly
and then describe some experimental approaches including tissue cultures,
used by the author’s group.
Amphibian retinal regeneration
Amphibians, both urodels and anurans, have long been the animals most
extensively studied for retinal regeneration with over a century (Fig. 1; Fig. 2)
(see reviews by Okada, 1991; Mitashov, 1997; Hitchcock et al., 2004).
Numerous studies on the newt retina regeneration have revealed that RPE cells
proliferate and differentiate to regenerate the retina following its surgical
removal (Hasegawa, 1958; Keefe, 1973a; 1973b; Levine, 1974, 1975; Reyer
1971; 1977; Stone, 1950a, 1950b). It is apparent that intracellular interaction
and distribution of cytoskeletons in RPE cells are largely affected during retina
regeneration (Fig. 1B, C) (Chiba and Saito, 2000; Umino and Saito, 2002), but
these are not yet well investigated, particularly at the initial stage of
regeneration. In spite of many morphological studies, including electron
microscopic observations and immunocytochemical staining, there have been
virtually no reports on the morphologic changes of RPE at the initial step.
Since upregulation of certain genes in RPE cells is considered to take place
right after retinectomy, more intensive studies are needed to examine the initial
changes of RPE cells at the molecular level. It is of interest to mention that
RPE cells become labeled almost simultaneously with BrdU at an interval of 45 days after retinectomy (Ikegami et al., 2002). Once they initiate cell division,
RPE cells produce retinal stem cells. Therefore, the initial period of 4-5 days is
a sort of ‘latent period’, in which several genes must be activated for the
sequential events of regeneration. After that period expression patterns of
several genes have been described such as Pax6 and Notch (Kaneko et al.,
1999; 2001). An attempt to find early-activated genes has been done and
several candidate genes have been described (Goto et al., 2006), although their
functions in retinal regeneration have not been determined.
Complete retinal regeneration in adult animals following surgical removal
of whole retinal tissue has been considered to take place only in certain urodele
amphibians. In contrast, it was considered that anuran amphibian species retain
the ability only in the larval stages and that during metamorphic stage, they
81
In vitro analysis of amphibian retinal regeneration
Newt retina regeneration
A
B
C
D
E
F
Figure 1. Newt retinal regeneration. Newt retinal regeneration at different stages
after the surgical removal of the retina is shown in the light micrographs from A to F.
The retinal pigment epithelial (RPE) cells appear to be irregularly arranged at day 5
(yellow arrow in B) which then become more epithelial at day 10 (arrow in C). At the
same time, RPE cells become de-pigmented. Then, a multi-stratified epithelium is
formed, separateing a pigmented single epithelium layer at the basal side (arrow in E).
In the neuroepithelial layer cells are randomly arranged. About one month later, a well
stratified retinal tissue and a RPE have developed (arrow in F). Hematoxylin and eosin
staining. (A, B) Day 5, (C) Day 10, (D, E) Day 14, (F) Day 28.
(from Araki, 2007).
quickly lose the ability to regenerate the retina, only retaining the ability to
repair partially-damaged retinal tissue (Lombardo, 1969). We recently showed
that X. laevis retain the ability to regenerate the retina even in mature animals
following the surgical removal (Fig. 2) (Yoshii et al, 2007). This finding leads
us to examine further the possibility that regeneration potency can be
potentiated more widely in other higher vertebrates.
In spite of a volume of descriptive studies on urodele RPE
transdifferentiation, we still have very little information on the cellular and
molecular mechanisms involved in amphibian retina regeneration (Eguchi,
2004; Okada, 2004). The finding of complete retinal regeneration in two
different amphibian species provide a new tool for the molecular analysis of
regulatory mechanisms. At the same time, X. laevis is a suitable animal for
transgenic analysis and this may enable us to search for genes and their
regulative expression responsible for retinal regeneration.
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Masasuke Araki
Xenopus retinal regeneration
A
B
*
C
D
*
Figure 2. Xenopus retinal regeneration. Retinal regeneration of adult X. laevis is shown
at different stages after the surgical removal of the retina. (A, B) An early stage of retinal
regeneration at day 7 after retinectomy. Arrows indicate retinal vasucular membranes
(RVM). The area indicated by asterisk in (A) is shown in (B) at a higher magnification. In
(B), a new flat epithelial layer consisting of pigmented epithelial cells is juxtaposed with
the retinal pigemented epitheliaum (RPE) (yellow arrow). Blue arrow indicates single
pigmented cell located between the RPE and RVM. (C, D) Retinal regeneration at day 20.
