1 Laboratoire d'Embryologie Moléculaire, Bat. 445 Université Paris
XI, 91405 Orsay, France
2 Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2
3DY, UK
3 Institut fuer Biochemie, Humboldtallee 23, 37073 Göttingen, Germany
* Author for correspondence (e-mail: muriel.perron{at}emex.u-psud.fr)
Accepted 13 January 2002
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SUMMARY |
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Key words: Retinal pigment epithelium, Retinal stem cells, Hedgehog pathway, Proximodistal axis, Xenopus
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INTRODUCTION |
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In contrast to these numerous studies on the function of the mature RPE,
little is known about its normal development and in particular about the
molecular mechanisms involved in RPE cell differentiation. During the initial
stages of vertebrate retinogenesis, cells of the optic vesicle adopt one of
two alternate cell fates. Cells in the distalmost part of the vesicle,
immediately beneath the surface ectoderm, undergo neural differentiation,
while cells in the proximal part differentiate into RPE. This patterning could
involve intrinsic cues such as the transcription factors Mitf and Otx2 that
are differentially expressed in the prospective neural and RPE domains of the
optic vesicle (Mochii et al.,
1998; Martinez-Morales et al.,
2001
). But the patterning of the optic vesicle also depends on
interactions with the overlaying surface ectoderm
(Hyer et al., 1998
). Surface
ectoderm-derived FGFs may play an important role in mediating at least part of
this interaction by promoting neural fate in the closely apposed
neuroepithelium of the distal optic vesicle
(Hyer et al., 1998
). By
contrast, the extra-ocular mesenchyme surrounding the proximal vesicle
promotes an RPE fate through an activin-like signal
(Fuhrmann et al., 2000
). In
this study, we present evidence for a role of Hedgehog genes in RPE cell
differentiation.
The Hedgehog genes encode secreted signalling proteins that mediate various
cell-cell interactions in both vertebrates and invertebrates. In vertebrates,
Sonic hedgehog (Shh) is involved in patterning the embryonic limb and spinal
cord, and has a role in tooth, lung and hair development (reviewed by
Ingham and McMahon, 2001). It
has been shown that Shh receptor is composed of at least two proteins: the
tumour suppressor protein Patched (Ptc) and the multipass membrane protein
Smoothened (Smo) (Murone et al.,
1999
). The binding between Shh and Ptc is thought to relieve
Ptc-mediated inhibition of the activity of Smo
(Denef et al., 2000
), leading
to the activation of transcriptional targets. In Xenopus, two Ptc
genes have been identified: Ptc1 and Ptc2
(Takabatake et al., 2000
;
Koebernick et al., 2001
).
Three zinc-finger motif transcription factors, Gli1, Gli2 and Gli3, also play
critical roles in the mediation and interpretation of Hh signals through the
activation and repression of Hh target genes
(Ruiz i Altaba, 1999
;
Koebernick and Pieler, 2002
;
Ruiz i Altaba et al., 2002
).
It has been shown that transcriptional targets of the pathway include
Ptc1, Ptc2 and Gli1 themselves
(Lee et al., 1997
;
Goodrich et al., 1999
;
Lewis et al., 1999
;
Pearse et al., 2001
). Several
lines of evidences converge to suggest that Shh is involved in early eye
development. Targeted gene disruption of Shh in the mouse leads to cyclopia,
with no optic stalk, suggesting that Shh is involved in the separation of the
eye fields and the formation of the optic stalk
(Chiang et al., 1996
). In
zebrafish, overexpression of shh expands the proximal retina (optic
stalk and RPE), at the expense of distal or neural retina
(Macdonald et al., 1995
;
Ekker et al., 1995b
). These
results suggest that Shh activity, emanating from the rostral midline, is
required for the proper formation of the proximodistal axis of the eye. After
the eye field separation, a source of Shh emanates from the eye primordium
itself in chick (Zhang and Yang,
2001b
). This source may play a role in the establishment of the
dorsoventral patterning of the eye during the transition from the optic
vesicle to the optic cup (Zhang and Yang,
2001b
).
