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First published online 19 July 2006
doi: 10.1242/dev.02474
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Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
* Author for correspondence (e-mail: cepko{at}genetics.med.harvard.edu)
Accepted 5 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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Key words: Wnt2b, ß-catenin, Retina, Wnt reporter, Ciliary body, Iris
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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; Livesey and Cepko,
2001; Zaghloul et al.,
2005). The eye field comprises groups of cells biased to develop
into various eye structures. It is specified in the anterior neural plate, and
evaginates to form the OV, which in turn invaginates to form the OC. The OC is
composed of two epithelial layers, with the outer layer becoming the retinal
pigmented epithelium (RPE) and the inner layer becoming the neural retina (NR)
(Chow and Lang, 2001;
Martinez-Morales et al.,
2004). A third region, the peripheral rim of the OC, forms two of
the peripheral support tissues, the ciliary body and the iris
(Beebe, 1986). These tissues
are composed of a non-pigmented inner layer, which is continuous with the NR,
and a pigmented outer layer, which is continuous with the RPE. The ciliary
body is divided into two regions, a non-folded, pars plana, and a folded, pars
plicata. In addition to the cells originating from the OC, and thus the neural
tube, neural crest-derived mesenchymal cells contribute to the formation of
the ciliary folds, forming connective tissues and ciliary muscles
(Beebe, 1986). The ciliary body
produces aqueous humor, which is crucial for maintaining the intraocular
pressure of the anterior chamber, and regulates the shape of the lens,
allowing it to focus on objects at various distances. In addition, the
pigmented layer of the ciliary margin (PCM) appears to harbor adult retinal
stem cells (Ahmad et al., 2000;
Moshiri et al., 2004;
Tropepe et al., 2000). Cells
in the pupillary margin have another interesting feature. These cells undergo
a unique form of transdifferentiation when they lose their pigment and
differentiate into the smooth muscle cells of the pupillary sphincter
(Link and Nishi, 1998;
Volpe et al., 1993). Several
genes, encoding secreted proteins, transcription factors and extracellular
matrix proteins, have been shown to play a role in the development of the
ciliary body and iris (Dhawan and Beebe,
1994; Hsieh et al.,
2002; Kubota et al.,
2004; Pressman et al.,
2000; Thut et al.,
2001). However, the fundamental molecular mechanisms and timing of
the specification of these tissues are unknown.
Wnt signaling has been implicated in cell cycle control in some regions of
the central nervous system, through control of the cell cycle machinery
(Megason and McMahon, 2002).
In other tissues, Wnts also provide instructive signals for cell fate
specification. For example, Wnt1 in neural crest stem cells determines sensory
neural fate in a ß-catenin-dependent manner
(Lee et al., 2004). Transient
expression of the stabilized or constitutively active (CA) ß-catenin in
cortical progenitor cells using the enhancer element of Nestin
produced a folded cortex phenotype, resulting from excess cell proliferation
(Chenn and Walsh, 2002).
However, recent studies using Nestin::Cre lines to activate or inactivate
ß-catenin showed that canonical Wnt signaling is required for the
establishment of the dorsal identity of the pallium during forebrain
development (Backman et al.,
2005). If a primary role for canonical Wnt signaling in the
developing forebrain is patterning, proliferation might be a secondary
effect.
The expression of Wnt signaling genes in the OC has been characterized.
Wnt2b, Fz4 and Lef1, among others, are expressed in the peripheral part of the
OC and lens (Jasoni et al.,
1999; Kubo et al.,
2003). Based upon the expression pattern, and on studies of
function, several roles have been ascribed to Wnt2b in the eye. Wnt2b
signaling was proposed to regulate proliferation in the lens, based upon its
expression in the proliferative lens epithelium
(Jasoni et al., 1999). In
addition, Wnt2b was shown to have a role in the formation of laminae in the
retina (Nakagawa et al.,
2003). In Xenopus, Frizzled 5 is expressed within the
ciliary marginal zone (CMZ), flanking the region containing stem cells, and
regulates the transition of progenitor cells to neural precursors via the
activation of Sox2 (Van Raay et al.,
2005). Early morphogenesis, including eye-field formation, also
requires the coordination of Wnt/ß-catenin signaling, mediated by
multiple Wnt ligands and receptors
(Cavodeassi et al., 2005).
