Developmental Brain Research 155 (2005) 49 – 59
www.elsevier.com/locate/devbrainres
Research report
Downregulation of Otx2 in the dedifferentiated RPE cells of regenerating
newt retina
Sanae Sakamia, Osamu Hisatomia, Shunsuke Sakakibaraa,1, Janice Liub,
Thomas A. Rehb, Fumio Tokunagaa,*
b
a
Department of Earth and Space Science, Graduate School of Science, Osaka University, Machikaneyama-chyo 1-1, Toyonaka, Osaka 560-0043, Japan
Departments of Biological Structure, Ophthalmology and Neurological Surgery, Health Science Center, University of Washington, Seattle, WA 98195, USA
Accepted 7 November 2004
Abstract
Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a
transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we
cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of
Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Mqller
glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly
expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented
monolayer (the outer layer) attached to Bruch’s membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and
CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers,
expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate
that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and
specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented
and the de-pigmented layers. This observation implicates the appearance of Mqller glial cells in an early phase of regenerating retina.
D 2004 Elsevier B.V. All rights reserved.
Theme: Development and regeneration
Topic: Regeneration
Keywords: Otx2; Regeneration; Dedifferentiation; Newt; RPE cell
1. Introduction
Although the adult mammalian retina has a potential
source for neural retina regeneration in the pigmented cells
of the ciliary margin, there is no evidence of utilization of
this source in vivo [42]. The Salamandriae (newt) retina has
* Corresponding author. Fax: +81 6 6850 5480.
E-mail address: tokunaga@ess.sci.osaka-u.ac.jp (F. Tokunaga).
1
Present address: Department of Anatomy and Developmental
Neurobiology, Kobe University School of Medicine, 7-5-1, Kusunoki-cho,
Chuo-ku, Kobe 650-0017, Japan.
0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.devbrainres.2004.11.008
been investigated for over a century as a model of neuronal
regeneration in the central nervous system (CNS)
[10,26,27,36,41]. Several studies using several species of
Salamandriae, Europian newt (Triturus), North American
salamander (Notophthalmus viridescens) and Japanese Firebellied newt (Cynops pyrrhogaster) revealed two potential
sources for neural retina regeneration: (1) cells in the region
of ora serrata and the ciliary part of the retina and (2) RPE
cells [13,17,19,41]. After surgical removal of whole neural
retina, remaining RPE cells proliferate and transdifferentiate
into virtually all kinds of retinal cells [29,41]. Morphological studies have demonstrated the essential steps of
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S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
transdifferentiation. The first step in transdifferentiation is
dedifferentiation of the RPE cells: these cells start to
proliferate, lose pigment granules and adopt a spherical
shape [17,19,41]. Dedifferentiated RPE cells have the
ability to reconstruct the entire neural retina; this process
is completed approximately 5 weeks after loss of the whole
neural retina [27,29]. Electrophysiological and immunohistochemical studies have suggested that the temporal order in
the formation of neural cell types is similar to that which
occurs during development [8,38]. Dedifferentiation of the
RPE cell is the first step of regeneration. However, there are
only a few reports about the characterization of this step at
the molecular level [20,41].
The homeobox gene Otx2 is essential for differentiation
of RPE cells and transactivation of the genes involved in
melanosome formation [23,24]. A recent study shows that
Otx2 transactivates the cone-rod homeobox gene Crx,
which is required for terminal differentiation and maintenance of photoreceptor cells [30]. To understand the role
of Otx2 during transdifferentiation of RPE cells, we
compared the expression of Cynops Otx2 with those of
RPE specific proteins—RPE-specific 65-kD protein, RPE65
and cellular retinol-binding protein, CRBP [12,35,37] in the
developing and the regenerating retina. In addition, we
investigated the expression pattern of RPE and Mqller glial
cell specific protein; cellular retinal-binding protein,
CRALBP [4].
We found that Otx2, RPE specific proteins (RPE65 and
CRBP) and CRALBP were uniformly expressed in the
monolayered pigmented cells (outer layer) attached to
Bruch’s membrane and the pigmented neural retina primordial (the inner layers). This observation suggests that the
RPE cells maintain the RPE characteristics during proliferation at the early regeneration stage. When regenerating
retina formed approximately 4 layers, the majority of cells
in the outer layer exited cell cycle and showed pigmentation,
while inner layers lost pigmentation. At this stage, normal
level of Otx2 expression was restricted to the outer layer.
RPE65 and CRBP expression was also restricted to the outer
layer. The differential expressions of Otx2 and RPE specific
markers between the outer and inner layers implicate an
essential role for Otx2 in patterning RPE cells and
presumptive neural retina. The expression of the RPE and
Mqller glial cell specific protein, CRALBP was observed
both in the inner and outer layers, suggesting appearance of
Mqller glial cells in de-pigmented presumptive neural retina
at the early stage of regeneration.
2. Materials and methods
2.1. Animals
Adult newts, C. pyrrhogaster, were obtained from local
agricultural fields. For fertilization, female newts were
injected twice with 100 U of serotropin (Teikokuzouki).