By this time, the laminar structure of the regenerating retina is partially developed at the
periphery (arrow in C). The area indicated by asterisk is shown in (D) at a higher
magnification. (D) A few RPE cells are found in the space between RPE layer and the
retinal epithelium as shown by blue arrows. Some cells extend from RPE layer to the
retinal layer (indicated by lower blue arrow). Blue-color arrowheads indicate pigmented
cells attached to the retinal layer. (from Yoshii et al., 2007).
RPE cell dynamics at the initial phase of transdifferentiation
To begin with this part, the author wants to tell the reason that we started
to study X. laevis retina regeneration and finally came to find that X. laevis can
regenerate the retina after complete retina removal.
In vitro analysis of amphibian retinal regeneration
83
Numerous review articles have indicated that anurans were considered not
to be able to regenerate the retina. Only at the larval stage was it believed to be
possible in certain species (Stroeva and Mitashov, 1983; Reh and Nagy, 1987).
As mentioned below, we had been working on the newt RPE using tissue
culture techniques and found that RPE cells do proliferate and differentiate
into neuronal cells under certain culture conditions (Ikegami et al., 2002;
Mitsuda et al., 2005). While we were working on the newt RPE, we considered
that the tissue culture technique could be used with X. laevis RPE to test
whether they can transdifferentiate into neural cells or not. The results showed,
as shown below, that X. laevis RPE cells (3 to 4 months after metamorphosis)
behave quite similarly to the newt RPE in vitro, suggesting that mature X.
laevis retain an ability to regenerate the retina. If X. laevis can not regenerate
retina in vivo, this might indicate that X. laevis RPE cells manifest their ability
of transdifferentiation only under culture conditions.
In our initial attempts to study retinal regeneration in X. laevis, we found that
immature retina was occasionally observed in retinectomized eyes at 30 days
after operation and we finally found that retina can regenerate if the retinal
vascular membrane is left in the posterior eye chamber (Fig. 2). In our
experiments, 120 animals in total, between 3 and 9 months after metamorphosis,
were surgically operated on to remove both the lens and retina (Yoshii et al.,
2007). At around post-operative day 30 (PD 30), both the retinal layer and lens
were found to have regenerated. Retinal regeneration was observed in
approximately 70% of operated animals (45 out of 65 animals operated on and
fixed after PD 15). In many cases with successful retinal regeneration, the lens
also regenerated. No profound difference was observed in the regeneration
process, regardless of the animal stages. In the rest of the animals (20 out of 65),
neither the lens nor NR could be seen, and the vitreous cavity was usually
occluded by incision closure in these unsuccessful cases. Shortly after
retinectomy (PD7-10), a pigmented cell layer became apparent in the vitreous
chamber, facing the original RPE layer (Fig. 2B). As a result, two pigmented
epithelial layers were observed, separated from each other, and facing each other.
During the subsequent period, this newly formed epithelial layer (inner layer)
appeared to undergo transdifferentiation into the neural retina. Cells in the two
pigmented epithelial layers are positively stained for both RPE65, a specific
marker of RPE, and Pax6, indicating that the pigmented cells in the inner layer
do not originate from the iris pigmented epithelial cells but from RPE cells
(Yoshii et al., 2007). RPE cells in the newly formed layer actively proliferate as
shown by BrdU uptake and intense staining for PCNA, a cell proliferation
marker. How could RPE cells form the new epithelial layer?
When the retina of Rana tadpoles is devascularized by complete removal
of the eyeball and restoring it into the orbit, new retina regenerates following
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Masasuke Araki
Figure 3a. Schematic diagram of retinal regeneration in X. laevis. The upper part in
(A) shows retinectomized eye cavity, and the lower shows intact one. Cells from two
origins regenerate the retina; ciliary marginal cells and the retinal pigmented epithelial
cells. The CMZ (ciliary marginal zone) partially remains after retinectomy with the
present surgical procedure, and CMZ stem cells initiate migration on the RVM (retinal
vascular membrane) to the posterior direction. (B) At the same time, some of RPE cells
leave RPE layer, migrate and attach to the RVM, where they form a new RPE layer as
indicated in (C). Numerous capillaries (indicated as C) are seen in RVM. RPE cells on the
RVM proliferate and transdifferentiate to neural retinal precursor cells (D, E). RPE cells
that were positively stained for RPE65 are shown by brown-color nuclei or pigmented
granules in the cytoplasm. (from Yoshii et al., 2006)
Figure 3b. RPE transdifferentiation in the newt. In the newt retina regeneration, RPE cells
become more loosely adhered each other soon after the retinectomy. At about day 5 cells
initiate proliferation and become de-pigmented and show more well packed epithelial structure.
