DEVELOPMENTAL DYNAMICS 209:387–398 (1997)
Basic Fibroblast Growth Factor (FGF-2) Induced
Transdifferentiation of Retinal Pigment Epithelium:
Generation of Retinal Neurons and Glia
D.S. SAKAGUCHI,1,2* L.M. JANICK,1 AND T.A. REH3
of Zoology and Genetics, Iowa State University, Ames, Iowa
2Signal Transduction Training Group, and Neuroscience Program, Iowa State University, Ames, Iowa
3Department of Biological Structure, University of Washington, Seattle, Washington
1Department
ABSTRACT
In the present study we report
that basic fibroblast growth factor (bFGF, FGF-2)
promotes the transdifferentiation of Xenopus laevis larval retinal pigment epithelium (RPE) into
neural retina. Using specific antibodies we have
examined the cellular composition of the regenerated retinal tissue. Our results show that, in
addition to retinal neurons and photoreceptors,
glial cells were also regenerated from the transdifferentiated RPE. These results were specific to
FGF-2, since other factors that were tested, including acidic FGF (aFGF, FGF-1), epidermal growth
factor (EGF), laminin, ECL, and Matrigel, exhibited no activity in inducing retinal regeneration.
These results are the first in amphibians demonstrating the functional role of FGF-2 in inducing
RPE transdifferentiation. Transplantation studies were carried out to investigate retinal regeneration from the RPE in an in vivo environment.
Sheets of RPE implanted into the lens-less eyes of
larval hosts transformed into neurons and glial
cells only when under the influence of host retinal factors. In contrast, no retinal transdifferentiation occurred if the RPE was implanted into
the enucleated orbit. Taken together, these results show that the amphibian RPE is capable of
transdifferentiation into neuronal and glial cellphenotypes and implicate FGF-2 as an important
factor in inducing retinal regeneration in vitro.
Dev. Dyn. 209:387–398, 1997. r 1997 Wiley-Liss, Inc.
Key words: retina; regeneration; Xenopus; Müller
cells; astrocytes
INTRODUCTION
During embryonic development the vertebrate retina
is generated from a single layer of neuroepithelial cells
which invaginates to form the two layered optic cup
(Coulombre and Coulombre, 1965). The outer layer
proliferates, forming the monolayered retinal pigment
epithelium (RPE), while the innermost layer continues
to divide and gives rise to the multilayered sensory
retina. Although sharing a common embryological origin, the presumptive RPE and retinal neuroepithelium
express unique sets of differentiated properties (Coulombre and Coulombre, 1965).
r 1997 WILEY-LISS, INC.
The RPE represents a relatively stable cell population, yet retains the ability to proliferate and in addition, possesses a remarkable growth potential when
exposed to pathological or culture conditions (Okada,
1980). Studies using a number of vertebrate model
systems have demonstrated that RPE cells can undergo
transdifferentiation, the process of phenotypic switching, whereby differentiated cells alter their identity to
become unique cell types (Coulombre and Coulombre,
1965; Reyer, 1977; Okada, 1980). Under the appropriate conditions transdifferentiation of the RPE into
neural retina can occur both in vivo (Stone, 1950;
Tsunematsu and Coulombre, 1981; Yasuda et al., 1981;
Klein et al., 1990; Kaneko and Saito, 1992) and in vitro
(Pittack et al., 1991; Opas and Dziak, 1994; Zhao et al.,
1995). The generation of new neural retina (i.e., regeneration) via transdifferentiation from the RPE occurs in
a number of amphibians and in embryonic chick and rat
(reviewed in Park and Hollenberg, 1993; Reh and
Pittack, 1995).
A number of studies have begun to investigate the
molecular nature of the factors controlling transdifferentiation. Studies by Reh and colleagues in amphibians
have provided evidence supporting the involvement of
factors associated with the extracellular matrix (ECM)
of the retina (Reh and Nagy, 1987; Reh et al., 1987;
Nagy and Reh, 1994). Recently, peptide growth factors
have also been implicated in the process of induction of
retinal regeneration, and a number of growth factors
and their receptors have been identified in ocular
tissues (reviewed in McAvoy and Chamberlain, 1990).
Among them, members of the fibroblast growth factor
(FGF) family have been implicated in a variety of
developmental processes such as mesoderm induction
(Slack et al., 1987) and neuronal differentiation and
survival (Walicke, 1988). The expression of the ligands
and receptors of the FGF family of growth factors
within the visual system is consistent with the idea
Grant sponsor: National Science Foundation; Grant number: IBN9311198; Grant sponsor: Iowa State University Biotechnology Council;
Grant sponsor: Iowa State University (SPIRG); Grant sponsor: The
Carver Trust.
*Correspondence to: D.S. Sakaguchi, Department of Zoology and
Genetics, 339 Science II, Iowa State University, Ames, IA 50011.