The developmental stages of embryos were determined
according to the criteria proposed by Okada and Ichikawa
[31]. Neural retinas and lenses were removed following the
procedure described by Kaneko and Saito [16]. Briefly, the
dorsal half of the eye was cut along the cornea–sclera
junction. The neural retinas were detached from the pigment
epithelium and removed after application of a gentle stream
of Ringer’s solution with a 27 G needle. Adult newts,
embryos and fertilized eggs were kept in aquaria at
approximately 23 8C, and handled according to the Guidelines for Animal Experimentation of Osaka University.
2.2. Cloning of Cynops Otx2 cDNA
The homeobox region of Cynops Otx2 cDNA was
amplified from an adult retinal cDNA by polymerase chain
reaction (PCR) using degenerate primers, OTX3 (5VGGAATTCGARMGNACNACNTTYAC) for sense priming
and OTXR1 (5V-GGGGATCCTGYTGYTGNCKRCANTT)
for antisense priming (R, A + G; M, A + C; Y, C + T; K, G +
T; and N, A + C + G + T). A newt retinal cDNA library
[40] was screened by high stringency plaque hybridization using the amplified fragments as a probe at 55 8C
with hybridization buffer containing 50% formamide, 5
SSPE, 5 Denhart’s solution, 0.5% SDS and 10%
dextran sulfate. It was then washed at 55 8C with 0.2
SSC containing 0.1% SDS. The 5V end of the Otx2 cDNA
was obtained by rapid amplification of cDNA ends
(RACE-PCR), using the ExpandTM High Fidelity PCR
system (Roche) with gene-specific primers OTX2-R1 (5VGTGCTAGGTGGTGGTGTG) and OTX2-R2 (5VAACTGCAGCCTCCACTCTGCTGTTGC), in combination with C-primer and C-Amp [15]. The nucleotide
sequence of the Cynops Otx2 cDNA has been submitted
to DDBJ/EMBL/GenBank databases with the accession
number AB052937. Molecular weight was predicted from
the primary structure using Compute pI/Mw tool at http://
kr.expasy.org/tools/pi_tool.html.
2.3. Preparation of an antiserum against Cynops Otx2
The cDNA containing the complete coding region of
Cynops Otx2 was inserted between the NcoI and BglII sites
of the pQE-60 plasmid vector and introduced into E. coli
cells (SG13009, Qiagen). Expression and purification of
recombinant Otx2 were carried out according to the
manufacturer’s instructions (The QIAexpressionistTM,
QIAGEN) with the following modifications: (1) the
expression was induced at OD600 = 1.0, then harvested at
30 8C for 1 h; (2) Ni-NTA-bound proteins were eluted with
elution buffer (100 mM Na2HPO4, 10 mM Tris, 8 M Urea,
250 mM imidazole, pH 6.3); (3) the eluted proteins were
dialyzed against elution buffer (pH 7.0) without imidazole,
then 3/4, 2/4 and 1/4-fold dilution of elution buffer without
imidazole (each for more than 6 h), and finally against PBS
(13.6 mM NaCl, 0.2 mM KCl, 0.8 mM Na2HPO4, 0.1 mM
S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
KH2PO4). The purified proteins were used to immunize
mice to produce the anti-Otx2 antiserum.
2.4. Western blotting
Homogenized neural retinas of adult newts (containing
approximately 100 Ag of protein) were separated by 12%
SDS polyacrylamide gel electrophoresis and transferred to
polyvinylidene fluoride (PVDF) membranes (Bio-Rad) at
1.7 mA/cm2 for 1 h in transfer buffer (25 mM Tris, 192 mM
glycine, and 20% methanol). The blotted membranes were
immersed in blocking solution (5% goat serum in TBST; 50
mM Tris, 150 mM NaCl and 0.1% Tween-20, pH 7.5) and
blocked overnight. The membranes were incubated with
anti-Otx2 diluted 1:5000 in reaction buffer (0.1% Tween-20
and 5% goat serum in PBS) for 1 h at 23 8C, and overnight
at 4 8C for further reaction, and then washed with TBST.
Next, the membranes were incubated with biotinylated antimouse IgG (VECTOR) diluted 1:2000 in blocking solution
for 1 h, and then washed with TBST. Finally, the
membranes were incubated with HRP-conjugated streptavidin (VECTOR) diluted 1:1000 in TBST for 1 h, and then
washed with TBST. Antibody binding was visualized with
diaminobenzidine. For a negative control, the antiserum was
pre-incubated with the recombinant Cynops Otx2 proteins,
and processed as described above.
2.5. Tissue preparation and histochemistry
Cynops eyecups with intact and regenerating retinas were
fixed with 4% paraformaldehyde in SPB (5% sucrose in
PBS) for 5 h at 4 8C. After removing the jelly coat, whole
embryos were fixed with 4% paraformaldehyde in SPB for 1
h on ice and further fixed for 5 h at 4 8C on a gentle shaker.
Fixed tissues were then frozen as described by [2] with the
following modifications: (1) tissues were handled at 4 8C in
all steps; (2) PBS solution was used instead of PB solution.