Hedgehog genes are expressed in the retina while many cells are undergoing
division and differentiation (Wallace and
Raff, 1999; Stenkamp et al.,
2000
; Takabatake et al.,
1997
). In Drosophila, hh controls retinal development by
propagating a wave of photoreceptor differentiation across the eye disc
(Greenwood and Struhl, 1999
;
Dominguez, 1999
;
Dominguez and Hafen, 1997
;
Levine et al., 1997
). A
similar wave of Shh influences neural differentiation in the zebrafish eye
(Neumann and Nuesslein-Volhard,
2000
). The Shh signal, secreted by early differentiated ganglion
cells, has distinct roles at different concentration thresholds. High levels
of Shh inhibit rather than promote ganglion cell differentiation in chick
retinas. Thus, Shh signals could modulate ganglion cell production and thereby
control the progression of the retinal neurogenic wave
(Zhang and Yang, 2001a
). In
vitro data published so far, however, suggest that the roles of shh
gene in retinal cell differentiation are very complex. Murine retinal cultures
show that Shh can regulate mitogenesis resulting in increased
photoreceptor differentiation (Levine et
al., 1997
) and Müller glia cell differentiation
(Jensen and Wallace, 1997
). It
has been shown in zebrafish that injection of a cocktail of shh
antisense oligonucleotides slows or arrests the progression of rod and cone
photoreceptor differentiation (Stenkamp et
al., 2000
). Shh, which is secreted by the axons of ganglion cells
also stimulates astrocytes proliferation in the optic nerve
(Wallace and Raff, 1999
). A
role in the retinal organisation has also been suggested since the retina of a
mouse carrying a conditional mutation in Shh display extensive laminar
disorganisation (Wang et al.,
2002
).
Other Hedgehog genes are also expressed in vertebrate retina. The zebrafish
Tiggywinkle hedgehog (twhh) gene is also expressed in
ganglion cells and in the RPE (Neumann and
Nuesslein-Volhard, 2000;
Stenkamp et al., 2000
). Twhh
and Shh belong to a same group if phylogenic relationships are taken into
account (Ingham and McMahon,
2001
). Indian hedgehog (Ihh) has been detected
outside the eye, in a layer adjacent to the RPE, and along the optic nerve in
the mouse (Wallace and Raff,
1999
), but the expression of Desert hedgehog
(Dhh) has not been described. We have therefore undertaken a study of
the role of all three Hedgehog genes in retinal cell differentiation in
Xenopus retina. Three members of the Hedgehog family have previously
been isolated in Xenopus, one homologue of Shh
(X-shh), one homologue of Ihh, banded hh (X-bhh),
and one homologue of Dhh, cephalic hedgehog (X-chh)
(Ekker et al., 1995a
;
Ingham and McMahon, 2001
). It
has been shown that Hedgehog genes are expressed in the Xenopus adult
neural retina (Takabatake et al.,
1997
) but their expression during Xenopus retinal
development is unknown. We therefore first studied the expression of these
genes in the developing retina. The expression of Patched (Ptc) genes
and Gli1 has been studied in the mouse retina
(Wallace and Raff, 1999
;
Wang et al., 2002
), but the
expression of the other components of the Hedgehog signalling pathway has
never been investigated during vertebrate retinogenesis. Therefore, in order
to highlight cells that receive Hh signals, we also undertook an analysis of
the expression of potential downstream components of the cascade, two
patched Xenopus homologues, the homologue of smoothened and
three Gli genes, at different stages of retinogenesis. We also investigated
the role of Hedgehog genes by activating or blocking the pathway.
Interestingly, our experimental approach led us to discover a new role for Hh
signalling in RPE cell differentiation. In addition, we found that Hh
signalling is important for the proximodistal axis throughout the optic
vesicle maturation.
