Department of Zoology, Miami University, Oxford, Ohio 45056, USA
Author for correspondence (e-mail:
delriok{at}muohio.edu)
Accepted 8 June 2004
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SUMMARY |
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Key words: Retina, Regeneration, Hedgehog, Fgf, Transdifferentiation
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Introduction |
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It has been demonstrated that postnatal chickens have embryonic-like
retinal stem cells located in the ciliary body (CB) and retina progenitor
cells in the ciliary marginal zone (CMZ) that are able to generate some
retinal neurons. The retinal stem cells in the ciliary body of the adult
chicken only proliferate in response to growth factors and the non-pigmented
epithelium in the CB has been shown to proliferate and give rise to retinal
neurons in response to injury (Fischer and
Reh, 2000; Fischer and Reh,
2003
) (for a review, see
Haynes and Del Rio-Tsonis,
2004
).
The focus of this report, however, is the embryonic chick, which also has
the capability to regenerate its retina. Unlike the adult chicken, the chick
can regenerate all of the retinal layers after they have been completely
removed by retinectomy during a small window in their development around
embryonic day 4 (E4; Hamburger and Hamilton stages 22-24.5)
(Coulombre and Coulombre,
1965). Members of the Fgf family such as Fgf1 and 2, are required
to induce retina regeneration after its removal. Other growth factors such as
TGFß, insulin, IGF1 and 2 and NGFß do not promote regeneration
(Park and Hollenberg, 1989
;
Park and Hollenberg,
1991
).
Interestingly, the embryonic chick retina can regenerate by two distinct
modes; via transdifferentiation of the retina pigmented epithelium (RPE), or
via activation of cells found in the anterior marginal region of the eye,
which includes the ciliary body (CB) and ciliary marginal zone (CMZ)
(Coulombre and Coulombre,
1965). The CB consists of two layers of cells, the pigmented and
non-pigmented ciliary epithelium (PE and CE, respectively). Because there are
no molecular markers that can clearly distinguish between the CB and the CMZ
during the early stages of development, we will refer to this region as the
CB/CMZ.
During transdifferentiation, the developing RPE loses its characteristic
phenotype, becoming depigmented and proliferates to form a neuroepithelial
layer that then differentiates into the various layers of the retina. This
Fgf2-stimulated transdifferentiation occurs primarily in the posterior part of
the optic cup and gives rise to a retina with reverse polarity, where the
outer nuclear layer (ONL) is located on the inner surface of the eye, and the
ganglion cell layer (GCL) is on the outer surface of the eye. The
transdifferentiated retina lacks the RPE layer, which does not replenish
itself during regeneration (Coulombre and
Coulombre, 1965).
The other mode of regeneration that gives rise to a fully differentiated
retina appears as a consequence of the activation of stem/progenitor cells in
the CB/CMZ. These cells have been shown to have activity up to E9
(Willbold and Layer, 1992).
The regenerated retina forms with normal polarity and can associate with
adjacent RPE that did not undergo transdifferentiation
(Coulombre and Coulombre, 1965
;
Park and Hollenberg, 1991
).
Until the present report, the only times both of the aforementioned embryonic
modes of regeneration were observed together in the same eye was during the
classic Coulombre and Coulombre studies
(Coulombre and Coulombre, 1965
)
where a piece of retina was used as a source of Fgf, and when Park and
Hollenberg (Park and Hollenberg,
1991
) used Fgf1 as a stimulus. We show that Fgf2 is able to
promote both types of regeneration.
All studies carried out to date dealing with retina regeneration in the embryonic chick have focused on identifying factors that are responsible for the induction of regeneration, and are based solely on histological observations during a limited number of time points. Here we present a comprehensive study of Fgf2-induced regeneration. We use several cell-specific markers to identify the different cell types that form during regeneration and compare the temporal and spatial nature of the two modes of retina regeneration with normal retina development. We also show for the first time that retina regenerating from the anterior region of the eye originates from retina stem/progenitor cells located in the CB/CMZ.
Along with studying the temporal and spatial nature of the regenerating
chick retina, we were also interested in identifying how key molecules control
or influence regeneration. Sonic hedgehog (Shh) has been shown to play an
important role in the development of the retina in a number of different model
organisms (Levine et al.,
1997; Greenwood and Struhl,
1999
; Neumann and
Nuesslein-Volhard, 2000
;
Stenkamp et al., 2000
;
Zhang and Yang, 2001a
;
Zhang and Yang, 2001b
;
Wang et al., 2002
;
Dakubo et al., 2003
;
Perron et al., 2003
;
Stenkamp and Frey, 2003
;
Shkumatava et al., 2004
). Shh
is of particular interest because it has been shown that ectopic Shh
expression in developing chick eyes causes retina to transdifferentiate into
RPE, and inhibition of the Shh pathway causes the RPE to transdifferentiate
into neuroepithelium (Zhang and Yang,
2001a
). Hedgehog signaling is also implicated in the specification
of the proximodistal axis and differentiation of RPE in developing
Xenopus embryos (Perron et al.,
2003
); it helps establish a proximodistal axis during zebrafish
eye development (Take-uchi et al.,
2003
) and is required for proper lamination and organization of
the retina (Wang et al., 2002
;
Shkumatava et al., 2004
). This
research points to Shh being involved during transdifferentiation of the
regenerating chick retina. On a different note, Shh has been shown to regulate
neural progenitor proliferation in vitro and in vivo
(Lai et al., 2003
). In
addition, the downstream effectors of the hedgehog pathway, Smoothened (Smo),
Gli2 and Gli3 are strongly expressed in retinal progenitor cells of the CMZ in
Xenopus (Perron et al.,
2003
), suggesting that the hedgehog pathway may play a role in
regeneration occurring from the CB/CMZ.