Cryosections of 5–10 Am thicknesses were prepared for
immunohistochemistry. In situ hybridization was carried out
as described in [22], using Cynops Otx2 cRNA containing
the whole coding region as a probe. Immunohistochemistry
was carried out as described previously [11], with the
following modifications: the sections were treated with
acetone for 5 min, 100% ethanol for 5 min, and then
rehydrated with a series of ethanol concentrations (95%–
50%), and washed with PBS. For antigen retrieval and
double staining with anti-PCNA, slides were put in 0.8 M
urea solution, heated in a microwave oven for 100 s and
then cooled to room temperature. The slides were then
washed 5 times with PBS. For a negative control, the
antiserum was pre-incubated with the recombinant Cynops
Otx2 protein. Cell nuclei were labeled with 50 Ag/ml
Hoechst 33258 (Molecular Probes) for 1 h. Primary
antibodies were diluted in PBST (0.3% Triton-X in PBS)
containing 5% goat serum and 0.01% NaN3 at the following
concentrations: mouse anti-Cynops Otx2 at 1:200, rabbit
51
anti-PCNA IgG (Oncogene) at 1:200, rabbit anti-RPE65
(gift from Dr. T.M. Redmonad, NIH, Bethesda, Maryland)
at 1:200, mouse anti-CRBP and rabbit anti-CRALBP (gift
from Dr. J.C. Saari, University of Washington, Seattle,
Wshington) at 1:400. Secondary antibodies: biotinylated
anti-mouse IgG (VECTOR) and biotinylated anti-rabbit IgG
(VECTOR) were diluted in PBST containing 5% goat serum
and 0.01% NaN3 at 1:200 as recommended by manufacturer’s protocol. Fluorophores-conjugated antibody and streptavidin were diluted in PBST containing 0.01% NaN3 at the
following concentrations as recommended by the manufacturer’s protocol: goat Alexa Fluor488-conjugated anti-rabbit
IgG (Molecular Probes) at 1:500, Cy3-conjugated streptavidin (Jackson) at 1:1000. Fluorescence was detected using
a confocal microscope (LSM 510 Pa and LSM 510 META,
Carl Zeiss).
2.6. RNA isolation and RT-PCR assay
The intact and regenerating (18, 24, 28 or 34 days after
the surgical removal of intact neural retinas) eye cups, as
well as livers, were prepared from adult newts and
immediately dissolved in ISOGEN (Nippon Gene). RNA
preparation was carried out according to the manufacturer’s
instructions (Nippon Gene). Total RNA was treated with
DNase I (Invitrogen) and then reverse-transcribed with MMuLV reverse transcriptase (Roche) using oligo(dT) primers
(Amersham). PCR was performed in 20 Al reaction mixtures
containing 0.4 pmol of b-actin or Otx2 primers, 1 mM
MgCl2 and 0.5 units of Ex Taq (Takara). All PCR
amplifications were performed at 94 8C (1 min), 60 8C
(2 min) and 72 8C (3 min) for 45 cycles. A cDNA fragment
encoding Cynops b-actin was amplified from a retinal
cDNA pool using degenerate primers h-actin f 5VATGGTNGGNATGGGNCAR (sense) and h-actin r 5VYTCNGCNGTNGTNGTRAA (antisense). The nucleotide
sequences of gene-specific primers for newt h-actin were 5VGGAATTCATTGAGCACGGCATTGTGACCAAC (sense)
and 5V-CGGGATCCGCATACCCTTCGTAGATTGGCACA
(antisense), and those for Cynops Otx2 were 5V-AACTGCAGGAAGCAACCACCTTACACCGTGAA (sense) and
5V-GGAATTCTGTGCTAGGTGGTGGTGTGAACTG
(antisense).
3. Results
3.1. Isolation of Cynops Otx2 cDNA and analysis of its
expression during regeneration by RT-PCR
We isolated Otx2 cDNA from C. pyrrhogaster retina
cDNA to characterize its expression. The deduced amino
acid sequence of Cynops Otx2 predicted a molecular mass
of approximately 31 kDa and showed a high degree of
identity to other known Otx2s (87–90%). Fewer amino acid
identities were observed with the OTX-related sequences,
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S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
Table 1
Identity of Cynops Otx2 and Otx-related sequences of other vertebrates
Species
Otx2
Otx5b
Otx5
Otx1
Otx3
CRX
Xenopus
Zebrafish
Mouse
Human
Xenopus
Xenopus
Zebrafish
Xenopus
Zebrafish
Mouse
Human
Zebrafish
Mouse
Human
Identity %
OTX tail
Full-length
Homedomain
WSP
motif
1
2
87
89
90
87
77
76
73
62
61
57
58
59
50
52
98
100
100
100
96
96
98
97
97
98
98
95
86
86
92
100
100
100
92
92
92
77
64
77
85
71
76
84
84
92
84
84
84
84
77
80
79
50
44
79
53
53
100
100
100
100
85
85
92
77
77
77
77
69
77
77
All Otx-related sequences of other vertebrates were obtained from DDBJ/
EMBL/GenBank databases: Xenopus Otx2;Q91813, zebrafish otx2;
NP_571326.1, mouse Otx2; NP_659090.1, human OTX2; NP_758840.1,
Xenopus Otx5b; CAB63872.1, Xenopus Otx5; BAA 86260.1, zebrafish
otx5; NP_851848.1, Xenopus Otx1; AAK31735.1, zebrafish otx1;
BAA05158.1, mouse Otx1; NP_035153.1, human OTX1; NP_055377.1,
zebrafish otx3; NP_571290.1, human CRX; NP_000545.1, and mouse
CRX; NP_031796.