E-mail: dssakagu@iastate.edu
Received 10 March 1997; Accepted 21 April 1997
388
SAKAGUCHI ET AL.
that FGFs play an important role in retinal development, and thus, may play an equally important role
during retinal regeneration (Gao and Hollyfield, 1992;
Bugra et al., 1993; Ishigooka et al., 1993; McFarlane et
al., 1995). Indeed, FGFs have been shown to promote
retinal regeneration in embryonic chick and rat RPE in
vitro (Pittack et al., 1991; Guillemot and Cepko, 1992;
Opas and Dziak, 1994; Zhao et al., 1995; Pittack et al.,
1997) and from embryonic chick RPE in vivo (Park and
Hollenberg, 1989, 1991). These studies have clearly
demonstrated an important role of acidic or basic FGF
(FGF-1 or -2) in inducing the RPE to regenerate neural
retina, including neuroepithelial cells and a variety of
neuronal phenotypes such as ganglion cells, amacrine
cells, and photoreceptors (Pittack et al., 1991; Guillemot and Cepko, 1992; Opas and Dziak, 1994; Zhao et
al., 1995). No other factors that have been tested,
including EGF, NGF, TGF-b, or activin have any demonstrated activity in inducing neural retinal regeneration
from the RPE (Pittack et al., 1991; Guillemot and
Cepko, 1992; Opas and Dziak, 1994).
To examine whether FGF-2 also serves to promote
the transdifferentiation of the amphibian RPE into
neural retina, we cultured the RPE from Xenopus laevis
tadpoles in the presence of FGF-2. A panel of cell-type
specific antibodies was used to begin characterizing the
underlying mechanisms involved in the transdifferentiation phenomenon. We have found that the RPE can
give rise to new retina including neurons and photoreceptors, and demonstrate for the first time, that FGF-2
stimulation also can lead to the production of retinal
glial cells. The FGF-2 induced transdifferentiation occurred only when the sheets of RPE were maintained in
suspension, whereas, adhesion to the substrate prevented the regenerative response. In addition, sheets of
RPE were implanted into the lens-less eyecups of larval
Xenopus to determine if transdifferentiation under
these conditions would also lead to the regeneration of
neurons and glial cells. Our results indicate that the
transplanted RPE was capable of transdifferentiation
into neurons and glia. In contrast, no transdifferentiation was observed when the RPE was implanted into
the enucleated orbit, indicating that retinal regeneration occurred only in the presence of ‘‘retinal-derived
factors.’’ Clues into the regulation of neuronal and glial
cell regeneration may lead to a better understanding of
cellular determination, and has important implications
for the development and regeneration of the nervous
system.
RESULTS
Retinal Pigment Epithelial Explant Cultures
We have examined the ability of Xenopus RPE explants to undergo transdifferentiation to neural retina
when cultured in the presence of FGF-2. An immunohistochemical analysis was performed in order to characterize the morphological transformation that occurred
within the FGF-2-treated RPE explants. Explants were
fixed, sectioned, and a panel of cell-type specific antibodies used to identify and characterize the cellular composition of the RPE regenerates (see Table 1). Retinal
pigment epithelial cells were identified with XAR-1, a
monoclonal antibody which recognizes a 64 kD protein
found specifically in the RPE cells of both pigmented
and albino amphibian eyes (Sakaguchi, 1990). Monoclonal antibodies XAN-1 and XAN-3 were used to identify
neurons and neuroepithelial cells (Sakaguchi et al.,
1989), while XAP-1 and XAP-2 were used to specifically
identify photoreceptors (Harris and Messersmith, 1992;
Leonard and Sakaguchi, 1995). An anti-glial fibrillary
acidic protein (anti-GFAP) antibody and 14H7, an
anti-vimentin (anti-VIM) antibody, were used to identify retinal glial cells (Müller cells and astrocytes;
Sakaguchi et al., 1989; Sakaguchi and Henderson,
1993).
Explanted sheets of RPE, when cultured on a shaker
platform (Nutator), remain suspended in the media and
usually failed to attach to the bottom of the culture
plate. When FGF-2 (20 ng/ml) was added to these
cultures every third day, dramatic morphological
changes were observed. Within 7 days in vitro (DIV),
some of the FGF-2-treated RPE explants began losing
pigmentation, and nonpigmented cells within the explants were observed extruding from the RPE. After 14
days of exposure to FGF-2, some of the RPE explants
exhibited relatively large regions of unpigmented cells
which were adjacent to, or intermixed with pigmented
cells (Fig. 1). Typically, RPE explants cultured for as
long as 30 DIV still retained a significant number of
pigmented cells. Under control conditions (i.e., without
the addition of FGF-2) the sheets of RPE formed
floating clumps or aggregates that retained their pigmentation. No obvious morphological changes were
observed in any of these RPE explants (Fig. 2). The
morphological transformation from the aggregated
sheets of RPE, to explants displaying both pigmented
and nonpigmented regions of cells, was observed only in
cultures that were treated with FGF-2. The ability of
FGF-2 to induce transdifferentiation was observed to
occur in a dose-dependent fashion (Table 2). Concentrations of FGF-2 higher than 20 ng/ml (up to 100 ng/ml)
failed to produce an increase in the percentage of
transdifferentiating RPE explants (data not shown).