Using a clonal assay in retinal reaggregation cultures, along with
overexpression studies, it was proposed that Wnt2b plays a role in the
maintenance of retinal progenitor cells (RPCs) in the CMZ of the chick retina
(Kubo et al., 2003). However,
three observations suggest that the canonical Wnt signaling pathway is
involved in other aspects of eye development as well. One observation is that
in situ hybridization (ISH) for Wnt2b shows a dynamic expression pattern,
suggesting an involvement in several aspects of eye development. Wnt2b is
exclusively expressed within the dorsal surface ectoderm (SE) at the OV stage,
and in both the SE and RPE at the early OC stage. At later stages, Wnt2b is
expressed solely in the iris epithelium, both in the pigmented and
non-pigmented layers, but not in the RPE. The second is that activation of
ß-catenin did not promote retinal progenitor cell proliferation in an
explant experiment (Ouchi et al.,
2005). The third observation is that reporter assays indicate that
the highest level of signaling through the canonical Wnt pathway occurs in the
RPE and in the tip of the OC (Liu et al.,
2003). These two regions have relatively low proliferation rates
compared with the retina (Kubota et al.,
2004), which has a high proliferation rate, but low Wnt reporter
activity, in keeping with Beebe's observation of a low rate of proliferation
in the cells at the anterior rim of the OC
(Beebe, 1986). Similarly,
wg or constitutively active armadillo, the fly homolog of
ß-catenin, do not induce excess proliferation when overexpressed in the
developing Drosophila eye. Rather, they induce the formation of
peripheral tissues, such as the head cuticle or pigment rim, surrounding the
eye (Baonza and Freeman, 2002;
Tomlinson, 2003;
Treisman and Rubin, 1995).
In this study, we investigate the function of Wnt2b/ß-catenin in the
developing chick eye. In contrast to two previous reports of Wnt2b showing an
increase in the proliferation of retinal cells in vitro
(Kubo et al., 2003;
Kubo et al., 2005), we do not
find an increase in retinal cell proliferation when Wnt signaling is activated
in the retina in vivo. Instead, we found that CA-ß-catenin
(Funayama et al., 1995) or
Wnt2b interferes with the maintenance of retinal progenitor identity, and
leads to the conversion of retinal cells into the peripheral fates of the
ciliary body/iris. Furthermore, loss-of-function studies involving the
expression of DN-Lef1 or a fusion of Lef1 with the engrailed repressor
(Lef1-En) showed an inhibition of peripheral marker expression and iris
hypoplasia without affecting retinal tissues. Together, our results suggest
that the Wnt2b/ß-catenin pathway plays a crucial role in specifying the
peripheral fates of the eye, in keeping with the role of wg in the
Drosophila eye.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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) were
removed with HpaI and EcoRI, and the remaining vector
containing eight tandem repeats of Lef1/TCF-binding sites or mutated
Lef1/TCF-binding sites was ligated to a DNA fragment encoding AP. The
resulting constructs are named SuperTopAP and SuperFopAP, respectively.
Viral injection/in vivo electroporation and analysis of phenotypes
Fertilized eggs were obtained from SPAFAS (CT) and incubated at 37°C to
Hamilton-Hamburg (HH) stage 10 (Hamburger
and Hamilton, 1951). Viral injection (RCAS:CA-ß-catenin and
RCAS:Wnt2b) and plasmid electroporation were carried out as described
previously (Schulte et al.,
1999). Retinas with morphological phenotypes (CA-ß-catenin
induced a thin/folded phenotype and Wnt2b a minor thinning) at E5.5 and E7.5
were further analyzed for gene expression changes by in situ hybridization,
followed by anti-viral gag antibody staining to visualize the areas of viral
infection. The number of retinas showing gene expression changes (see Results)
was more than 80% of RCAS:CA-ß-catenin- and RCAS:Wnt2b-injected embryos
manifesting the morphological phenotypes described above. Gene expression
changes were correlated with viral infection in an individual animal, which
was possible as infection was not complete, and this was considered the best
internal control for effects of introduced genes. In addition, retinas
infected with RCAS or RCAS:GFP, or untreated retinas, were used as controls.