Otx5b, Otx5, Otx1, OTX3 and CRX (50–77%) (Table 1). To
characterize the temporal expression of Otx2 in the
regenerating eye, we performed RT-PCR at the several
stages during retinal regeneration (18 days, 24 days, 28 days
and 34 days after surgical removal of neural retinas) and in
the intact eyes. The relation between anatomically assigned
regeneration stages and days after the surgical removal of
the neural retina is shown in Fig. 1 [13]. For all samples
from the regenerating and intact eyes, specific amplification
of the Otx2 cDNA fragment was observed, but it was not
detected in the negative control using liver cDNA as
template (Fig. 2, upper panel). Without reverse-transcrip-
tion, the regenerating eyes did not show any signal (data not
shown), eliminating the possibility of genomic DNA
contamination. As a positive control, all samples showed
b-actin-specific amplification (Fig. 2, lower panel). These
results indicate that Otx2 expression is maintained during
retina regeneration.
3.2. Otx2 expression in both the mature retina and the
RPE in newt
To localize the Otx2 expression in the regenerating
retina, we prepared anti-Otx2 polyclonal antibodies against
the full-length recombinant Otx2 protein. The anti-Otx2
antiserum was immunoreactive to a single band with
apparent molecular mass of 36 kDa in retinal homogenate
(Fig. 3-1). This molecular weight was close to that observed
for mouse Otx2 [1]. This reactivity was blocked by preincubation of anti-Otx2 with the full-length recombinant
Otx2 protein (Fig. 3-2). Thus, this anti-Otx2 antiserum is
monospecific to the Cynops Otx2.
Previous studies of other vertebrates have localized Otx2
to both the neural retina and the RPE [1,3,30,43]. We find a
similar pattern of expression in the newt. In the neural
retina, Otx2 is expressed in both the INL and the ONL
(Figs. 4A–C); the cells in the INL are in the outer half of the
layer, possibly bipolar cells (arrow, Fig. 4A). In the ONL,
the cells expressing Otx2 are presumably photoreceptors
(arrowhead, Fig. 4A). The Otx2 immunoreactivity is
confirmed by in situ hybridization, which also shows the
strongest signal in the INL (arrow, Fig. 4C), and a lower
level of signal in the ONL (arrowhead, Fig. 4C). The
labeling in the RPE was obscured by the pigment granules.
Immunofluorescence microscopy showed Otx2 expression
in the RPE cells (Figs. 4D and E). Arrows point to Otx2immunoreactive RPE cell nuclei co-stained with Hoechst
33258 (blue) (arrows, Fig. 4D). The Otx2 immunoreactivity
in RPE cells can be seen by co-labeling with antibody
Fig. 1. Schematic diagram of the relation between anatomically assigned regeneration stages and time course after surgical removal of neural retina.
(1) Approximately 2 to 3 cell layered regenerating retina, consisting of various degrees of pigmented cells. (2) Approximately 4 cell layered regenerating retina,
consisting of two distinct layers; the pigmented monolayer (the outer layer) and the presumptive neural retina (the inner layers). (3) The inner layer (the
presumptive neural retina), consisting of approximately 7 cell layers. Synaptic layers not present at this stage. (4) Regeneration complete morphologically.
Corresponding figures in this study are listed under the line. BM, Bruch’s membrane; OL, outer layer; IL, inner layer; RPE, retinal pigment epithelium; ONL,
outer nuclear layer; INL; inner nuclear layer; GCL, ganglion cell layer. (Schemes drawn from Hasegawa, 1958 [13] and our observations.)
S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
Fig. 2. Expression of Otx2 in regenerating eyes at several stages. RT-PCR
was carried out using reverse-transcribed total RNA from liver and several
stages of regenerating eyes, including RPE (lane 1: 18 days; lane 2: 24
days; lane 3: 28 days; lane 4: 34 days after surgical removal of neural retina;
lane 5: adult intact eye; lane 6: liver).
against RPE cell specific proteins, RPE65 (arrows, Fig. 4E).
However, Otx2 is not expressed in the proliferating nuclear
antigen (PCNA) positive cells at the CMZ (green; arrow,
Fig. 4F), but is expressed by the RPE cells underlying the
CMZ (arrowhead at upper side, indicated by the square and
the inset marked with an asterisk, Fig. 4F) and pigmented
cells underlying the paras ciliaris (arrowhead at lower side,
indicated by the square and the inset marked with two
asterisks, Fig. 4F). Around the CMZ, a majority of cells
showed weak PCNA immunoreactivity. This suggests that
Otx2 is expressed immediately after the cells have withdrawn from the cell cycle.