The transformation was specific to FGF-2-treated pigment epithelial sheets, since those treated with equal
or higher concentrations of FGF-1, EGF, laminin, ECL,
or Matrigel were similar to the control, untreated RPE
explants (0% exhibiting transdifferentiation; see Table
2). Furthermore, FGF-2-treated explants which attached to the culture substrate, also failed to exhibit
any dramatic morphological changes.
Figures 1 and 2 show sections of FGF-2-treated (Fig.
1A,B) and control (Fig. 2A,B) RPE explants labeled
with XAR-1, the RPE-specific monoclonal antibody.
Nonpigmented areas within RPE explants incubated in
FGF-2 were clearly visible in many cases (Fig. 1A,C). As
the RPE explants underwent transdifferentiation, they
389
FGF-2 INDUCED TRANSDIFFERENTIATION OF RPE
TABLE 1. Antibodies Used for Immunohistochemistry*
Antibody
XAR-1
Specificity
in the
retina
RPE
XAN-1
XAN-3
XAP-1
Dilution
1:20
Source
D.S. Sakaguchi
Antibody
host
Mouse
Neurons
Neurons
Photoreceptors
1:20
1:20
1:20
D.S. Sakaguchi1
D.S. Sakaguchi1
D.S. Sakaguchi1
Mouse
Mouse
Mouse
XAP-2
Photoreceptors
1:20
D.S. Sakaguchi1
Mouse
Anti-VIM
(14H7)
Glial cells
1:20
Devel. Studies
Hybridoma Bank
Mouse
Anti-GFAP
Glial cells
1:250
ICN Immunobiology
Mouse
References
Sakaguchi, 1990
Sakaguchi and
Henderson, 1993
Sakaguchi et al., 1989
Sakaguchi et al., 1989
Harris and Messersmith, 1991;
Leonard and
Sakaguchi, 1995
Harris and Messersmith, 1991;
Leonard and Sakaguchi, 1995
Dent et al., 1989
Sakaguchi and Radke,
1996
Sakaguchi and
Henderson, 1993
Sakaguchi and Radke,
1996
*XAR-1, Xenopus Anti-Retinal pigment epithelium antibody -1; XAN-1 and -3, Xenopus AntiNeuronal antibodies -1 and -3; XAP-1 and -2, Xenopus Anti-Photoreceptor antibodies -1 and -2;
Anti-VIM, anti-vimentin antibody; Anti-GFAP, anti-glial fibrillary acidic protein antibody.
1The XAN-1 and -3, and the XAP-1 and -2 antibodies have been donated to the Developmental
Studies Hybridoma Bank.
began to lose their pigmentation, as well as XAR-1
like-immunoreactivity (IR). In contrast, the untreated
or non-FGF-2-treated explants retained their pigmentation, as well as XAR-1-IR (Fig. 2B) even up to 30 days in
culture. The nonpigmented cells in the FGF-2-treated
RPE explants were immunoreactive with the XAN-1
(Fig. 1D) and XAN-3 (data not shown) antibodies that
label neural cells. In contrast, control and non-FGF-2treated RPE explants exhibited no detectable IR with
these antibodies, and thus confirmed the absence of
neurons and neuroepithelial cells (Fig. 2D). Figure 3
shows sections of RPE explants treated with FGF-2 for
21 DIV and labeled with the XAN-1 (Fig. 3B) or the
XAP-1 antibody (Fig. 3D and F), an antibody that
specifically labels photoreceptors in the Xenopus retina.
XAN-1-IR was observed in 73% of FGF-2-treated (20
ng/ml) RPE explants (Table 2). The XAN-1-IR cells
often resembled a columnar neuroepithelium (Fig. 3B),
similar to the neural retina at the rim of the eye by the
mitotically active ciliary margin. XAP-1-IR cells were
observed in 53% of the FGF-2 (20 ng/ml) stimulated
RPE regenerates (Table 2). In a few preparations
XAP-1-IR was observed deep within the transdifferentiating RPE explant, in small discrete clusters of cells
(data not shown). However, the XAP-1-IR more often
was expressed towards the outer layers at the periphery of the regenerating RPE explants, as illustrated in
Figures 3D and F. The XAP-1-IR cells, although lacking
pronounced outer segments, in some cases exhibited
elongated morphologies resembling newly developing
photoreceptors (Fig. 3E, inset). In addition, XAP-1-IR
was also observed in processes within the explant
(arrows in Fig. 3F).