RPE cells do not leave the epithelium and within the epithelium, cells at the most basal side
(attaching to the Bruch’s membrane) become pigmented. More apically located cells now
form a stratified epithelial structure. IML: inner limiting membrane. (from Araki, 2007).
In vitro analysis of amphibian retinal regeneration
85
retina degeneration (Reh and Nagy, 1987). In that regeneration, it was observed
that RPE cells detach from the epithelium, phagocyte degenerating retinal cells
during migration and finally re-attach to the retinal vascular membrane. A
partial retinal regeneration was reported in adult Rana when a radial incision or
partial retinal removal was made (Lombardo,1969). These studies indicate that
some anurans retain the ability to regenerate the retina in oculo under certain
conditions.
Fig. 3 summarizes the results of X. laevis retinal regeneration. During
regeneration processes, the RPE layer always remains pigmented (that is, RPE
cells do not undergo depigmentation) and the newly formed epithelium (the
regenerating NR) emerges on the remaining retinal vascular membrane.
Epithelial cells constituting this layer are considered to originate from RPE
cells and partly from cells in the CMZ. Numerous isolated pigmented cells are
found in the vitreous space between the inner epithelial layer and the RPE,
seemingly having detached from the RPE layer and migrated to the inner layer.
RPE cells anchored by the retinal vascular membrane initiate cell proliferation
and transdifferentiate into retinal cells. They finally regenerate the whole retina
structures. It is particularly interesting to draw attention to the fact that no
retinal regeneration occurs at the original RPE layer. It appears that for retinal
regeneration RPE cells must detach from the RPE layer, be deprived of cellcell interaction by migrating alone and must attach to the retinal vascular
membrane.
A retinal vascular membrane (RVM) consisting of the inner limiting
membrane (basement membrane) and capillaries is considered to supply an
anchoring base for migrating RPE cells, because when the RVM was
intentionally removed, no pigmented layer was formed, resulting in no
regeneration. The RVM and Bruch’s membrane are both positively stained for
laminin. In contrast to these observations in anurans, the newt RPE cells
always transdifferentiate within the epithelium, as if they remained firmly
fixed to each other or to the basement membrane. As the cells in the epithelium
undergo transdifferentiation, they become depigmented and proliferate. It is
possible that asymmetrical cell division gives rise to two daughter cells, one
adhering to the basement membrane and the other free from the membrane.
The latter cell type then develops into retinal stem cells to regenerate the
retinal structure. An important question remains to be solved in the anuran case
why RPE cells at the original RPE layer do not transdifferentiate in situ;
instead they only do so after migrating to the RVM. Some ECM components,
characteristic to RVM, appear to be needed for RPE cell transdifferentiation
and formation of retinal stratified structure. For the moment, we do not know
about the molecular nature characteristic of the RVM. Alternatively, any
molecular component in the anuran RPE basement membrane may maintain
the properties characteristic of RPE cells.
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Masasuke Araki
In vitro approach to RPE transdifferentiation
Numerous culture techniques have been utilized in amphibian RPE to
elucidate the cellular and molecular mechanism involved in
transdifferentiation. Intraocular implantation (vitreous chamber culture of
tissues) was applied to ocular tissues such as the iris pigmented epithelium and
RPE and it has been shown that pigmented epithelium from tadpoles of X.
laevis and Rana temporaria transdifferentiate to the retina (Lopashov and
Sologub, 1972; Bosco, 1988; Arresta et al., 2005). It is also reported that these
tissues from adult anurans can transdifferentiate into the retina under a certain
condition (Sologub, 1977; Bosco, 1988). Although vitreous chamber culture is
suitable to ensure retinal histogenesis (formation of sublayers), artificial
culture methods are more widely used for the cell and molecular analysis of
RPE transdifferentiation (Mitsuda et al., 2005). Reh and his colleagues
developed cell and tissue culture methods for anuran RPE and obtained
evidence that extracellular matrix molecules like laminin and heparan sulfate
proteoglycans play a crucial role in larval Rana transdifferentiation (Reh et al.,
1987; Nagy and Reh, 1994). RPE cells from the newt were also cultured after
tissues were dissociated, and voltage-gated inward currents were observed with
time in culture (Sakai and Saito, 1997). Under dissociate culture condition,
however, newt RPE cells do not manifest neuronal morphology. This was also
the case in RPE cells from chick embryos; bFGF fails to induce neuronal
transdifferentiation when RPE is cultured as dissociated cells (Pittack et al.,
1991). These results indicate that cell-cell communication in RPE cells are a
pre-requisite for transdifferentiation.