3.3. Downregulation of Otx2 in the presumptive neural
retina during development
We next explored Otx2 expression in the developing
retina of newt to compare it with other vertebrates. The
spatiotemporal expression pattern of Otx2 is similar in
Cynops and other vertebrates [3,30,43]. The strong immunoreactivity for anti-Otx2 was observed at the anterior
portion of the neural tube at St.26 (Fig. 5A) and then
restricted to the presumptive RPE of the optic cup at St.31
(Fig. 5C). In contrast to the presumptive RPE, weak and
uniform immunoreactivity was observed in the presumptive
neural retina of the optic cup (Fig. 5C). Later, Otx2
immunoreactivity was observed in the subpopulation of
cells in developing neural retina, and some of those cells
showed co-localization of Otx2 and PCNA at St.39 (Figs. 5E
and G). Following development, Otx2 immunoreactivity was
restricted to a similar part of the intact retina. But a
subpopulation of PCNA positive cells at the anterior portion
of retina, the presumptive CMZ co-expresses Otx2 at St.44
(Fig. 5I). Such co-localization was also observed in the CMZ
at St.54, which had morphologically mature retina (Fig. 5K).
53
expressing Otx2 and PCNA were uniformly observed in 2
cell layered regenerating retina, consisting of various
degrees of pigmented cells attached to, or detached from,
Bruch’s membrane (Figs. 6A and B). Approximately half
the population of cells showed Otx2 immunoreactivity
(Fig. 6C). A similar expression pattern was maintained
until regenerating retina became 3 cell layered (Figs. 6E–H).
Here, we tentatively call the monolayer attached to Bruch’s
membrane the outer layer and other layers that will later
form neural retina the inner layers. 19 days after surgical
removal of the neural retina, the entire inner layer loses
pigment granules and the outer layer becomes a morphologically distinct pigmented monolayer (Fig. 6I). In the
inner layer, immunoreactivity of Otx2 became weaker
(Fig. 6K) and most of the cells maintained PCNA
immunoreactivity (Fig. 6L). Only a few cells showed a
substantial immunoreactivity for Otx2 (Fig. 6K). In the
outer layer, the cells maintained Otx2 expression, while a
majority of them were negative for PCNA (Fig. 6J). 26 days
after surgical removal of the neural retina, the inner layers
were composed of approximately 7 cell layers (Fig. 6M). In
the inner layers, a subpopulation of cells was immunoreactive for both Otx2 and PCNA (Fig. 6N), similar to the
pattern at developmental stage 39 (Fig. 5G). This is
consistent with the observation that regeneration and
development show similarities in many respects at later
stages [8,38]. 30 days after surgical removal of the neural
retina, regenerated retina showed stratified organization
(Fig. 6O). The expression patterns of Otx2 and PCNA were
similar at this stage of regeneration and in the intact retina;
however there were some exceptions. In the regenerating
retina, cells immunoreactive for both PCNA and Otx2
3.4. Downregulation of Otx2 in the inner layers of the
regenerating retina
Next, we characterized Otx2 expression in the dedifferentiating RPE cell at the early phase of regeneration. 14
days after surgical removal of the neural retina, cells co-
Fig. 3. The anti-Otx2 antiserum specificity in Western blotting. Lane 1: preincubation of anti-Otx2 with PBS, lane 2: pre-incubation of anti-Otx2 with
full-length recombinant Otx2 proteins. Both lanes contained homogenates
of neural retinas of adult newts.
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S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
Fig. 4. Expression of Otx2 in the intact retina. (A, D, E and F). Otx2 (red) immunofluorescence in the intact retina. (A) Otx2 immunoreactivity observed in the
RPE cells, the ONL (arrowhead) and the outer half of the INL (arrow). (B) Bright-field image of panel A. (C) In situ hybridization was performed using 35Slabeled Otx2 cRNA. The hybridization signal was shown by white dots in a dark-field image. Strong hybridization signal was observed in the INL (arrow) and
weak hybridization signal forming a line was observed at the ONL (arrowhead). High background in RPE cells prevented the detection of Otx2 mRNA in the
RPE cells. (D) High magnification Otx2 image indicated by the square in panel A was merged with nuclei staining (Hoechst 33258, in blue) and bright-field
image. Otx2 (in red) localized to nuclei was observed in pink color (blue plus red). RPE cell nuclei were indicated by arrows. (E) Same field image of panel D,
RPE65 immunofluorescence (in green) surrounded the Otx2 immunoreactive RPE cell nuclei (in red). Each arrow in panels D and E indicate same RPE cell
nucleus. (F) Immunofluorescent localization of Otx2 (in red) and PCNA (in green) in the CMZ of adult retina. Co-localization of Otx2 and PCNA was observed
in the RPE cell nuclei underlying the CMZ (upper arrowhead, indicated by the square marked with an asterisk) and the pigmented cells underlying the paras
ciliaris (lower arrowhead, indicated by the square marked with two asterisks), but not observed in CMZ (arrow). Upper inset marked with an asterisk shows the
same field indicated by the square marked with an asterisk. PCNA immunofluorescent image merged with bright-field image showed PCNA
immunofluorescent in RPE cell nuclei underlying the CMZ (in green). Lower inset marked with two asterisks shows the same field indicated by the
square marked with two asterisks. Otx2 immunofluorescent image merged with bright-field image showed Otx2 immunofluorescent in the pigmented cells
underlying the paras ciliaris (in red). (G) Bright-field image of panel F. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL; inner nuclear layer;
GCL, ganglion cell layer; CMZ, ciliary marginal zone; PC, paras ciliaris. Scale bar, 100 Am.
persist in neural retina proximal to the optic nerve head and
throughout the CMZ (Fig. 6P).