Although previous studies using embryonic chick and
rat RPE have characterized neuronal cell types that
have been produced during transdifferentiation (Pittack et al., 1991; Guillemot and Cepko, 1992; Opas and
Dziak, 1994; Zhao et al., 1995), these studies did not
investigate the possible differentiation of glial cells
following the FGF-2 treatment. Thus, we have carried
out an immunohistochemical analysis using anti-glial
fibrillary acidic protein (GFAP) and anti-vimentin (VIM)
antibodies to determine whether the FGF-2 induced
transdifferentiation also resulted in the differentiation
of retinal glial cells from Xenopus RPE. The anti-GFAP
and anti-VIM antibodies label both Müller glial cells
and astrocytes in the Xenopus retina (Sakaguchi et al.,
1989; Sakaguchi and Henderson, 1993; Sakaguchi and
Radke, 1996). By 21 DIV extensive IR for both the
anti-GFAP and -VIM antibodies was observed in nonpigmented cells of the regenerated RPE. Figure 4
illustrates anti-GFAP- (Fig. 4A,B) and anti-VIM-IR
(Fig. 4C–F) in transdifferentiating RPE explants. In a
number of RPE explants (Fig. 4F) these glial cell
markers labeled radially oriented cells characteristic of
Müller glia of the vertebrate retina (Fig. 5E). These
elongated cells also possessed endfeet which appeared
to form inner and outer limiting membranes characteristic of the normal retina (Fig. 4D,F). It remains to be
determined if astrocytes were also generated under
these conditions, since the anti-GFAP and -VIM antibodies label both Müller glia and astrocytes in the Xenopus
390
SAKAGUCHI ET AL.
Fig. 1. Photomicrographs of tissue sections from RPE explants treated for 14 DIV with FGF-2 (20 ng/ml).
A,C: Differential interference contrast (DIC) images of the sectioned RPE explants. B,D: The corresponding
fluorescence images. A,B: XAR-1 immunoreactivity of the RPE cells. The asterisk indicates a region of newly
generated, nonpigmented cells within the RPE explant. C,D: XAN-1 labeling of neuroepithelial cells and
neurons throughout the regenerating RPE explant. Abbreviation: RPE, Retinal pigment epithelium. Scale
bars 5 100 µm.
retina. In contrast, immunoreactivity for these glial cell
markers was never observed in the control or non-FGF2-treated RPE explants (Fig. 2, Table 2). These results
are the first describing the regeneration and differentiation of retinal glia following FGF-2 induced transdifferentiation in the RPE.
Transdifferentiation of Transplanted Retinal
Pigment Epithelium
A number of investigators have examined the transdifferentiation of RPE following transplantation into
the eyecup of host amphibians (Rana temporaria: Lopashov and Sologub, 1972; Sologub, 1977; Lopashov, 1983;
Xenopus laevis: Sologub, 1977; Cioni et al., 1986; Bosco,
1988). Based on histological analysis, these studies
revealed that, under the appropriate transplantation
conditions, the RPE was capable of transdifferentiating
into neural retina. However, no detailed molecular
analyses were carried out to specifically identify the
regenerated retinal cell types. To determine if neurons
and/or glial cells are specifically regenerated under
these conditions we have transplanted sheets of RPE
into the lens-less eyes of Xenopus larva, and investigated the ability of retinal-derived factors to stimulate
transdifferentiation of the RPE. Only cases in which
the transplanted RPE could be identified were examined in this analysis. Sheets of RPE implanted into the
lens-less eye of tadpoles, transdifferentiated and produced non-pigmented retinal tissues that were immunoreactive with the XAN-1 and anti-VIM antibodies,
indicating that neural and glial differentiation had
occurred (Fig. 5). However, no obvious laminar organization was observed within the regenerated retinal
tissues under these conditions. Fifteen RPE transplants were examined with the XAN-1 antibody and
five of the implants (33%) displayed significant XAN1-IR (Fig. 5A–C). Twenty RPE implants were examined
with the anti-vimentin antibody and 11 (55%) of these
implants displayed anti-VIM-IR (Fig. 5D–G). Thus,
46% of the RPE transplants displayed some transdifferentiation based on immunoreactivity for the neural or
glial cell antibodies. In contrast, when implanted into
the enucleated orbit, the RPE failed to undergo transdifferentiation. The implanted RPE retained its pigmentation and formed aggregates, and all of the RPE implants examined (10) with the XAN-1 or anti-VIM
antibodies failed to display any immunoreactivity for
the neural or glial cell markers. These results show
that neurons and glial cells were produced from regenerating RPE.
FGF-2 INDUCED TRANSDIFFERENTIATION OF RPE
391
Fig. 2. Photomicrographs of tissue sections from control RPE explants after 21 DIV. A,C,E: DIC
photomicrographs of the sectioned RPE explants. B,D,F: The corresponding fluorescence images. A,B: XAR-1
labeling of the RPE cells of the explant. C,D: The control RPE explants were not labeled with the XAN-1
antibody. E,F: The control RPE explants were not labeled with the anti-GFAP antibody. The asterisk in E
indicates a central region within the RPE explant devoid of cells. Scale bars 5 100 µm.