In the current study, we show that Shh acts in a similar way during retina
regeneration as it does during development. We overexpressed Shh using an
Rcas-Shh retrovirus and inhibited the Hedgehog (Hh) pathway using a potent
synthetic and less toxic form of cyclopamine, KAAD (3-keto, N-amino-ethyl
aminocaproyl dihydrocinnamoyl). We demonstrate that an overexpression of Shh
allows the RPE to maintain its phenotype and inhibits transdifferentiation
normally seen under Fgf2 stimulation. In fact, almost all regeneration under
these circumstances comes from the ciliary region, and only RPE that is
closest to the source of Fgf2 transdifferentiates. Furthermore, we show, for
the first time, that Shh alone is able to initiate regeneration from the
CB/CMZ independently of Fgf2. Conversely, KAAD-mediated inhibition of the
hedgehog pathway enhances the transdifferentiation of RPE into retina, with
almost all of the RPE giving rise to new retina, and only the most anterior
pigmented epithelium, including the ciliary body, not transdifferentiating.
Our studies are in agreement with those of Zhang and Yang
(Zhang and Yang, 2001b),
demonstrating that overexpression of Shh causes a reduction in RGC production
by negatively regulating undifferentiated ganglion progenitor cells, or
controlling their cell population via cell death, while inhibition of the
hedgehog signaling pathway increases RGC differentiation.
Our studies offer evidence that Shh regulates cells that are not fully committed, as demonstrated by transdifferentiation of the RPE, as well as being responsible for activation of retinal progenitor cells in the CB/CMZ.
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Materials and methods |
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Preparation of Fgf2, KAAD and PD173074
Heparin-coated polyacrylamide beads (Sigma, St Louis, MO, USA) were washed
three times in phosphate-buffered saline (PBS). Fgf2 (R&D Systems, Inc.,
Minneapolis, MN, USA) was resuspended in 1x PBS at a concentration of 1
µg/µl. Heparin beads were then incubated in Fgf2 for at least 2 hours
before use. A 1 mM KAAD (Totonto Research Chemicals, Ontario, Canada) stock
was prepared in 100% ethanol. Affi-gel Blue beads (BioRad, Hercules, CA, USA)
were washed in PBS and dehydrated through a series of ethanol washes of
increasing concentration. KAAD stock solution was added to the Affi-gel Blue
beads to a final working concentration of 100 µM. The Fgfr inhibitor
PD173074 (a kind gift from Pfizer, New York, NY, USA), was resuspended in DMSO
at a concentration of 100 mM and incubated in ethanol-dehydrated Affi-gel Blue
beads.
Surgical procedures
A window was made in the egg using forceps and microsurgical removal of the
retina was carried out as previously described, at about Hamburger and
Hamilton stage 24 (Coulombre and Coulombre,
1965; Park and Hollenberg,
1989
). Briefly, a fine tungsten wire was used to make an incision
in the dorsal part of the eye. Microdissection scissors were then used to make
a semi-circular cut so that access to the optic cup was possible via the
dorsal portion of the anterior eye. Using fine forceps, the retina was teased
loose, taking extra care not to damage the RPE. An Fgf2-coated heparin bead
was placed into the eye. The eggs were covered with tape and placed back into
the incubator until 1, 3, 5, 7 or 11 days after surgery at E5, E7, E9, E11 or
E15, respectively, when the eyes were collected. The eyes were then processed
for histology, immunohistochemistry, in situ hybridization or TUNEL assay.
Tissue fixation and sectioning
Tissues processed for histological sectioning were fixed in Bouin's
fixative for at least 24 hours and embedded in paraffin wax. Tissues used for
immunohistochemistry were fixed in 4% formaldehyde, cryoprotected in 30%
sucrose and embedded in OCT freezing medium (Sakura Finetek, Torrance, CA,
USA). Tissues used for in situ hybridization were processed similarly, with
the exception of using 4% paraformaldehyde as a fixative. All eyes used for
histology and immunohistochemistry were sectioned at 10 µm, whereas the
ones used for in situ hybridization were sectioned at 14 µm.
Antibodies
The following antibodies were obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD and maintained by The
University of Iowa, Department of Biological Sciences, Iowa City, IA 52242,
USA. Anti-Pax6 1:10; anti-Napa 73 1:100; anti-visinin (7G4) 1:100; anti-BrdU
(G3G4) 1:100; anti-AMV3C2 1:100, anti-vimentin, 1:100. Anti-Brn3a, 1:100, was
purchased from Covance Research Products, Inc. (Denver, PA, USA). Polyclonal
p-ERK antibody (1:100) was purchased from Cell Signaling Technology Inc.