While we were searching for the key substances involved in retinal
regeneration in the newt, we utilized a tissue culture method originally
developed for tissue culture of embryonic chick RPE (Araki et al., 1998;
2002); RPE sheets were isolated and cultured on culture filter membrane
without dissociation. This method enabled us to analyze the cellular
mechanisms involved in transdifferenetiation in the Silver homozygote mutant
of the quail under culture conditions. Intraocular administration of FGF2 into
retinectomized chicken eye chambers induced the remaining RPE to
transdifferentiate into retina at an early stage of development (Coulombre and
Coulombre, 1970; Park and Hollenberg, 1989). In the Silver homozygote, RPE
cells autonomously transdifferentiate to neural retina during embryonic
development, resulting in a double NR at the posterior region of the eye (one
original and the other newly formed retinal layer from RPE). The RPE
transdifferentiation begins at about 4 days of incubation and occurs only at the
posterior central region of the eye (Araki et al., 1998 and 2002; Mochii et al.,
1998a and 1998b). We were interested to learn whether tissue interaction between
RPE and the neighboring choroid tissue has any role in transdifferentiation.
In vitro analysis of amphibian retinal regeneration
87
With our culture method, tissues consisting of RPE and choroid cells were
cultured and the results were compared with that obtained by single cultures of
isolated RPE. It was shown that RPE with the choroid tissue transdifferentiate
to retina, while isolated RPE did not (Araki et al., 2002), suggesting that the
mesenchymal tissues play a crucial role in RPE transdifferentiation. Similar
tissue interaction also appears to play an important role in the amphibian
transdifferentiation.
Tissue culture study of RPE from different amphibian
species enables us to compare their transdifferentiation
ability under the same condition
The newt RPE was cultured under the artificial culture condition described
above, and it was found that RPE cells transdifferentiate into various retinal
cells including photoreceptors when RPE and the choroid were cultured
together (Ikegami et al., 2002). When RPE was removed from the choroid by a
treatment with dispase and was cultured alone, RPE cells neither proliferated
nor differentiated into neural cells and remained epithelial (Fig. 4). The RPE
sheets were then co-cultured with individual choroids by separating these two
tissues by a membrane filter. The results showed that RPE cells became
depigmented and proliferated, suggesting that some diffusible substances from
the choroid are involved in RPE-choroid interaction (Mitsuda et al., 2005).
Among various growth-promoting factors examined, FGF2 had a drastic effect
on RPE cell proliferation and neuronal differentiation. Addition of FGF
signaling pathway inhibitors like SU5402 or U0126 suppressed RPE
proliferation and neuronal differentiation. These results indicated that the
choroid plays an essential role in newt retinal regeneration. FGF2 appears to
induce some neurogenic genes including Pax6 upregulated in RPE cells. It is
still unknown whether FGF2 gene is upregulated in the choroid or not when
the retina is removed. Our preliminary search for FGF2 protein by specific
antibody against newt FGF2 showed that numerous cells in the choroid
become immunoreactive (data not shown).
As mentioned above, while we were working on the newt, we were also
interested in the anurans. Our culture method provides a good experimental
system to compare the RPE properties of the two animal species (Fig. 4; Fig. 5).
The RPE-choroid tissues from mature X. laevis were laid on a filter membrane
and cultured for 30 days. At around Day 6 in vitro, RPE cells migrated out
from the periphery of the explants and began to proliferate as indicated by
BrdU labeling. By Day 30, numerous migrated cells extended long branching
processes, positively stained for neural markers such as acetylated tubulin and
neurofilament.