DISCUSSION
In vertebrates the reprogramming of specialized cells
into a new pathway is relatively rare. However, the
vertebrate eye has provided a unique system in which
to approach the study of the programming of differentiation events. In the present study we have evaluated the
ability of FGF-2 to stimulate the transdifferentiation of
Xenopus RPE in vitro. The results presented here show
that FGF-2 induced the transdifferentiation of the RPE
into retinal neuroepithelium which subsequently generated neurons, as well as glia. These results are the
first in the amphibian retina demonstrating a role
of FGF-2 in inducing retinal regeneration from the
RPE. In addition, these results confirm those obtained
by others using embryonic chick and rat RPE, and
support an important role of FGF-2 in regulating
cell phenotype during development and regeneration of
the neural retina. Furthermore, the present results
extend these earlier studies by demonstrating that
regeneration of retinal tissue from the RPE not only
leads to the differentiation of neurons, but also of glial
cells.
392
SAKAGUCHI ET AL.
TABLE 2. Transdifferentiation of RPE Explants*
Neurons and
neuroepithelial
PhotoGlial
Reagent
cells1
receptors2 cells3 Totals
Culture medium
0/10
0/5
0/5
0/20
(0%)
(0)
(0)
(0)
BSA Control
0/5
0/5
0/5
0/15
(0)
(0)
(0)
(0)
FGF-2 (bFGF)
0/5
0/5
0/5
0/15
[1 ng/ml]
(0)
(0)
(0)
(0)
FGF-2
2/5
1/5
2/5
5/15
[5 ng/ml]
(40)
(20)
(40)
(33)
FGF-2
11/15
8/15
13/20 32/50
[20 ng/ml]
(73)
(53)
(65)
(64)
FGF-1 (aFGF)
0/5
0/5
0/5
0/15
[20 ng/ml]
(0)
(0)
(0)
(0)
EGF
0/5
0/5
0/5
0/15
[20 ng/ml]
(0)
(0)
(0)
(0)
Laminin
0/10
0/5
0/5
0/20
[10 µg/ml]
(0)
(0)
(0)
(0)
ECL
0/7
0/7
0/6
0/20
[10 µg/ml]
(0)
(0)
(0)
(0)
Matrigel
0/6
0/5
0/4
0/15
[10 µg/ml]
(0)
(0)
(0)
(0)
*Numbers represent the number of explants and the percentage (%) of RPE explants exhibiting transdifferentiated phenotypes.
1Neuron and neuroepithelial cell phenotypes determined using XAN-1 or XAN-3 antibodies.
2Photoreceptor phenotype determined using XAP-1 or XAP-2
antibodies.
3Glial cell phenotype determined using anti-GFAP or antiVIM (14H7) antibodies.
Regeneration of the retina has been shown to occur in
a variety of vertebrates (reviewed in Hitchcock and
Raymond, 1992; Reh and Pittack, 1995). Two fundamentally different mechanisms appear to mediate these
regenerative events. The RPE of amphibians, and
embryonic chick and rats can undergo retinal regeneration by transdifferentiation, a process that involves a
change in cell fate of the RPE cells into neural precursors. In contrast, in teleost fish an intrinsic source of
neuronal progenitors act as a source of new retinal
neurons following damage, and recent studies in adult
goldfish have revealed that the RPE does not transdifferentiate in this species (Knight and Raymond, 1995).
Although retinal regeneration has been extensively
studied for many years, only recently have investigators begun to examine the cellular and molecular basis
of the regenerative process in the retina. Basic FGF
stimulated transdifferentiation has been observed to
promote retinal regeneration in embryonic chick RPE
both in vitro (Pittack et al., 1991; Opas and Dziak,
1994) and in vivo (Park and Hollenberg, 1989). More
recently, FGF-stimulated transdifferentiation of the
embryonic rat RPE has also been demonstrated (Zhao
et al., 1995). Using a number of cell-type specific
antibodies, these investigators have clearly demonstrated changes in expression of neuronal and retinal
molecules induced by FGF-2 in the transdifferentiating
RPE. Neuroepithelial cells have been identified with
antibodies directed against N-CAM or neuron specific
enolase (Pittack et al., 1991; Opas and Dziak, 1994;
Zhao et al., 1995). While markers for differentiated
neurons has permitted the identification of ganglion
cells (Pittack et al., 1991; Zhao et al., 1995), amacrine
cells (Zhao et al., 1995), and photoreceptors (Zhao et al.,
1995).