(Beverly, MA, USA). Other antibodies were kind gifts: anti-Chx10, 1:1000 and
anti-Shh, 1:1000, from Dr Thomas Jessell, Columbia University, New York, NY,
USA and anti-Mitf, 1:100, from Dr Makoto Mochii, Himeji Institute of
Technology, Hyogo, Japan. Secondary antibodies include goat anti-mouse FITC,
goat anti-rabbit Cy3, goat anti-mouse biotin (Jackson Immunoresearch
Laboratories Inc, West Grove, PA, USA), goat anti-rabbit 586, goat anti-rabbit
Alexa 546, goat anti-mouse Alexa 488 and streptavidin-conjugated Alexa Fluor
350 (Molecular Probes, Eugene, OR, USA).
Immunohistochemistry
A general immunohistochemical protocol was used. Frozen sections were
washed in 1x PBS and blocked for 1 hour. When antibodies against
transcription factors were used, a 5-minute 1% saponin (Sigma, St Louis, MO,
USA) wash was used followed by three washes in 1x PBS. Primary
antibodies were diluted in blocking solution and sections were incubated
overnight either at room temperature or 4°C, followed by washes in 1
x PBS and incubation with secondary antibodies for at least 1 hour.
Coverslips were then placed on the slides using Vectashield mounting medium
(Vector Labs, Burlingame, CA, USA).
p-ERK immunohistochemistry
Retinectomies were performed as described and collected 4 hours after an
Fgf2-coated bead or control heparin bead was placed in the eye. Animals that
received KAAD beads had these beads placed in their eyes for 2 hours prior to
the Fgf2 beads. Animals that were infected with Rcas-Shh were given a
subretinal injection of Rcas-Shh on E3.5 approximately 12-16 hours before the
surgery. Four hours after the administration of the Fgf2, the embryos were
processed for immunohistochemistry.
In situ hybridization
Shh and Ptc1 probes were kind gifts from Dr Cliff Tabin
(Harvard University, Boston, MA, USA). All probes were prepared using a Dig
RNA labeling kit (Roche Applied Sciences, Indianapolis, IN, USA) and
hydrolyzed to 0.1 kb. Tissue sections were prepared as described above and in
situ hybridization was performed using the manufacturer's protocol with
modifications (Roche Molecular Biochemicals: Nonradioactive In Situ
Hybridization Application Manual, 2nd edition).
Retroviral production
Replication competent Rcas (A) retrovirus engineered to express Shh along
with control Rcas construct expressing GFP were generous gifts from Dr Cliff
Tabin (Harvard University; Boston, MA, USA), Dr Teri Belecky-Adams (IUPUI,
Indianapolis, IN, USA) and Dr Ruben Adler (Johns Hopkins University,
Baltimore, MD, USA). Retroviral stocks were prepared by transfecting cultured
DF1 chicken fibroblasts (ATCC, Manassas, VA, USA) with retroviral DNA using
lipofectamine reagent (Gibco Invitrogen Corp., Carlsbad, CA, USA). Retrovirus
was collected and concentrated using a Millipore ultra collection device
(Millipore, Billerica, MA, USA) and titered by diluting concentrated stocks
and infecting cultured embryonic chicken fibroblasts followed by
immunohistochemistry using the AMV3C2 antibody against a viral coat protein to
determine CFUs/µl (colony forming units/µl).
Microinjection
108-109 CFUs/µl of the Rcas-Shh or of control
Rcas-GFP were mixed with Fast Green dye and injected into the eye cavity,
after the retina had been removed, with a pulled glass capillary pipette using
a mouth piece. Expression of virus was confirmed by immunohistochemistry.
Quantitative real-time PCR
Total RNA was isolated using the RNA II isolation kit (BD Biosciences, Palo
Alto, CA, USA). 1.5 µg of RNA was then reverse transcribed using Improm II
reverse transcriptase (Promega, Madison, WI, USA). Real-time PCR was carried
out on a Rotor-Gene 3000 Thermocycler using Sybrgreen as a fluorophore
(Molecular Probes, Eugene, OR, USA). For each sample, Ptc1 primers were used
to determine Ptc1 levels and GapDH was used as an internal control. A dilution
series of cDNA was used to run a standard curve for each primer set and for
each sample in order to determine the amount of Ptc1 and GapDH cDNA in each
sample. Once these levels were determined, the quantitative value of Ptc1 was
divided by the value of GapDH for each sample in order to determine the
relative amount of Ptc1 cDNA. RNA was isolated from nine eyes for each sample
quantified. Each PCR reaction was carried out three times separately, in order
to ensure accuracy of results. Statistics for the relative levels of
Ptc1 RNA include the average of the three separate PCRs normalized to
the Fgf2-treated eyes. Student's t-test was used to determine
statistical significance.
Ganglion cell counts
Ganglion cells were detected using the Brn3a antibody at E11 with the
immunohistochemical methods described above. Three separate eyes for each
treatment group (KAAD/Fgf2, Fgf2 and Rcas-Shh/Fgf2) were used and ganglion
cells were counted from 100 µm x 100 µm areas at random. The
number of random areas counted for each experimental group were as follows:
KAAD/Fgf2, n=56; Fgf2, n=96; Rcas-Shh/Fgf2, n=88.