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Masasuke Araki
Organtypic culture of isolated newt RPE sheet
A
C
D
E
G
I
B
F
J
H
K
Figure 4. Organotypic culture of isolated newt RPE sheet. (A ~ E) Isolation of newt
RPE sheet from the choroid and culturing of isolated RPE. (A) A tissue consisting of
RPE and choroid (sclera had been removed) stained for Azan. (B) Dispase treatment
removed RPE from the choroid. The Bruch’s membrane stained in blue color is found
at the choroid (arrow). (C) Isolated RPE sheet. No other cells from the choroid were
found. (D) Isolated RPE sheet cultured on the membrane for 2 days showed an
epithelial feature which remained on Day 30 as seen in (E). Scale bar in A is 100 µm
and is applied to B, and C. Scale bar in D is 100 µm and is applied to E. (F ~ K)
Isolated RPE sheets were cultured with FGF-2 and examined for cell proliferation and
neural differentiation. Numerous cells extended long neurite-like processes (F) and
some of them were stained either for Syntaxin1A (I), acetylated tubulin (J) or
neurofilament 200kD (K). While RPE cells cultured without FGF-2 did not labeled for
BrdU (G), numerous cells were labeled for BrdU when cultured with FGF-2, indicating
they were proliferating in the culture (H). Scale bar in F is 100 µm. Bar in G is 250 µm
for B and 50 µm for C. Bar in I is 50 µm and is applied to E and F.
(from Mitsuda et al., 2005).
RPE sheets were then isolated by treating RPE-choroid tissues with
dispase and cultured (Fig. 5). By Day 3, RPE cells had extended out from the
periphery of the sheet but still remained as a simple epithelial layer. At around
Day 30, RPE cells remained pigmented and no neuronal differentiation could
be seen, although cells had proliferated, as shown by BrdU labeling (Figs. 8B, C).
89
In vitro analysis of amphibian retinal regeneration
Organotypic culture of singly isolated Xenopus RPE
Day 3
Day 30
B
C
D
E
F
Control
A
FGF
2
+IGF
1
Figure 5. Organotypic culture of isolated Xenopus RPE sheet. (A, D) Day 3 and (B,
C, E, F) Day 30. RPE sheet was separated from the choroid and cultured on a filter
membrane. (A, B, C) show cultures without FGF2 and IGF1, and (D, E, F) show
cultures with both factors. (C, F) show BrdU labeling and (E) shows acetylated tubulin
staining. In control cultures (A, B), RPE cells proliferated but remained mostly
pigmented, while addition of FGF2 and IGF1 induces RPE cells to proliferate,
depigment and differentiate into neural cells, as shown by acetylated tubulin
immunocytochemistry (E). Arrows in B indicate the peripheral edge of the epithelial
sheet. Scale bar in A is 20µm and is applied to D. Scale bar in B is 30µm and is applied
to C, E, F. (from Yoshii et al., 2007).
When cultured in the presence of FGF-2 and IGF-1, some of the RPE cells
became de-pigmented, extended long processes, and were positively stained
for acetylated tubulin. These observations suggest that RPE cells from mature
X. laevis proliferate and differentiate into neural cells under culture condition
in a similar manner to the newt.
Lens regeneration of the newt has been a paradigm of experimental tissue
regeneration, and recent studies have shown that FGF2 triggers lens
regeneration in the iris epithelium of the newt eye (Hayashi et al., 2004). After
the lens removal, lens regeneration appears to proceeds in two steps, the FGF2dependent proliferation of iris pigmented epithelium followed by expression of
Wnt2b and Frizzled4 in the dorsal half of the iris (Hayashi et al., 2006).
Whether activation of Wnt or other signaling pathway sencondarily occurs
during amphibian retina regeneration remains to be studied, particularly in X.
laevis regeneration, where RPE cells in a newly formed epithelial sheet on
RVM must have a different set of gene expression from RPE on Bruch’s
membrane.
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Masasuke Araki
Further application of the method: 3D-reconstruction
of retinal tissues
The culture method described above has developed an experimental
system to analyze the cellular dynamics and to compare them among different
animal species. Although RPE cells do proliferate and differentiate into
neuronal cells under the culture conditions, retinal histogenesis could not be
realized in that condition. Instead, cells extend over the filter membrane.
Another type of culture is needed for the understanding of in vivo retina
regeneration. Reaggregation culture technique has been utilized to develop in
vitro histogenesis of retinal structure using embryonic retinal cells (Layer et
al., 2002), although this method has not yet applied to amphibian regeneration
study. By culturing Xenopus embryonic RPE with rotation culture method,
neurons and glial cells differentiate in cell aggregates that show partially
organized structure (Sakaguchi et al., 1997).