In embryonic rat RPE explants, FGF-2 stimulated
neuronal differentiation with a similar temporal and
spatial distribution compared with the normal development of the retina (Zhao et al., 1995). The formation of
ganglion cells and amacrine cells, two of the earliest
produced cell types; and the appearance of rod photoreceptors, the last terminally differentiated retinal neurons, provided evidence that the multilayered tissue
formed following FGF-2 treatment may lead to the
production of a complete retinal epithelium (Zhao et al.,
1995). The in vivo transdifferentiation of the chick RPE
also results in the formation of a highly laminated
retinal structure (Park and Hollenberg, 1989). However, the newly regenerated retina was inverted, with
the photoreceptors differentiating along the inner surface of the retina near the vitreous, and the ganglion
cells differentiating in the outer portion of the eye (Park
and Hollenberg, 1989, 1991). In addition, the ability of
the embryonic chicken and rat RPE to regenerate
neural retina was restricted to early stages of ocular
development, prior to the irreversible commitment of
the RPE to the pigmented cell fate (Stroeva, 1960; Park
and Hollenberg, 1989; Pittack et al., 1991; Zhao et al.,
1995). In some cases, FGF-2 treatment of Xenopus RPE
explants induced retinal tissue that appeared to be
organized in a laminar fashion. The presence of a
laminar organization, with photoreceptors in an outer
layer surrounding neural tissue, or with radially oriented Müller cells spanning the width of the regenerated neuroepithelium, resembled the normal cellular
organization of the in vivo retina.
In this analysis we have found that FGF-2 can
redirect the fate of the RPE into neural retina. However, if the RPE explants adhered and flattened onto
the culture substrate the FGF-2-induced transdifferentiation was prevented. These results support the idea
that cytomechanics of cell-substratum interactions can
influence the extent to which a neural or glial phenotype can be expressed by the transdifferentiating RPE
(Pittack et al., 1991; Reh et al., 1991; Opas and Dziak,
1994). Indeed, several studies have shown that adhesion to the substratum and the spread nature of a cell
can modulate its responsiveness to soluble factors
(Pittack et al., 1991; Reh et al., 1991; Schubert and
Kimura, 1991; Sutton et al., 1991; Opas and Dziak,
1994). Furthermore, studies have revealed that cells
displaying a flattened and spread morphology often do
not enter, or withdraw from the differentiation pathway
(Ingber et al., 1990; Opas and Dziak, 1990). It is also
possible that cytoskeletal changes associated with formation of focal adhesions during the flattening may
influence gene expression involved in transdifferentia-
FGF-2 INDUCED TRANSDIFFERENTIATION OF RPE
393
Fig. 3. Photomicrographs of tissue sections from RPE explants
treated for 21 DIV with FGF-2 (20 ng/ml). A,C,E: DIC photomicrographs of
the sectioned RPE explants. B,D,F: The corresponding fluorescence
images. A,B: XAN-1 labeling of RPE regenerated neural tissue. C–F:
XAP-1 labeling of photoreceptor-like cells regenerated from the RPE. E,F:
Higher magnification images of C and D, respectively. The inset in E is a
higher magnification image from the region marked by the arrow. In the
inset, the asterisks indicate three XAP-1 immunoreactive cells that
morphologically resemble immature photoreceptors. The arrows in F
illustrate XAP-1-IR processes within the regenerated retina. Abbreviation:
RPE, retinal pigment epithelium. Scale bars for A,B,E,F 5 50 µm and for
C,D 5 100 µm.
tion. Thus, it is possible that RPE explants that adhere
and flatten upon the substrate are in some way less
responsive to FGF-2, preventing their transdifferentiation into neural retina.
In this analysis, 64% of the RPE explants exhibited
transdifferentiation following treatment with FGF-2 at
a concentration of 20 ng/ml. It is unlikely that higher
concentrations of FGF-2 would increase the percentage
of transdifferentiating RPE explants, since explants
exposed to concentrations as high as 100 ng/ml still
failed to exhibit a higher percentage of regeneration.
Another possibility is that the RPE cells of those
explants that did not transform were irreversibly committed to the pigment epithelium phenotype. Although,
previous studies in chick and rat embryos revealed a
gradual decline in the ability of the RPE to regenerate
retina, such a dramatic shift appears not to be the case
in amphibians (Lopashov and Sologub, 1972; Sologub,
1977). Although there is a decline in the regenerative
capacity of adult RPE, nevertheless, both larval and
394
SAKAGUCHI ET AL.
Fig. 4. Basic FGF-induced regeneration of retinal glia. Photomicrographs of tissue sections from RPE explants treated for 21 DIV with
FGF-2 (20 ng/ml). A,C,E: DIC photomicrographs of the sectioned RPE
explants. B,D,F: The corresponding fluorescence images. A,B: AntiGFAP labeling of glia cells. C–F: Anti-VIM labeling of glial cells. E,F:
higher magnification images of C and D, respectively. The arrows in F
illustrate anti-VIM-IR within radially oriented cells resembling Müller glial
cells. Abbreviation: RPE, Retinal pigment epithelium. Scale bars for
A–D 5 100 µm and for E and F 5 50 µm.