3.5. Downregulation of RPE65 and CRBP in the inner
layers of the regenerating retina
To compare the spatiotemporal expression of Otx2 with
those of RPE cell specific proteins, we followed the
expression of RPE65 and CRBP during regeneration. We
also investigated the expression of CRALBP, which is
localized in RPE and Mqller glial cells [4]. These proteins
are required for retinoid processing for vision [35,37]. In the
intact retina, immunoreactivities for RPE65 and CRBP were
observed in RPE (Figs. 7A and B) and CRALBP was
observed in RPE and Mqller glial cells (Fig. 7C). In the
regenerating retina, immunoreactivities for RPE65, CRBP
and CRALBP were uniformly observed in the 2 to 3 cell
layered regenerating retina (Figs. 7D–F), consisting of
various degrees of pigmented cells (Figs. 7G–I). There is
no correlation between localization of those proteins and
pigmentation of the cells (Figs. 7D–I). Concomitantly with
the entire loss of pigment from the inner layers (Figs. 7M
and N), immunoreactivities for anti-CRBP and anti-RPE65
disappeared from the inner layers (Figs. 7J and K).
Correlation between the downregulation of Otx2 and
RPE65 was also confirmed by double staining (Fig. 7M
inset). Only a few cells in the inner layers showed a
substantial immunoreactivity for anti-RPE65 (Fig. 7J) and
subsequently disappeared at later stages (data not shown).
Thus, RPE cell specific proteins dramatically decreased in
the inner layers at the same time when Otx2 was downregulated. In the outer layer, strong immunoreactivity of
anti-CRBP was observed in the majority of cells (Fig. 7K).
In contrast, RPE65 positive cells were rare (Fig. 7J). These
observations suggest that some of outer layer cells maintain
some RPE characteristics during early stages of regeneration. In contrast to RPE65 and CRBP, strong immunoreactivity for anti-CRALBP was observed in ramifications
of the inner layers, when the entire inner layer lost pigment
(Figs. 7L and O). Another Mqller glial cell marker, glial
fibrillary acidic protein (GFAP) antibody reacted with the
inner layers at the same stage (data not shown), suggesting
that precocious Mqller glial cells appeared in the early
regenerating retina. When the inner layers became approximately 7 cell layers, anti-CRALBP immunopositive cells in
the inner layer showed distinct Mqller glial cell morphology
(Fig. 7O inset).
4. Discussion
We have cloned the newt ortholog of Otx2 and examined
its expression during development and regeneration of the
retina. We found that Otx2 is expressed in a mature retina
with a similar pattern as has been reported for other
vertebrates. Previous studies have shown that Otx2 is
S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
55
Fig. 5. Expression of Otx2 in the developing retina. (A, C, E, G, I and K) Otx2 immunofluorescence (in red) in developing retina. (A) Strong Otx2
immunoreactivity observed in the anterior portion of neural tube, including forebrain (f b) and midbrain (mb) primordial and optic vesicle (OV, the retina
primordial). (B) Bright-field image of panel A. (C) At St.31, strong Otx2 immunoreactivity observed in the outer layer of optic cup, the presumptive RPE
(arrow). (D) Bright-field image of panel C. (E–G, I and K) Immunofluorescent localization of Otx2 (in red) and PCNA (in green) in the later phase of neural
retina development. (E–G) Co-localization of Otx2 (in red) and PCNA (in green) in the neural retina was observed in orange color (red plus green, arrows).
(I and K) Co-localization of Otx2 and PCNA in a subpopulation of cells at the anterior portion of neural retina (presumptive CMZ at St.44 and CMZ at St.54)
was observed in orange or yellow color (red plus green). (H, J, and L) Bright-field image of panels E–G, I and K, respectively. f b: fore brain, OV: optic vesicle.
mb: midbrain, pRPE: presumptive retinal pigment epithelium, pNR: presumptive neural retina, le: lens. Scale bar, 100 Am.
expressed in RPE cells. The Otx genes are important
regulators for differentiation of RPE cells. Otx1 / ;
Otx2+/ mice failed to specify pigmented epithelium and
this phenotype is associated with an expansion of the
prospective neural retina [23]. OTX2 transactivates the
genes involved in melanosome formation-melanosome
glycoproteins QNR71, the melanosome enzyme Tyrosinase
and the Tyrosinase-related Protein TRP-1—by directly
binding to the promoter region of these genes [24].
Overexpression of OTX2 in avian embryonic neural retinal
cells induces a pigmented phenotype [24]. Thus, OTX2
functions in the specification of RPE cells. Previous studies
have also shown that Otx2 is expressed in photoreceptors
and bipolar cells [1,3,43]. Otx2 appears to have multiple
functions in the developing retina. Inactivation of the Otx2
gene, under control of the Crx promoter, shows a complete
loss of photoreceptor cells, while retroviral mediated overexpression biases progenitor cells towards a rod photoreceptor fate [30]. Other transgenic studies in Xenopus have
shown that Otx2 promotes bipolar cell fate [43]. Moreover,
Otx1 / ;Otx2+/ mice have defects in both RPE development and retinal ganglion cells [23].