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MATERIALS AND METHODS |
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Xenopus MITF-A cloning
A partial Xenopus Mitf cDNA was cloned using degenerate PCR
primers and stage 21-24 RNA as a template. Conserved regions between human,
mouse, chicken and hamster Mitf-1 were used to design degenerate primers:
5DegMi1 ATG GAY CCN GCN YTN CAR ATG; 3DegMi1 TGC GCN CKN GCY TGC AT; 5DegMi2
ATG GAY CCN GCN YTN CAR ATG; and 3DegMi2 ARD ATN GTN CCY TTR TTC CA. To make
cDNA, 1 µg of total RNA and random hexamers were used in a standard 20
µl reaction with M-MLV reverse transcriptase (Promega). PCR amplification
of Xenopus Mitf was carried out in a solution containing 2 µl of
the cDNA template, 100 ng of the primers, 5DegMi1 and 3DegMi1, and 10% DMSO in
the standard PCR solution with AmpliTaq Gold® DNA polymerase (Applied
Biosystems). The cycling conditions were as follows: one cycle of 94°C, 9
minutes; five cycles of 94°C for 30 seconds, 37°C for 4 minutes and
72°C for 1 minute; five cycles of 94°C for 30 seconds, 45°C for 4
minutes and 72°C for 1 minute; and 35 cycles of 94°C for 30 seconds,
50°C for 4 minutes and 72°C for 1 minute. A nested PCR reaction was
then carried out using 2 µl of the PCR product and degenerate primers,
5DegMi2 and 3DegMi2 in exactly the same solution and conditions as the PCR
reaction above. To generate a larger amount of the resulting PCR product,
reamplification was carried out using 2 µl of the second PCR product, the
same primers and a standard Taq protocol (Roche Applied Science). The
clones were ligated into pGEM®-T Easy vector (Promega) and sequenced. The
deduced amino acid sequence of three clones was 73% identical to human Mitf
(138-247 amino acids; protein reference number I38024). Thus, these cDNAs
contained sequence common to heart, neural retina and RPE Mitf clones
(Mochii et al., 1998). In
order to isolate a sequence unique to retinal pigment epithelial Mitf, or
Mitf-A, the Xenopus Mitf sequence data was used to design primers for
a 5' RACE reaction, MixGSP-5'RACE CTT CGC CTT CTT TCA ATG AGG TTG
TG and 3Mix262 ATT GTC CTT CTT TTG CCG TTC. 5' RACE was carried out
using stage 35-36 total RNA as template and the SMART RACE cDNA Amplification
kit (Clontech). After the initial amplification with MixGSP-5'RACE and
the universal primer mix (UPM) provided in the kit, we used 1.5 µl of this
reaction with the nested universal primer (NUP) and 3Mix262 from the kit in a
second standard PCR reaction with AmpliTaq Gold. Cycling conditions were 35
cycles for 1 minute each at 94, 58 and 72°C. A single
750 bp product
was obtained, subcloned into pGEM-T-easy and sequenced. The isolated cDNA
contained an open reading frame 68% identical to the corresponding region of
Human Mitf-A (protein reference number T14752) suggesting that the isolated
cDNA is Xenopus Mitf-A.
In situ hybridisation
Digoxigenin (DIG)-labelled antisense RNA probes were generated for
Pax2, Pax6, X-bhh, X-chh, X-shh, X-Ptc-1, X-Ptc-2, X-Smo, Gli1, Gli2,
Gli3, Mitf, Xotx2, Brn3.0 and Vax2, according to the protocol of
the manufacturer (Roche). Whole-mount in situ hybridisation was performed as
described previously (Shimamura et al.,
1994), with the following change: to visualise expression in the
RPE, embryos were bleached (Broadbent and
Read, 1999
) just before the proteinase K step. After NBT/BCIP
(Roche) staining, embryos were vibratome sectioned (50 µm). For double in
situ hybridisation, we generated a fluoresceine X-Smo probe according
to the protocol of the manufacturer (Roche). X-Smo expression was
first revealed with NBT/BCIP, then we inactivated the remaining alkaline
phosphatase by incubating the embryos 30 minutes in PBS-EDTA 10 mM at 60°C
and we removed the anti-fluorescein antibody bound to the fluorescein-labelled
probe by incubating the embryos in Glycine 0.1 M-HCl pH2.2 for 10 minutes.