adult frog RPE are capable of regenerating retina
under the appropriate conditions (Lopashov and Sologub, 1972; Sologub, 1977). Another possibility is that
the transdifferentiation requires a disruption of the
normal cytoarchitectural relationships of the RPE with
the surrounding basal lamina. There are several lines
of evidence from in vitro and in vivo studies implicating
the ECM in regulating retinal regeneration from the
RPE. Retinal pigment epithelial cells from Rana tadpoles will regenerate retinal cells in vitro when grown
on a laminin-coated substrate, while other ECM compo-
nents such as fibronectin and collagen failed to stimulate regeneration (Reh et al., 1987). In addition, intraocular injection of an antibody directed against a
laminin-heparan sulfate proteoglycan (HSPG) complex, that specifically blocked the association of the
RPE with the vitreal basement membrane, inhibited
retinal regeneration in Rana tadpoles (Nagy and Reh,
1994). It is also possible that FGF-2, which normally
interacts with specific HSPGs and therefore would be
localized to the vitreal vascular basement membrane,
was inhibited by the blocking antibody and prevented
FGF-2 INDUCED TRANSDIFFERENTIATION OF RPE
395
Fig. 5. Photomicrographs of tissue sections of Xenopus RPE transplants following transdifferentiation within the lens-less eyes of tadpole
hosts. The transplanted RPE is marked by the asterisk. A,D,F: DIC
photomicrographs of the sectioned RPE transplants. B,C,E,G: The
corresponding fluorescence images. A–C: XAN-1 labeling of transplanted
RPE and host retina. The arrow in B and C indicates XAN-1-IR in the
non-pigmented tissue which transdifferentiated from the transplanted
RPE. D–G: Anti-VIM labeling of transplanted RPE after 28 DIV. F,G:
Higher magnification images of the RPE transplant in D and E, respectively. The arrowheads in G show examples of anti-VIM-labeled Müller
cells within the host retina. The arrows in E and G indicate anti-VIM-IR within
the transdifferentiated RPE. Abbreviations: RPE, Retinal pigment epithelium;
INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell
layer. Scale bars for A,B,D,E 5 10 µm; C 5 30 µm; F,G 5 50 µm.
the appropriate interactions required for retinal regeneration. The regeneration process may be regulated by
a balance between repressive and inducing interactions. Thus, effective transformation would require the
elimination of the repressive action of the cytoarchitectural relationships of the RPE cells with surrounding
cellular and ECM constituents, and the influence of
inducing factors, such as FGF-2.
A number of studies have shown that the RPE of
larval and adult frogs will transform into neural retina
when cultivated under the influence of retinal cells
(Lopashov and Sologub, 1972; Sologub, 1977; Cioni et
396
SAKAGUCHI ET AL.
al., 1986; Bosco, 1988). In these earlier studies characterization of the transdifferentiated retina was carried
out using conventional histological procedures and no
molecular identification of the actual regenerated cellphenotypes was carried out. Our results showing that
sheets of RPE transplanted into the lens-less eyecup
are capable of transdifferentiation into neural retina
and glial cells, similar to our in vitro analysis, lends
support to the idea that some retinal-derived factor,
perhaps FGF-2, may be involved in regulating the
transdifferentiation observed in vivo. In addition, these
results provide evidence that the transdifferentiation of
the RPE results in the regeneration of not only neurons,
but also of glial cells. The use of the antibody markers
was critical for the identification of the neural or glial
nature of the regenerated tissue, since no obvious
morphological differentiation into distinct retinal layers was observed in our studies.
Studies from a number of vertebrate species have
shown that, under the appropriate in vitro and in vivo
conditions, the RPE has a remarkable capacity to
regenerate neural retina. Elucidation of the molecular
mechanisms involved in the transdifferentiation process may further our understanding of the molecular
basis of cell determination.
EXPERIMENTAL PROCEDURES
Animals
Xenopus laevis embryos were produced in the laboratory by human chorionic gonadotropin (United States
Biochemical Corporation, Cleveland, OH) induced
spawnings and reared in 10% Holtfreter’s solution (6
mM NaCl, 0.06 mM KCl, 0.09 mM CaCl2, and 0.02 mM
NaHCO3) until they reached appropriate stages for
experimental analysis. Animals were maintained at
room temperature and staged according to the normal
Xenopus table (Nieuwkoop and Faber, 1956).