The counts from all square regions from all eyes in an experimental group were
then used to determine the average number of ganglion cells per 10,000
µm2 of regenerated retina. Student's t-test was
performed to assess statistical significance.
DiI and BrdU labeling
DiI cell labeling paste (Molecular Probes, Eugene, OR, USA) was used to
track cells of the CB/CMZ during regeneration. Retinectomies were performed as
described above. This exposed the lens and surrounding CB/CMZ region. Using a
glass micropipette, DiI cell labeling paste was carefully transferred onto the
surface of cells in the CB/CMZ area only. Embryos were collected either a few
minutes after the surgery, to ensure that only cells in the CB/CMZ were
labeled and that there was no transfer of DiI onto the RPE, or at E5 or E7 to
track the cells that regenerate from the CB/CMZ. Regenerating eyes were
labeled with BrdU (Roche, Indianapolis, IN, USA) by micropipetting 1 µl of
10 mM BrdU solution into the optic cup. The eyes were collected 1-3 days
post-retinectomy and processed for immunohistochemistry as described
above.
DiI anterograde labeling to follow axon fibers
Retinectomies were performed as described above and E11 regenerating and
control eyes were fixed with 4% formaldehyde overnight. The eyes were then
injected with 2-3 µl of DiI (Molecular Probes, Eugene, OR, USA). The
embryos were incubated in PBS at 37°C for 10 days. The eyes were sectioned
and DiI labeling was observed using confocal microscopy.
TUNEL
Cell death was detected by the TUNEL assay using the in situ cell death
detection kit, TMR Red (Roche, Indianapolis, IN, USA) as per the
manufacturer's instruction. Three separate eyes for each treatment group
(KAAD/Fgf2, Fgf2 and Rcas-Shh/Fgf2) were used and cell death in 100 µm
x 100 µm random regions was quantified. The number of random areas
counted for each experimental group are as follows: KAAD/Fgf2, n=59;
Fgf2, n=49; Rcas-Shh/Fgf2, n=49. The counts from all square
regions from all eyes in an experimental group were then used to determine the
average cell death per 10,000 µm2 of regenerated retina.
Student's t-test was performed to assess statistical
significance.
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Results |
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All retinal cell types are present in the regenerating retina
To examine regeneration of all cell types, and their laminar organization
within the regenerating retina, we used cell-specific markers for each of the
cell types found in the retina, as well as the RPE and the nerve fiber layer
(NFL). The following antibodies were used for double-labeling experiments:
Napa73 and Brn3a to detect the NFL and GCL, respectively; Pax6 to detect
horizontal, amacrine and ganglion cells along with Chx10 to detect bipolar
cells. In addition, co-expression of these two cell markers identifies
progenitor cells. Visinin and Mitf detect photoreceptors and RPE,
respectively. Vimentin is used to identify Müller glia.
The E7 developing retina shows some differentiated ganglion cells and nerve
fibers (Fig. 3A1), along with a
photoreceptor layer that is not well defined (compare
Fig. 3A3 with D3). At this
stage in development, the INL and ONL are not completely defined, as shown by
the disorganized cell layers in Fig.
3A2. Also at this time in development some of the cells in these
layers of the retina still contain undifferentiated progenitor cells, as
indicated by co-expression of Pax6 and Chx10, while other cells are undergoing
cell specification and differentiation. Müller Glia are uniformly
distributed throughout the retina (Fig.
3A4). The regenerated retina that originates both from
transdifferentiation (Fig.
3B1-B4) and from the CB/CMZ
(Fig. 3C1-C4) at this stage
shows no Brn3a expression, a marker for differentiated ganglion cells,
however, there are some Napa73-expressing cells. These cells are probably
ganglion cells that have not fully differentiated
(Fig. 3B1,C1). Regeneration has
produced a Pax6/Chx10 neuroepithelium, which presumably, has not yet become
committed to differentiate into the retinal layers
(Fig. 3B2,C2).
Transdifferentiating retina shows visinin-positive cells in the presumptive
photoreceptor layer and no Mitf expression
(Fig. 3B3), consistent with
downregulation of Mitf upon transdifferentiation
(Mochii et al., 1998a;
Mochii et al., 1998b
). CB/CMZ
regeneration shows strong Mitf expression in the RPE, but shows no
photoreceptor differentiation at this point
(Fig. 3C3). No obvious
differences are observed in the organization of the glia
(Fig. 3A4,B4,C4).
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Whole-mount eyes were prepared according to the method of Fischer and Reh
(Fischer and Reh, 2003) and
analyzed for patterns of regenerative activity in all four quadrants
(dorsal-ventral, nasal-temporal) with no obvious differences (not shown). We
also examined the E11 regenerating eyes to determine if the regenerating
retina was forming an optic nerve. Using DiI anterograde labeling we found
that the regenerating retina was sending projections out of the eye, however,
these projections were very disorganized and did not resemble a normal optic
nerve (Supplemental Fig. S1,
http://dev.biologists.org/cgi/content/full/131/18/4607/DC1).
Hh patterns are unchanged during regeneration
Because Shh plays many important roles during normal eye development, from
patterning to differentiation, we looked at the expression of Shh and its
receptor, Patched 1 (Ptc1) during normal retina development and during
regeneration.