Embryos were then washed five times in PBS. X-bhh expression was then
revealed with vector red (Vector Laboratories), which can be visualised both
in bright field and under fluorescence. Embryos were then vibratome sectioned
(50 µm). In situ hybridisation on cryostat sections (12 µm) was
performed as previously described (Perron
et al., 1998
).
BrdU staining
BrdU was injected intra-abdominally, and the animals were allowed to
recover for 2-8 hours postinjection. BrdU was detected using the BrdU
labelling kit (Roche) after a 45 minute treatment in 2 N HCl. For double
staining, the mRNA was first detected by whole-mount in situ hybridisation (as
described above). Embryos were then cryostat sectioned and BrdU
immunostained.
Cyclopamine treatment
Cyclopamine (Toronto Research Chemicals and a gift from William Gaffield)
or N-aminoethyl aminocaproyl dihydrocinamoyl cyclopamine (KAAD-cyclopamine;
Toronto Research Chemicals) was resuspended in 95% ethanol as previously
described (Sukegawa et al.,
2000) at a concentration of 5 mM. Embryos were incubated in the
dark in 20-100 µM of this cyclopamine solution diluted in MBS 0.1x
(Sive et al., 2000
). Control
embryos were incubated in MBS 0.1x containing an equivalent dilution of
95% ethanol. These solutions were changed daily.
Immunohistochemistry
Immunohistochemistry was performed on 4% paraformaldehyde fixed tissues.
Cryostat sections (12 µm thick) were incubated with primary antibodies
(monoclonal anti-RPE antibody XAR1, a gift from Don Sakaguchi; monoclonal
anti-rhodopsine R2-12, a gift from N. Colley, monoclonal anti-tubulin, Sigma),
and visualised using anti-mouse fluorescent secondary antibodies (Alexa,
Molecular Probes).
In vivo lipofection
pCS2-GFP vector (a gift from D. Turner) was transfected into the
presumptive region of the retina of stage 18 embryos as previously described
(Holt et al., 1990;
Dorsky et al., 1995
). Embryos
were fixed at stage 41 and cryostat sectioned (10 µm). GFP-positive cells
were counted and cell types were identified based upon their laminar position
and morphology, as previously described
(Dorsky et al., 1995
).
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RESULTS |
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We found that expression of X-chh starts to be detected in the RPE slightly later than X-bhh, from stage 35-36 onwards (Fig. 2E). This expression spreads out in the RPE in a very similar way to X-bhh. X-chh is, however, also detected in the hindbrain (Fig. 1E,F). In the retina of stage 42 embryos, the expression of both X-bhh and X-chh is maintained in the RPE but is still completely excluded from the most peripheral RPE, overlaying the CMZ (Fig. 2D,F).