During development, Otx2 is expressed in pigmented
epithelium and only at low levels in the retinal progenitors.
Although Otx2 is expressed throughout the optic vesicle, it
is downregulated in the neural retinal part of the optic cup,
but still expressed in the presumptive pigmented epithelial
layer. Later in development, when neurons are becoming
postmitotic, Otx2 is strongly expressed in the postmitotic
neurons in the inner and outer retinal layers. Previous
studies on chicken development have shown that OTX2 is
expressed transiently in many types of newly differentiating
neurons in the retina [3], but not in the mitotically active
progenitor cells. A similar observation was made in the
mouse retina. However, we did find some PCNA immunoreactive cells that also were labeled with Otx2. Considering
that PCNA is not degenerated rapidly after mitosis in
proliferating HeLa cells [28], PCNA may have still
remained soon after the terminal mitosis. However, in the
Xenopus retina, Otx2 is expressed in mitotically active,
BrdU labeled cells at the CMZ [33,43]. Therefore, the
expression of Otx2 in at least some retinal progenitors may
be a common feature of amphibians that is not shared by
higher vertebrates.
56
S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
Fig. 6. Expression of Otx2 and PCNA in regenerating retina. (A–D) Same field of approximately 2 cell layered regenerating retina. (A) Bright-field image. The
majority of the cells in the inner layer contain some pigments. (B) Co-localization of Otx2 and PCNA in both the outer and the inner layer cells was observed in
yellow green or yellow color (red plus green). (C) Immunofluorescent localization of Otx2 (in red). (D) Immunofluorescent localization of PCNA (in green).
(E–H) Same field of approximately 3 cell layered regenerating retina. (E) Bright-field image. The majority of the cells in the inner layers still contain some
pigments. (F) Co-localization of Otx2 and PCNA in both the outer and inner layer cells was observed in yellow green or yellow color (red plus green). (G)
Immunofluorescent localization of Otx2 (in red). (H) Immunofluorescent localization of PCNA (in green). (I–L) Same field of approximately 4 cell layered
regenerating retina. (I) Bright-field image. At this stage, entire inner layers lost pigment. (J) Otx2 immunofluorescence (in red) in the majority of the outer layer
cells was not co-localized with PCNA (in green). (K) Immunofluorescent localization of Otx2 (in red) in the inner layers was weaker than that of the outer layer
except for a few cells. (L) Immunofluorescent localization of PCNA (in green) was observed in the majority of cells in inner layers, but apparent in the outer
layer. (M–N) Same field of multi-layered regenerating retina. Inner layers were composed of approximately 7 cell layers. (M) Bright-field image. (N) Colocalization of Otx2 and PCNA in the subpopulation of inner layer cells was observed in orange or yellow green color (red plus green). (O–P) Same field of
stratified regenerating retina. Retina regeneration close to complete morphologically. (O) Bright-field image. (P) Co-localization of Otx2 and PCNA was
observed in yellow or yellow green color (red plus green) in the subpopulation of neural retinal cells near the optic nerve head and the CMZ. ON: optic nerve.
Scale bar, 100 Am.
During retina regeneration, we found that Otx2 and RPE
specific proteins (RPE65 and CRBP) were uniformly
expressed in both the inner and outer layers until the entire
inner layer lost pigment. Early in regeneration, the inner and
outer cells are not distinguishable by their Otx2, RPE65 and
CRBP expression pattern. In the intact retina, melanin
synthesis is not detectable except for the basal part of the
iris. After the formation of the inner layer, which consist of
cells containing various degrees of pigments, the cells in the
outer layer start melanin synthesis, but the inner layer cells
do not initiate melanin synthesis [41]. This suggests that
although the outer and the inner layers uniformly express
Otx2 and other RPE cell markers, they can be distinguished
by a difference in melanin synthesis. Only when the
regenerating retina formed approximately 4 layers did the
majority of monolayered cells attach to Bruch’s membrane
(the outer layer), withdraw from the cell cycle and acquire a
normal expression level of Otx2. At the stage when strong
CRBP expression was detected exclusively in the outer
layer, some of the outer layer cells expressed RPE65, and
the pigment granules disappeared from the entire inner
layer. At a similar stage, the outer and the inner layer show
discernible compositions of subcellular organelles [18], and
Neural Cell Adhesion Molecules (N-CAM) appeared in the
inner layers [27]. Taken together, downregulation of Otx2 in
the inner layers is consistent with the timing of morphological changes and upregulation of neural markers.