In situ hybridization studies show that during normal development (at E11) Shh expression is localized to the ganglion cell layer, whereas Ptc1 expression is found in part of the INL and in the ONL in the posterior retina (Fig. 4A1,A3). Immunohistochemistry studies show that Shh protein is present at high concentration closer to the ganglion cells, and is diffused throughout the retina in a gradient-like fashion (Fig. 4A2). In the anterior region of the eye, where the CB/CMZ is located, Shh mRNA is expressed (Fig. 4B1) along with Ptc1 (Fig. 4B3). Shh protein is also found in the CB/CMZ (Fig. 4B2).
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Transdifferentiated retina had similar expression patterns to that of developing retina, with Shh message detectable in the presumptive GCL, and Ptc1 message mostly present in the INL and ONL (compare Fig. 4E1,E3 with A1,A3). Shh protein was also observed in a gradient, in which the expression was strongest close to the GCL (Fig. 4E2).
Function of Shh during retina regeneration
In order to determine a possible role for Shh during retina regeneration,
we overexpressed Shh using a retrovirus, Rcas-Shh, or inhibited the hedgehog
pathway using KAAD.
Shh overexpression was confirmed using in situ hybridization for the Shh transcript (Fig. 5A1) as well as by assaying the changes in Ptc1 expression, a downstream effector of the Hh pathway (Fig. 5A3). To confirm that ectopic Shh transcript was being translated into protein, we performed immunohistochemical studies using an antibody against Shh (Fig. 5A5). Both the in situ hybridization and the immunohistochemistry show similar ectopic expression patterns of Shh that are characterized by heavy `patches' of expression throughout the retina. We also confirmed infection of the virus using the AMV 3C2 antibody against a viral coat protein (Fig. 5A6). This method of detecting viral infection was also indicated by patches of immunopositive retina.
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Shh plays a role in RPE maintenance during regeneration
Eyes were treated with Fgf2 alone, Rcas-Shh/Fgf2 or KAAD/Fgf2 and examined
at E7, E11 or E15 (Fig. 6).
There were no obvious differences in laminar organization between the three
treatments. At E7, all three treatments gave rise to a neuroepithelium
(Fig. 6B,C,E,F,H,I). By Ell,
regenerating retina from the CB/CMZ in the KAAD/Fgf2- and Fgf2-treated eyes
had a GCL, IPL and INL, however, the OPL and ONL had not yet differentiated
(Fig. 6K,N). The
Rcas-Shh-treated eyes appeared to be more differentiated as the OPL and ONL
were visible (Fig. 6Q). In
contrast, the transdifferentiated retina in all treatment groups had all
retinal layers present (Fig.
6L,O,R). All layers were well defined at E15
(Fig. 6T,U,W,X,Z). Although
there were no obvious defects in cellular location and organization during Shh
manipulation, eyes treated with KAAD/Fgf2 after retinectomy at stage 24 showed
an increased domain in transdifferentiation from the posterior towards the
anterior when compared to the Fgf2-treated eyes (compare
Fig. 6A,J,S with D,M,V). It
appears that in the KAAD-treated eyes, only the anterior-most pigmented cells,
including the pigmented ciliary epithelium, remained untransdifferentiated
(Fig. 6A,J,S). Eyes treated
with KAAD alone showed no regeneration from the CB/CMZ or from
transdifferentiation (not shown).
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In order to determine if the Shh pathway is playing a similar role during normal development, E3.5 eyes were injected with Rcas-Shh subretinally so that the viral infection was localized between the retina and RPE. Eyes were given approximately 12 hours for the virus to be expressed. At E4, an incision was made in the extraocular mesenchyme behind the RPE and an Fgf2-soaked bead was placed next to the RPE in both Rcas-Shh-injected eyes and control eyes that received no Rcas virus. When the eyes were examined two days later, 80% (n=5) of the Fgf2-treated eyes showed some transdifferentiation, whereas 80% (n=5) of control eyes showed some transdifferentiation, whereas the Rcas-Shh-treated eyes showed no transdifferentiation (n=6; Fig. 7A).
|
Eyes were collected 4 hours later and immunostained for the active form of ERK, phospho-ERK. ERK is activated through the Fgf pathway, thus ERK phosphorylation can be used to determine Fgf signaling activity. By examining phospho-ERK activation it is evident that the Fgf pathway is being stimulated by Fgf2, compared to the control, which did not have an Fgf2 bead (Fig. 7B). In eyes treated with KAAD and Fgf2, phospho-ERK immunoreactivity was similar to levels of phospho-ERK immunoreactivity with Fgf2 alone. It is very noticeable, however, that overexpression of Shh inhibits ERK phosphorylation, as almost no immunoreactivity was detected, suggesting it is having a negative effect on Fgf signaling (Fig. 7B).
These results indicate that the Hh pathway is involved in RPE maintenance by regulating Fgf2-stimulated signaling upstream of ERK in the RPE.
Shh activates stem/progenitor cells in the CB/CMZ via a Fgfr signaling pathway
Interestingly, we observed regeneration from the CB/CMZ region when Shh was
overexpressed and ectopic Fgf2 was absent. In 60% of eyes treated with
Rcas-Shh alone (n=15) there was robust regeneration from the CB/CMZ
(not shown; similar to that seen in Fig.