Xenopus downstream components of the hedgehog pathway are
expressed in the retinal pigment epithelium and in retinal stem cells
To know what cell types in the retina receive the Hh signals produced from
RPE cells, we investigated the expression of downstream components of the
hedgehog cascade. We therefore performed in situ hybridisation
experiments at different stages of the developing retina with the following
Xenopus probes: X-Patched-1 [X-Ptc-1
(Koebernick et al., 2001;
Takabatake et al., 2000
)],
X-Patched-2 [X-Ptc-2
(Takabatake et al., 2000
)],
X-Smoothened [X-Smo
(Koebernick et al., 2001
)],
Gli1 (Lee et al.,
1997
), Gli2 [also called Gli4
(Marine et al., 1997
;
Ruiz i Altaba, 1998
)] and
Gli3 (Marine et al.,
1997
). We found that all these genes are expressed in the
developing eye, although more or less strongly depending on the stage of
development (Figs 1,
3). For example, in tadpole
embryos, X-Ptc-1, X-Ptc-2 and Gli1 expression is strong in
the brain but very faint in the retina
(Fig. 1I-L,O,P), whereas
expression of X-Smo, Gli2 and Gli3 is strong enough in the
retina to be detectable in whole embryos
(Fig. 1M,N,Q-T). On
cross-sections, we found that X-Ptc-1 is faintly expressed in the
periphery of the retina at stages 28 and 34, and that its expression decreases
with development and only a very faint expression remains in the RPE of stage
42 embryos (Fig. 3A-C).
X-Ptc-2 and Gli1 are both expressed in the presumptive RPE
and later in the RPE itself (Fig.
3D-I). X-Smo, Gli2 and Gli3 are expressed in the
presumptive RPE and in the periphery of the optic vesicle at stage 28 and 34
(Fig. 3J-V). At stage 42, these
complex expression patterns become restricted to the most peripheral region of
the CMZ containing retinal stem cells, and in the RPE surrounding this region.
To confirm that these genes are indeed expressed in the peripheral pigmented
epithelium, we performed in situ hybridisation experiments on poorly bleached
embryos using a short coloration reaction in order to visualise both the blue
staining and the remaining light brown pigmentation. We found that these genes
are indeed expressed in pigmented cells surrounding the CMZ
(Fig. 3R and data not shown).
Therefore, some downstream components of the Hh cascade are expressed in the
RPE in a pattern complementary to that of X-bhh and X-chh.
By comparing expression of X-Smo/Gli2/Gli3 with that of
X-bhh/X-chh at stage 42 (Fig.
3L,P,U with Fig.
2D,F), it seems that there is a gap between the hh
expression domain and the X-Smo/Gli2/Gli3 expression domain in the
RPE. To investigate whether these expression patterns overlap or not during
development, we performed double in situ hybridisation experiments on stage 38
embryos with both a X-Smo and a X-bhh probe. In between
these two expression domains, cells do not seem to express any of these genes
at high levels, suggesting that this might be an intermediate zone
(Fig. 4A-C).
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It has recently been discovered that some mouse RPE cells can self renew
and also generate multipotent neural precursors in vitro, two properties of
stem cells (Tropepe et al.,
2000). These RPE cells are located in the ciliary margin of the
mouse retina, the pigmented ciliary margin. We therefore wondered whether the
peripheral pigmented epithelial cells in Xenopus could proliferate in
vivo. Long pulses (8 hours) of BrdU incorporation allowed us to show that
indeed some of these RPE cells were BrdU positive
(Fig. 4G-I). However, no
BrdU-positive cells were detected in the more central RPE where X-bhh
and X-chh are expressed (data not shown). Therefore, only peripheral
RPE cells are still proliferating. We then combined in situ hybridisation
using Gli3 as a probe, the expression of which is representative of
the Hh signalling pathway, with BrdU staining to ask if the dividing cells
express components of the Hh pathway. We found that indeed some BrdU positive
cells in the peripheral pigmented epithelium are included in Gli3
expression domain (Fig. 4J-L).
X-Smo, Gli2 and Gli3 are thus all expressed in a domain
containing young and occasionally dividing RPE cells.
X-Shh, from the rostral midline, is involved in the establishment of
the proximodistal axis of the retina
It has previously been reported that overexpression of Shh or a
dominant-negative form of PKA (dnPKA) in zebrafish leads to
development defects in the eye that suggest involvement in proximodistal
patterning (Ungar and Moon,
1996; Ekker et al.,
1995a
; Macdonald et al.,
1995
). When we overexpressed dnPKA or shh in
Xenopus embryos, we also found such defects
(Fig. 5 and data not shown).