We have developed a new method to ensure the 3D reconstruction of
retinal structure regenerating from the RPE monolayer sheet under artificial
conditions. In our preliminary experiments, retinal vascular membranes were
isolated from X. laevis eyes and were then put over the RPE sheet cultured on
the filter. In this method RPE cells differentiate to a multi-cellular layered
structure. On substituting an artificially reconstructed gel form for the retinal
vascular membrane, a complete retinal structure developed from RPE sheets
(data not shown). Morphological examination of the cultured RPE indicates
that RPE cells actually transdifferentiate into retinal stem cells to reconstruct
the whole retinal structure including ganglion cells, amacrine cells, Müller
cells and rods. In the development of the retina, the inner limiting membrane
including the basement membrane functions as an anchorage for retinal cells
and is essential for the survival of the ganglion cells (Halter et al., 2005).
Disruption of basement membrane leads to dramatic aberrations in retinal
histogenesis (Halfter et al., 2001). The present 3D reconstruction method for
RPE culture provides the RPE cell-derived retinal stem cells with physical
cues for retinal histogenesis.
By this method it may be possible to understand the whole processes of
amphibian retinal regeneration under culture conditions. The extrinsic factors
that surround RPE and the cellular and molecular mechanisms involved in
transdifferentiation and histogenesis of regenerating retina are of particular
interest. At the same time the method enables us to make an artificially
reconstructed retina, although we have not yet determined whether the retinal
ganglion cells extend axons and make a proper connection with the target
region of the brain.
In vitro analysis of amphibian retinal regeneration
91
Conclusion and perspectives: A hypothetical mechanism
underlying RPE transdifferentiation
Obviously tissue interaction plays an important role in RPE
transdifferentiation as shown by the culture studies and also in vivo observations.
The choroid makes a direct contact with RPE and appears to play a crucial role
in the initial step of transdifferentiation. The choroid will be the source of FGF2,
a trigger signal for transdifferentiation. It needs to be determined in the further
studies which cell type is responsible for the synthesis of FGF2 and by what
mechanism the signal is transduced to the RPE cells. Apparently, RPE cells
never initiate any morphological and cell dynamic changes unless the retina is
surgically removed. The retina-RPE interaction might be necessary for RPE cells
to maintain the RPE-specific phenotypes by continuous expression of certain
transcription factors like Mitf. It is speculated that retinectomy in the eye or
isolation of RPE tissues for culture will downregulate some RPE specific genes.
At the same time, FGF2 in the choroid is transmitted to RPE cells. This, in turn,
upregulates several transcription factors, including Pax6 that are necessary for
retinal regeneration. FGF2 has been shown previously to upregulate genes for
cell growth and neurogenesis (Azuma et al., 2005; Spence et al., 2007). The most
important question is what is the signal by which the retina exerts its effect on
RPE. This question will be addressed using the culture system.
Removal of the retina may induce alterations in cell-cell communication of
RPE cells. Only few studies have been done on the morphological changes in
RPE cells right after retinectomy, such as junctional complex and cell adhesion
molecules. Following retinectomy in X. laevis, RPE cells detach from the
epithelium and initiate migration. This apparently deprives RPE cells of direct
cell communication. In vitro studies show that RPE cells do not proliferate
when newt RPE is isolated and cultured as an epithelial sheet, while they begin
proliferation when cultured on the choroid. RPE cells transdifferentiate to
neuronal cells only after they migrate out from the periphery of the graft as
single cells. All these observations clearly indicate that cell communication
form of RPE cells regulate their dynamic state.
Although RPE cells behave seemingly differently at the initial stage of
regeneration between urodele and anuran amphibians, many common features
are noticed in retinal regeneration of the two animal species and culture studies
indicate that there are no fundamental differences in the cell dynamics
particularly at the initial step of transdifferentiation. The seemingly different
cell behavior between the newt and Xenopus RPE cells observed in vivo
regeneration is probably not due to intrinsic cell properties but reflects some
differences in the extrinsic cues such as ECM components. It is becoming
increasingly important to compare retinal regeneration among a variety of
animal models. The culture technology will provide an excellent approach to
92
Masasuke Araki
address the important question of what mechanisms are involved in different
vertebrates; in certain animal species complete retina regeneration occurs,
while in other species like mammals it never happens in vivo. In future studies
we will be able to answer this question, why does it happen?
Acknowledgments
The author is most grateful to all of his colleagues in Developmental
Neurobiology Laboratory at Nara Women’s University, who made a great deal
of contributions in performing our regeneration study, particularly IkegamiUeda, Mituda, Yoshii, and Kuriyama are sincerely acknowledged. The author
also thanks Dr. Edith McGeer (Univ. of British Columbia, Canada) for her
valuable comments on the manuscript. A part of this work was funded by a
Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
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