6G in one set of experiments and 89% in another set (n=9;
Fig. 7C), and no regeneration
via transdifferentiation. Variation in infectivity may help to explain why
only 60%-89% of Rcas-Shh-treated eyes were able to regenerate a retina.
To ensure that the Rcas virus was not contributing to regeneration, we infected eyes with Rcas-GFP after removing the retina. These eyes showed no regeneration (not shown).
Since we observed that Hh modulates the Fgf signaling pathway in the RPE,
and that Shh is sufficient to promote regeneration in the CB/CMZ, we wanted to
see if the Hh pathway was working through a Fgf pathway in the CB/CMZ. To do
this, we removed the retina from eyes at E4 and injected Rcas-Shh and added
PD173074, a potent Fgfr antagonist (Bansal
et al., 2003). As a control to ensure that PD173074 was having the
desired effect of antagonizing the Fgfr, we removed the retina at E4 and added
Fgf2 + PD173074. Controls were done for both experiments substituting DMSO for
PD173074. Eyes were allowed to regenerate for 3 days before being processed
for histology and assayed for regeneration. Eyes treated with Fgf2 + PD173074
showed a significant decrease in regeneration compared with controls
(
2, P<0.025), as only 33% (n=11) had
regenerating retina, indicating that PD170374 was efficiently antagonizing
Fgfr and inhibiting Fgf signaling. Rcas-Shh + PD173074 eyes also showed a
significant decrease compared with controls, with only 30% of eyes showing
regeneration from the CB/CMZ (n=10) (
2,
P<0.01). Rcas-Shh control eyes (with DMSO) showed approximately
89% regeneration from the CB/CMZ (n=9) and Fgf2 control eyes (with
DMSO) showed 100% regeneration from the CB/CMZ (n=4)
(Fig. 7C).
Shh controls ganglion cell populations during retina regeneration
When analyzing these treated eyes for retinal cell markers, all three
treatment groups, Rcas-Shh/Fgf2, KAAD/Fgf2 or Fgf2 alone, had patterns similar
to those of Fgf2-treated eyes at E7 (Fig.
3B1-C4), having a mostly undifferentiated neuroepithelium (data
not shown). By E11 and E15, both Rcas-Shh/Fgf2 and KAAD/Fgf2-treated eyes also
resembled Fgf2-treated eyes (Fig.
8A1-K3): all eyes were positive for cell markers for NFL, GCL,
amacrine cells, bipolar cells, horizontal cells, photoreceptors and RPE, (the
RPE is not present in panels showing transdifferentiation;
Fig. 8D3,E3,F3,J3,K3). One
recognizable difference between Rcas-Shh/Fgf2 and KAAD/Fgf2 treatments was
that the KAAD/Fgf2-treated eyes at E11 appeared to have more ganglion cells
than the Fgf2 eyes and Rcas-Shh/Fgf2-treated eyes appeared to have fewer
ganglion cells than the Fgf2 only-treated eyes (compare
Fig. 8A1,D1,B1,E1 and
C1,F1).
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Discussion |
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Transdifferentiation occurs when the RPE loses its characteristic pigmented
phenotype, dedifferentiates, proliferates and forms a neuroepithelium that
eventually gives rise to all of the layers of the neural retina. However, once
the RPE undergoes transdifferentiation it is lost in that area of the eye and
does not replenish itself. In vivo transdifferentiation does not take place
after about E4.5 (Coulombre and Coulombre,
1965) (data not shown). It has been well documented that signals
from both the surface ectoderm during eye induction and signals from the
extraocular mesenchyme are required for proper development of the RPE and
neural retina (Nguyen et al., 2000;
Fuhrmann et al., 2000
).
Specifically, the extraocular mesenchyme is required for induction and
maintenance of RPE-specific genes such as Mitf, Wnt13 (now known as
Wnt2b) and the matrix protein MMP115, which may help to repress
retina-specific genes (Fuhrmann et al.,
2000
; Jasoni et al.,
1999
). Reduction of Mitf expression is sufficient for
spontaneous transdifferentiation of the RPE into neural retina in vitro and in
vivo in the Quail Mitf mutant
(Mochii et al., 1998a
;
Mochii et al., 1998b
), and
overexpression of Mitf is able to cause a hyperpigmented phenotype in RPE
cells, as well as rendering the RPE cells unable to respond to Fgf2 in culture
(Mochii et al., 1998a
). Early
in eye development, when the optic cup is differentiating into retina and RPE
domains, Fgf1 and Fgf2 signals from the surface ectoderm are required
(Hyer et al., 1998
;
Pittack et al., 1997
) for
proper segmentation of retina and RPE. Studies on Fgf receptor type 1 (Fgfr1)
and Fgf receptor type 2 (Fgfr2) expression in the developing chick eye show
that the retina has the highest levels of these receptors at around E4, with
levels dropping off until about E18. Specifically, there is a trend in the RPE
for lower levels of Fgf receptor expression after E4. Expression that remains
in the eye after E4 tends to be highest in the ganglion cells and
photoreceptors (Tcheng et al.,
1994
; Ohuchi et al.,
1994
). Reduction of Fgf receptor expression in the RPE may help
explain why the RPE loses its ability to respond to Fgf2 and to
transdifferentiate in vivo after E4.5. It may be that since the RPE has the
ability to transdifferentiate in vivo until E4.5, there is a transitional
stage at which RPE must be responsive to the Fgf signals from the surface
ectoderm for proper development, while at the same time the RPE starts to lose
this responsiveness in order to become a fully committed and differentiated
RPE. It has also been reported that constitutive activation of the Fgf
downstream signaling molecule, MEK1, activates the MAPK signal transduction
molecule ERK and induces transdifferentiation of RPE to retina in developing
chick eyes. This transdifferentiation is correlated with a downregulation of
Mitf in the RPE caused by proteolysis stimulated by ERK phosphorylation of
Mitf (Galy et al., 2002
). Our
findings directly link Fgf signaling and regulation of RPE-specific factors,
and indicate that loss of Fgf responsiveness may be required for the RPE to
fully differentiate. In addition, Mochii et al.