The ventral region of the retina is transformed in a large optic stalk. This
is illustrated by an increased expression of Pax2, an optic stalk
marker, and a decreased expression of Pax6, a neural-retina marker
(Fig. 5). It is noticeable that
the dorsal Pax6 expression remains largely unaffected. Similarly, the
morphology of the dorsal neural retina, as well as the dorsal RPE, retains a
normal morphology. In Xenopus, the dorsal retina is derived from more
distal region of the optic cup than the ventral retina. This result thus
suggests that Shh signalling in Xenopus is also mediated by PKA and
may also be involved in the establishment of the proximodistal axis of the
retina.
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Hedgehog signalling in the retina is involved in RPE cell
differentiation
As we were most interested in later roles for the Hh pathways, we decided
to incubated Xenopus embryos in cyclopamine solution only from the
late neurula stage after the eye fields have clearly separated (stage 20).
When we analysed the phenotype of these embryos at stage 40, we indeed did not
see cyclopic embryos. However, we observed two obvious major developmental
defects. The spinal cord was not as straight as in control embryos, and the
pigmentation was abnormal, notably in the RPE
(Fig. 8A,B). Indeed,
pigmentation around the lens (the peripheral pigmented epithelium) was
completely missing, and the remaining pigment was less dark than in control
embryos.
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We then further investigated the effect of cyclopamine on RPE
differentiation using the antibody XAR1 as a RPE marker. We found that the
staining was strongly reduced or even completely absent in the RPE of
cyclopamine-treated embryos (Fig.
8O,P). When embryos were incubated in cyclopamine from the
two-cell stage, the same absence of XAR1 staining was detected (data not
shown). Although the strength of this phenotype was dose dependent, the XAR1
staining was already severely reduced in embryos incubated in 20 µM of
cyclopamine. This result suggests that the Hh pathway is involved in RPE
differentiation. To investigate this further, we performed in situ
hybridisation on cyclopamine-treated embryos with several other RPE markers.
We first wanted to look at an early marker of RPE cell differentiation, such
as Mitf (Mochii et al.,
1998). We therefore cloned by RT-PCR the Xenopus
homologue of Mitf (see Materials and Methods). In wild-type embryos,
Mitf is expressed in the developing RPE
(Fig. 8Q). We found that the
expression of Mitf was decreased in cyclopamine-treated embryos. This
effect was the most dramatic in the periphery of the retina where no
expression at all was detected, although some expression was visible in the
central differentiating RPE (Fig.
8R). We then looked at the expression of other RPE markers,
including Xotx5, X-bhh and X-chh. Xotx5 expression is mostly
affected in the ventral part of the cyclopamine-treated retina in the RPE
layer while its expression in the photoreceptor layer seems normal
(Fig. 8S,T). The intensity of
the expression of both X-bhh and X-chh is reduced in
cyclopamine embryos. This is more striking in the periphery of the retina than
in the central RPE, as if the effect follows a gradient of severity from the
periphery to the centre (Fig.
8U-X). As a conclusion, although the expression of the various RPE
markers we used were differentially affected by the cyclopamine treatment, all
were reduced, strongly suggesting that Hh signalling is essential for the
proper RPE differentiation.
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DISCUSSION |
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Hh genes are expressed in different cell types during
retinogenesis
We found that X-shh is expressed in newborn ganglion cells. This
is consistent with previous data in mouse and zebrafish
(Wallace and Raff, 1999;
Stenkamp et al., 2000
). We
found that X-bhh and X-chh are expressed in the retina from
stage 35 onwards, in the RPE but not in the neural retina. This expression
seems to be maintained as detected by RT-PCR in the Xenopus adult
retina (Takabatake et al.,
1997
). Ihh has also been detected, using RT-PCR, in the
rat RPE (Levine et al., 1997
).