What factors are responsible for the differential expression of Otx2 in the inner and outer layers? One candidate is
bFGF. bFGF represses Otx2 expression of cultured gastrula
stage animal cap ectoderm of Xenopus [21]. bFGF promotes
transdifferentiation of RPE cells in neural retinal cells both
in vivo and in vitro in embryonic chicken [32,34] and in
vitro in Xenopus larvae [39]. In vitro studies have shown
that components of the extracellular matrix (ECM) can
support the maintenance of the RPE cell phenotype [5]. In
the intact retina, the basal surface of the RPE is covered by a
collagenous ECM of Bruch’s membrane. Thus, during retina
regeneration, Bruch’s membrane can be a potential cue to
maintain RPE cell phenotype in the outer layer, while the
inner layer may lose Otx2 expression via a bFGF dependent
pathway. Further experimentation is required to substantiate
this hypothesis.
A final interesting observation made in this study was that
Mqller glia appears to be generated very early in the
regeneration process. This is in contrast to the development
of the retina, where Mqller glial cells are generated at the late
phase of retinal histogenesis [7]. In the regenerating retina,
we found strong expression of both CRALBP and GFAP
throughout the regeneration process. It is possible that the
S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
57
Fig. 7. Expression of RPE65, CRBP and CRALBP in intact and regenerating retina. (A, D and J) Immunofluorescent localization of RPE65 (in green). (B, E
and K) Immunofluorescent localization of CRBP (in red). (C, F and L) Immunofluorescent localization of CRALBP (in red). (A–C) Immunofluorescent images
were merged with bright-field images. (A and B) In the intact retinas, immunofluorescence of RPE65 and CRBP was localized in RPE. (C) In the intact retina,
immunofluorescence of CRALBP was localized in both RPE and Mqller glial cell. (D–F) Immunofluorescence of RPE65, CRBP and CRALBP was localized
in both the outer and the inner layers consisting of various degrees of pigmented cells. (G, H and I) Bright-field image of upper panels. (J) Immunofluorescent
localization of RPE65 in some outer layer cells. A few cells immunofluorescent for RPE65 were observed in the inner layer. (M) Bright-field image of panel J.
(M inset) Partial image taken from the same section of panels J and M, viewed at same magnification. Immunofluorescence of Otx2 (in red) in inner layer was
weaker than that in the outer layer cells and RPE65 immunofluorescence in the inner layers almost disappeared. Only some cells in the inner layers showed
normal level of Otx2 immunofluorescence but this was not co-related with RPE65 immunofluorescence. At this stage, the plasma membrane showed a nonspecific background signal, which could not be removed by pre-absorption of primary antibodies using purified Otx2 (data not shown). (K) Strong CRBP
immunofluorescence (in red) was observed in the majority of outer layer cells. (N) Bright-field image of K. (L) Immunofluorescent localization of CRALBP (in
red) in both the outer and inner layers. (O) Bright-field image of panel L. (O inset) Immunofluorescent localization of CRALBP in both the outer and inner
layers, at the stage when approximately 7 cell layered inner layers were formed. Scale bar, 100 Am.
appearance of Mqller glial cells at an early stage in retinal
regeneration may be important for the process. Mqller glial
cells can positively feedback into bFGF expression in vitro
[6], and thus, during regeneration, Mqller glial cells are a
potential source of bFGF for inner layer transdifferentiation.
5. Conclusions
In this study, we have cloned the newt ortholog of Otx2
and examined its expression in retina during development
and regeneration. We found that Otx2 is expressed in a
mature retina with a similar pattern as it was reported for
other vertebrates. During development, Otx2 is expressed in
both PCNA immunoreactive progenitors and in postmitotic
neurons. During retina regeneration, we found that Otx2,
RPE65 and CRBP expression was downregulated in the
inner layers when the entire inner layer lost pigment. After
the formation of approximately 7 layered inner layers, the
expression pattern of Otx2 in the retina was similar to that
during development. Thus, dedifferentiation of RPE cells are
possibly stepwise, and losses of RPE markers such as
RPE65 and CRBP are coupled with commitment of the
inner layer into neural retina. This finding will help us
understand the order of events—the proliferation of partially
dedifferentiated RPE cells and the commitment of the inner
cells to the neural retina. Recently, lipofection method was
successfully applied to transfer genes efficiently into
cultured newt pigmented iris cells and retinal pigment
epithelium cells [9,14]. Furthermore, electroporation method
is now available for expression and repression of genes in
the rodent retina in vivo [25]. In the future, modification of
such transient transfection methods will allow us to study
the function of Otx2 gene during neural retina regeneration.
Acknowledgments
The authors thank Prof. Takehiko Saito (Tsukuba
University) for his helpful suggestions about the operation,
58
S. Sakami et al. / Developmental Brain Research 155 (2005) 49–59
Dr. Toshikazu Nakamura, Dr. Hiroshi Funakoshi and Mr.
Kenji Nakamura (Osaka University) for their technical
assistance on operating the confocal microscope. We are
grateful to Mr. Shintaro Yamamoto (Osaka University) for
kind help in obtaining newts. We also thank Dr. T.M.
Redmond (National eye institute) for providing us with antiRPE65 antibody, and Dr. J. Saari (University of Washington) for anti-CRBP antibody and anti-CRALBP antibodies. This research was supported by grants from the
Special Coordination Funds for Promoting Science and
Technology (SCF), by SUNBOR and by a Grant from Aid
for Scientific Research from the Japanese Ministry of
Education, Science, Sports and Culture.
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