(Mochii et al., 1998a
)
indicated that levels of Mitf are not solely responsible for regulation of
transdifferentiation. They show that there are higher concentrations of Mitf
present in the RPE at E5 than E9. However, E5 RPE is able to
transdifferentiate in vitro after only a short period in culture, whereas E9
RPE takes much longer. They suggest that the ability to transdifferentiate may
be regulated by the sensitivity of cells to extracellular signals.
A role for Hedgehog in RPE specification and differentiation in the regenerating retina
Our results indicate that the Hh pathway plays an important role in the
differentiation of the RPE. Inhibition of the Hh pathway using the potent
teratogen KAAD causes the transdifferentiation domain in the regenerating eye
to expand from the posterior to the anterior of the eye cup so that in most
cases only the anterior-most pigmented epithelium remains intact.
Additionally, ectopic expression of Shh inhibits transdifferentiation of the
RPE into neural retina (NR), and only in a few cases did we observe
transdifferentiation in these eyes. The transdifferentitation we did observe
in Rcas-Shh/Fgf2-treated eyes occurred in an Fgf2 concentration-dependent
manner, as only RPE closely associated with the source of Fgf2
transdifferentiated, most likely because these areas were in contact with the
highest concentration of Fgf. We also show that the RPE maintenance role of
Shh occurs during normal development. Our results support observations by
Perron et al. (Perron et al.,
2003), indicating that in Xenopus, Hedgehog signaling
plays a clear role in proximodistal axis specification and RPE
differentiation, and that inhibition of Hh signaling with cyclopamine causes
RPE differentiation defects.
The transcription factor Otx2 is able to regulate RPE differentiation
independently of Mitf, but has also been shown to bind and transactivate Mitf
(Martinez-Morales et al.,
2003). Zhang and Yang (Zhang
and Yang, 2001a
) have shown that ectopic Shh expression is able to
cause misexpression of Otx2. It is possible that downregulation of Otx2 by
inhibiting the Hh pathway is subsequently causing a loss of transactivation of
Mitf, leading to its downregulation. In addition, activation of the Fgf
pathway may be causing degradation of the Mitf protein. Combining this, with
the indirect Hh downregulation of Mitf transactivation may have an additive
effect for reducing Mitf protein and mRNA levels, explaining the enhanced
transdifferentiation domain observed in our experiments. Although further
studies are needed, our results suggest that the hedgehog pathway may play a
role in retina regeneration by negatively interacting with the Fgf pathway to
inhibit transdifferentiation, as is seen by our phospho-ERK immunostaining,
where Rcas-Shh decreased ERK activation.
The connection between the Hh pathway and Mitf is seen in Xenopus,
where it has been shown that inhibiting the Hh pathway also leads to a
decrease in Mitf expression in the RPE
(Perron et al., 2003). However
in that study there was no correlation between Otx2 regulation and Mitf
expression. Other studies have shown that Otx2 is negatively regulated by Fgf8
during development of the optic vesicle
(Crossley et al., 2001
), and
that when a bead soaked with Fgf8 is implanted into the extraocular mesenchyme
of a developing chick eye, it will induce transdifferentiation of the RPE into
retina (Vogel-Hopker, 2000). There is also a possibility that a downstream
target of Shh signaling may play a more direct role in regulation of the Fgf
signaling pathway.
Shh induced regeneration from the CB/CMZ is dependent on the Fgf signaling pathway
A specific role for Shh in stimulating and enhancing regeneration from the
CB/CMZ still needs to be elucidated. However, from our studies, it is clear
that Shh overexpression is sufficient to cause stem/progenitor cells to give
rise to a regenerated retina through the Fgf pathway. Our results show that
Rcas-Shh promoted regeneration from the CB/CMZ in 60% and 89% (two different
experiments) of eyes in the absence of Fgf2. It has been observed that Hh can
act through the Fgf pathway in different contexts. For example, Fgf2 or Shh
promote neocortical precursors to differentiate into oligodendrocyte
progenitors in culture. Fgf2-induced differentiation of neocortical precursors
is independent of the Hh pathway. However, Hh-stimulated differentiation is
dependent on constitutive activity of Fgfrs, which maintain a basal level of
active MAPK. Inhibition of Fgfrs in these necortical precursors inhibits
Shh-stimulated differentiation into oligodendrocyte precursors
(Kessaris et al., 2004). Our
experiments inhibiting Fgfrs using the potent Fgfr antagonist PD173074 show
that the mitogenic activity stimulated by Shh in the CB/CMZ is dependent on
Fgf receptor signaling, and may require basal Fgfr activation, as seen in the
Kessaris et al. study. Further experiments are needed to dissect the mechanism
by which Shh acts on the Fgf pathway to exert its effects.