However, it has been reported that Dhh is expressed in the rat neural
retina and not in the RPE, by RT-PCR
(Levine et al., 1997
). It
seems therefore that the regulation in the retina of X-chh in
Xenopus and of Dhh in mammals may have diverged during
evolution.
Complementary expression of Hh pathway genes in the central and the
peripheral RPE
Our data demonstrate a molecular difference between peripheral and central
RPE cells in tadpole retina. Indeed, expression of X-bhh and
X-chh is restricted to the central RPE cells, while X-Smo,
Gli2 and Gli3 expression reveals a narrow peripheral annulus in
the RPE. In addition, we found a zone between these two domains of expression
that do not express any of these genes strongly. This therefore led us to
propose a molecular subdivision of the RPE into three zones. From most
peripheral to most central RPE, we found Hh downstream genes in the first
zone, then these genes were inactivated, whereas Hh genes are not yet
activated in zone 2. Next, Hh and Xotx5 genes were strongly activated
in zone 3 (see Fig. 9A). This
subdivision may not reflect distinct cell types as much as it does a gradient
of differentiation from the periphery to the central RPE, similar to the
gradient of differentiation that occurs in the CMZ
(Perron et al., 1998).
Antibodies against the different components of the pathway may be necessary to
reveal such a gradient at a protein level. Previous data suggest that all RPE
cells were not equivalent. Layer and Willbold have found that peripheral RPE
behave differently from the central RPE in culture
(Layer and Willbold, 1989
).
Indeed, it has been shown that retinal and pigmented cells have the ability to
generate histotypic in vitro retina in culture. However, the sequence of
layers is identical with that of in situ retina only if the pigmented cells
are derived from the eye periphery (Layer
and Willbold, 1989
). In addition, our BrdU experiments suggests
that pigmented cells in the peripheral epithelium are `younger' than RPE cells
in the central region as some peripheral RPE cells are still dividing while
all central RPE cells are postmitotic in stage 42 embryos. This is consistent
with the conventional idea of how the RPE grows. The central area
differentiates earlier than the marginal zones
(Stroeva and Mitashov, 1983
).
The proliferative state of the RPE has, however, led to debates (reviewed by
Stroeva and Mitashov, 1983
).
In chick, it was assumed that mitotic activity had ceased completely in the
RPE of a 4-day-old embryo, while others have found mitosis later in the
embryonic RPE, but it was thought that by day 14, there were no dividing cells
in the RPE. Recently, however, Fisher and Reh have re-examined the mitotic
state of the retinal margin of hatched chicks
(Fischer and Reh, 2000
;
Fischer and Reh, 2001
).
Surprisingly, they found a proliferative margin similar to the CMZ of
amphibians, suggesting the presence of stem cells. In addition, consistent
with our results, they found the presence of proliferative cells in the
peripheral pigmented epithelium (Fischer
and Reh, 2001
). It is interesting in this context to note that
retinal stem cells have been found in the pigmented ciliary body located in
the margin of adult mouse retina. Although these cells do not have the
capacity to regenerate in vivo, they can proliferate in vitro and
differentiate into retinal-specific cell types, including rod photoreceptors,
bipolar neurones and Müller glia
(Tropepe et al., 2000
;
Ahmad et al., 2000
). Therefore,
the presence of retinal stem cells in the peripheral pigmented epithelium
might be conserved in amphibians, chick and mammals. It is interesting to note
that X-smo, Gli2 and Gli3 are strongly expressed only in
retinal stem cells of the CMZ (the most peripheral part of the CMZ) and in the
peripheral pigmented epithelium. These genes are therefore the first markers
of this retinal stem cell region. Indeed, genes that have been shown to be
expressed in retinal stem cells so far in Xenopus CMZ are also still
expressed in differentiating cells of the retina, such as Pax6,
Xoptx2 or Rx1 (Perron et
al., 1998
; Zuber et al.,
1999
). However, genes expressed only in the CMZ, such as the bHLH
gene Xath5, are not expressed in the most peripheral region
containing stem cells (Kanekar et al.,
1997
; Perron et al.,
1998
). It would be interesting now to look at the expression of
Smo or Gli genes in mammalian retina to see whether they
also represent specific markers of pigmented ciliary margin retinal stem
cells. It is now necessary to ask whether these peripheral pigmented
epithelium cells in Xenopus retina do indeed self-renew and behave as
retinal stem cells in vivo.