Retinal Pigment Epithelial Explant Cultures
Eyes were surgically removed from anesthetized
(1:10,000 MS-222, Sigma, St. Louis, MO) stage 47 to 53,
Xenopus laevis tadpoles. The eyes were rinsed and
incubated in sterile Hank’s buffered salt solution (1.2
mM CaCl2, 5.37 mM KCl, 0.44 mM KH2PO4, 0.81 mM
MgSO4, 0.23 mM Na2HPO4, 137.9 mM NaCl, 9 mM
D-glucose, 0.04 mM Phenol Red in double processed
tissue culture water [Sigma] supplemented with 2%
fungibact and 1% penicillin/streptomycin [Sigma]). The
eyes were then dissected to remove the RPE from the
neural retina using fine watch maker forceps. The
anterior one-quarter to one-third of the RPE, the region
nearest the ciliary margin, was cut away and discarded
to ensure removal of this mitotically active zone. In
many fish and amphibians the eyes continue to grow
throughout the life of the animal by the addition of cells
at the ciliary margin (Beach and Jacobson, 1979),
therefore removal of this proliferating zone eliminates
this possibility. The sheets of RPE were then transferred to a final rinse in 60% L15 culture medium
(Sigma) supplemented with 5% fetal bovine serum in
double processed tissue culture water. The pieces of
RPE were then placed into individual wells in a 24-well
nontissue culture-treated plastic plate (Falcon #8111)
and maintained as stationary cultures or placed onto a
Nutator (Clay Adams Model# 1105) shaking device at
22°C and cultured for up to 35 days. Basic and acidic
FGF (FGF-2 and FGF-1) and EGF were obtained from
U.S. Biochemicals; laminin was obtained from Life
Sciences-Gibco BRL (Gaithersburg, MD), ECL from
Upstate Biotechnology Inc. (Lake Placid, NY), and
Matrigel from Collaborative Research Inc. (Bedford,
MA). To maintain the appropriate concentration, the
reagents were once again added to the wells when the
cultures were fed every 3 days. At the end of the
incubation period, the cultures were fixed in 4% paraformaldehyde in 0.1 M PO4 buffer or Dent’s fixative (20%
dimethyl sulphoxide (DMSO) and 80% methanol; (Dent
et al., 1989) for 2 hr.
Retinal Pigment Epithelial Transplants
Stage 45–50 Xenopus larva were used for the RPE-toeye transplants. The host tadpoles were anesthetized
(1:10,000 MS-222 in 10% Holtfreter’s solution), and
placed into shallow depressions made in the dental-wax
base of a dissecting dish. The hosts were immobilized
and restrained using minuten pins, taking care to
minimize any damage to the host tissues. Sharpened
watch-makers forceps were then used to carefully tear
the overlying epidermis and a small cut was made in
the sclera surrounding the eye in order to remove the
lens. In some cases, a retinectomy was also performed
to remove the neural retina following the lens removal.
For this procedure the neural retina was carefully
excised through the tear at the front of the eye. Only
those animals, in which we were confident that no
additional surgical trauma was produced, were used as
hosts for the RPE implants. Donor RPE was isolated as
described above and implanted into the lens-less or
enucleated orbit of the host animal. These animals
were allowed to recover and grow for an additional
28–30 days and then prepared for histological analysis.
Our analysis was restricted to those specimens in
which the host retina remained intact, and the presence of an obvious RPE implant could be identified.
Immunohistochemistry
Following fixation, the tissues were rinsed in 0.1 M
PO4 buffer, cryoprotected in 30% sucrose in 0.1 M PO4
buffer for 1 to 12 hr, and then embedded in OCT
medium (Miles, Elkhart, IN). The frozen tissues were
then sectioned at 12 µm using a cryostat (American
Optical, Buffalo, NY) and the sections mounted on
Superfrost/Plus glass microscope slides (Fisher Scientific, Fair Lawn, NJ). Tissue sections were rinsed in
phosphate buffered saline (PBS: 140 mM NaCl, 3 mM
KCl, 5 mM Na2HPO4, and 2 mM KH2PO4) and blocked
in 5% goat serum, 0.2% BSA, 0.03% Triton X-100 in
FGF-2 INDUCED TRANSDIFFERENTIATION OF RPE
PBS, and incubated in primary antibody diluted with
blocker for 2 hr at room temperature or overnight at
4°C (for primary antibodies and dilutions used, see
Table 1). They were then rinsed three times with PBS
and incubated with appropriate secondary antibodies
(Fisher Scientific) conjugated to FITC or RITC (diluted
1:200 in blocking solution) for 90 min at room temperature. Sections were again rinsed three times with PBS
and mounted under glass coverslips in Gelmount
(Fisher). Negative controls were run in parallel during
the immunohistological processing by omission of the
primary or secondary antibodies. No antibody staining
was observed in the controls.
Tissue sections were examined with a Nikon Microphot FXA photomicroscope equipped with epifluorescence and photographed using Kodak TMAX 400 black
and white film or Kodak Ektachrome 400 color slide
film or Fujicolor 400 color negative film. For some
figures, negatives and slides were digitally scanned and
the figures prepared using Adobe Photoshop Version 3.0
and Macromedia Freehand Version 5.5 for the Macintosh. Outputs were generated on a Tektronix phaser
continuous tone color printer.
ACKNOWLEDGMENTS
The authors wish to thank T.D. Folsom and M.H.
West Greenlee for critical reading of the manuscript.
The 14H7, anti-VIM monoclonal antibody was obtained
from the Developmental Studies Hybridoma Bank,
maintained by the Department of Pharmacology and
Molecular Sciences, Johns Hopkins University School
of Medicine and the Department of Biology, University
of Iowa, under contract NO1-HD-2-3144 from the
NICHD. This manuscript, designated by Iowa State
University as J-17314 of the Iowa Agriculture and
Home Economics Experiment Station, Ames, IA, project
number 3205, was supported by Hatch Act and State of
Iowa funds.
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