Shh is sufficient to activate stem/progenitor cells in the CB/CMZ
Combined, our results indicate that Shh has at least two different
functions within the eye. The first is in RPE maintenance and is tied to the
Fgf pathway. The second is in regulating and stimulating regeneration from the
CB/CMZ. It has been demonstrated in Xenopus that Smoothened, Gli2 and
Gli3, downstream targets of the Shh pathway, are highly expressed in
stem/progenitor cells located in the CMZ and peripheral pigmented epithelium,
implicating the hedgehog pathway in stem/progenitor cell maintenance
(Perron et al., 2003). In
addition, recent studies in the post-hatch chick show that Shh is present in
the CMZ and that this region responds to ectopic Shh by proliferating (T. A.
Reh, personal communication). Also, mice with a single functional allele of
Ptc1 develop a CMZ-like structure not present in wild-type mice. This CMZ-like
region expresses genes present in the CMZ of lower vertebrates and responds to
injury by proliferating and giving rise to retinal neurons
(Moshiri and Reh, 2004
). These
studies, in combination with the results presented here, indicate that the Hh
pathway is essential for CB/CMZ development and maintenance and is probably
involved in proliferation and generation of new neurons from this area.
A role for Hedgehog in regulation of ganglion cells, during retina regeneration
It is known that Hedgehog signaling is responsible for driving a wave of
retinal differentiation in a variety of species. Drosophila Hedgehog
plays a crucial role in patterning the compound eye
(Greenwood and Struhl, 1999),
and vertebrates including zebrafish and chick require waves of Shh expression
for proper differentiation of retinal cell types
(Neumann and Nuesslein-Volhard,
2000
; Stenkamp et al.,
2000
; Zhang and Yang,
2001b
; Stenkamp and Frey,
2003
; Shkumatava et al.,
2004
). Specifically, Zhang and Yang
(Zhang and Yang, 2001b
) have
shown in the developing chick retina that Shh is required for proper ganglion
cell differentiation. Shh is needed to drive the differentiation wave of
ganglion cells from the posterior to the anterior, while at the same time,
ganglion cells that have already differentiated secrete Shh behind the wave of
differentiation in order to inhibit surrounding progenitor cells from
differentiating into more ganglion cells. Differentiation of progenitor cells
into ganglion cells is inhibited if levels of Shh are ectopically increased,
and ganglion cell production is increased if Shh levels are decreased or if
the Hh pathway is inhibited (Zhang and
Yang, 2001b
). Our results support these previous studies
indicating that overexpression of Shh inhibits ganglion cell production in the
regenerating retina, whereas inhibition of the Hh pathway increases ganglion
cell number.
Our tunnel assay results show that the decreased number of ganglion cells
in the Rcas-Shh-treated eyes are, in part, due to increased cell death. It has
been shown that Shh is able to inhibit progenitor cells from differentiating
into ganglion cells (Zhang and Yang,
2001b). One possible reason for the increased cell death is that
cells that do not differentiate may receive intrinsic or extrinsic cues to
undergo apoptosis. It is unlikely that the ectopic expression of Shh is
directly responsible for the increased apoptosis, since it has been shown that
in the absence of a Shh signal, ectopic Ptc1 is able to induce apoptosis in
the ventral spinal cord of embryonic chicks, and that a Shh signal is able to
prevent the cell death from occurring
(Thibert et al., 2003
).
It has recently been suggested that ganglion cell production ends in a
given region of the retina because of cell-cell interactions, and not because
of loss of competence by progenitors to produce ganglion cells
(Silva et al., 2003).
Interestingly, in that study ganglion cell generation was observed to be
complete by E11. Our studies showed that there is an increase in cell death in
the Rcas-Shh regenerating eyes at E11, supporting the idea that there is some
environmental cue that is responsible for initiating apoptosis in
undifferentiated cells. In fact, Mayordomo et al.
(Mayordomo et al., 2003
) have
shown that the ganglion cell population is modulated via a caspase 3-dependent
cell death during early retina development. In addition, we saw no statistical
difference between Fgf2- and KAAD-treated regenerating eyes, offering further
evidence that the Hh pathway is not directly responsible for the apoptosis
observed.
Conclusion
We demonstrate that retina regeneration by transdifferentiation as well as
by the activation of stem/progenitor cells from the CB/CMZ can give rise to
all the cell types of the retina. Furthermore, we show that Shh controls the
domain of transdifferentiation and plays a role in the ability of RPE cells to
transdifferentiate. This activity seems to be Fgf2 dependent. Shh is also able
to induce regeneration from the CB/CMZ, which requires Fgfr activity, as
antagonizing the receptor inhibits this type regeneration. Shh also seems to
control the number of ganglion cells that are produced in the regenerated
retina.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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