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A role for Hh genes in RPE cell differentiation
We have shown that blocking Hh signalling by cyclopamine, after the eyes
have separated, does not lead to any obvious defect in neural retinal
histogenesis. Rather, we found that cyclopamine induces severe RPE defects
(see Fig. 9B). As we obtained
RPE defects with a low dose of cyclopamine and with KAAD-cyclopamine, which
has been shown to be less toxic but more potent, we could rule out a possible
toxic effect. Similar to our results, when the Shh signal was perturbed only
after the optic cup formation in chick embryos, using a blocking antibody,
pigmentation was also affected, the ventral part being lost
(Zhang and Yang, 2001b). We
also found that the ventral part of the RPE is often more affected but we
found that this was mainly due to a proximodistal axis defect. The RPE
differentiation defect we have observed, however, occurs both in the dorsal
and the ventral parts. One explanation for this difference may be that
cyclopamine blocks all Hh signals while the anti-Shh antibody blocks only Shh
signalling. Our expression data of Hh genes during normal retinogenesis is
consistent with a role in RPE differentiation, X-shh being expressed
in early RPE tissue, while X-bhh and X-chh are expressed
later in RPE cells. As cyclopamine acts via Smoothened
(Taipale et al., 2000
;
Chen et al., 2002
), our data
strongly suggest that the effect of Hh genes on RPE differentiation involves
the Patched-Smoothened-Gli signalling cascade. Again, this is consistent with
our finding that Ptc, Smo and Gli genes in Xenopus are
indeed expressed in RPE precursors. Ptc1 is also expressed in RPE in
chick optic vesicle (Zhang and Yang,
2001b
). In tadpole embryos, Hh genes are expressed in
differentiated RPE cells while cells that receive the signal are expressed in
the young RPE cells including dividing cells of the peripheral pigmented
epithelium. Moreover, our results suggest that Hh signal might instruct these
cells to differentiate into mature RPE tissue. We could therefore make a
parallel with what happens in Drosophila eye disc where
differentiated photoreceptors behind the morphogenetic furrow express
hh and instruct precursor cells to differentiate, allowing the
progression of the furrow (reviewed by
Burke and Basler, 1997
).
Long-range effect of Hh signalling
In tadpole retina, we found that some cells, in between the domains of
expression of X-bhh/X-chh and of XSmo/Gli2/Gli3, do not seem
to express any of these genes, thereby leaving a gap between cells secreting
Hh and cells that potentially mediate the signal. This would be consistent
with a long-range morphogen action that has been demonstrated in several other
tissues (reviewed by Ingham and McMahon,
2001). In the vertebrate limb for example, Shh protein can spread
for many cell diameters (Lewis et al.,
2001
). In tadpole embryos, we found a very low expression of
Gli1, X-Ptc-1 and X-Ptc-2 in the RPE. As these three genes
are transcriptionally regulated by the Hh pathway, this may reflect a weakly
active pathway in tadpole embryos consistent with the low rate of de novo RPE
cell production at this stage, compared with earlier development where we have
detected a stronger expression of these three genes in the differentiating
RPE. Altogether, these results suggest that Hh signalling, probably involving
X-shh most at early stages and both X-bhh and X-chh at later stages, is
required for generating RPE cells during retinogenesis in an ongoing process
of central-to-peripheral axis formation in the growing eye.
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ACKNOWLEDGMENTS |
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