For electroporation (pMIW III:GFP+SuperTopAP±RCAS:CA-ß-catenin,
RCAS:DN-Lef1and RCAS:Lef1-En), the DNA concentration was 
400 to 800
ng/µl in PBS; 400 ng/µl of pMIW III:GFP plasmid was included to
visualize the area of transduction. Embryos were examined 24-48 hours after
electroporation for the presence of the GFP in or around the retina, and gene
expression changes (Collagen IX) and ciliary body/iris phenotype were analyzed
at E4.5 and E14-E16, respectively, using in situ hybridization, followed by
antibody staining (anti-GFP or anti-viral gag) to visualize the areas of viral
infection. Experimental protocols with embryos were approved by the
Institutional Animal Care and Use Committee at Harvard University.
Cell proliferation assay
E5 chick retinas were electroporated in a chamber containing 400 ng/µl
of pCAG:CA-ß-catenin DNA and 400 ng/µl of pCAG:GFP, and cultured for
16-18 hours in explant culture medium (10% FCS, 45% HAMS F12 nutrient, 45%
DME, 200 mM L-Glutamine, 10 mM Penicillin/Streptomycin, 10 mM Hepes). To label
dividing retinal progenitors in S-phase, 5 µl of [3H]thymidine
(NEN) was added to 1 ml of culture medium (final concentration of 5 µCi/ml)
6 hours prior to harvesting. The areas with GFP+ cells were cut out
under a fluorescence microscope (Leica Fluo III) and dissociated by trypsin or
papain. Autoradiography was carried out as described previously
(Morrow et al., 1998). For in
ovo labeling, 3 or 6 days after in ovo viral injection into the OV at stage
10, 0.3 ml of 5 mg/ml BrdU was injected to the yolk 30 minutes prior to
harvesting. Centrally located thin and folded areas were examined.
ISH and AP staining
Section ISH was performed as previously described
(Murtaugh et al., 2001).
Probes were labeled with digoxigenin (DIG), and AP-conjugated anti-DIG
antibody (1:2500, Roche) was used for detection with NBT/BCIP (5 mg/ml).
Probes used were Collagen IX (Dhawan and
Beebe, 1994), Bmp7 (Oh et al.,
1996), WFDC1 [J. Trimarchi and C.L.C., unpublished; ChEST719l20
(MRC geneservice)], Chx10 (Chen and Cepko,
2000), Notch1 (Austin et al.,
1995), Wnt2b (Jasoni et al.,
1999) and Lef1 (Kengaku et
al., 1998). For the Wnt reporter assay, staining for AP activity
was performed in NTMT, as described
(Murtaugh et al., 2001).
IHC and imaging
IHC was performed as described previously
(Dyer and Cepko, 2001). The
primary antibodies used were 
-BrdU (Sigma) at 1:200, -pH3
(Upstate Biotechnology) at 1:200, -ß-Tubulin III (Upstate
Biotechnology) at 1:250, 3C2 mAb (Developmental Studies Hybridoma Bank; DSHB)
at 1:50, -p27 (DSHB) at 1:200, -Collagen IX (DSHB) at 1:50,
-Pax6 (DSHB) at 1:30, -GFP (Molecular Probes) at 1:200,
-Visinin mAb (DSHB) at 1:30 and -SMA (Sigma) at 1:200. Secondary
antibodies used were Cy2- or Cy3-conjugated anti-mouse or anti-rabbit
antibodies (Jackson Immunoresearch Laboratories) at 1:250. For BrdU labeling,
sections were separately treated with 0.1% trypsin for 10 minutes and,
subsequently, with 2N HCl for 30 minutes, which destroys the fluorescence of
GFP. Also, IHC of -GFP was performed to detect the area of SuperTopAP
electroporation after AP reaction, which obscures or destroys the GFP
fluorescence. For nuclear counterstaining, DAPI (blue) was added to the final
wash solution at 0.0005%.
TUNEL assay
After antibody staining, retinal sections were subjected to fluorescent
detection using an in situ cell death detection fluorescence kit (Roche),
according to the manufacturer's instructions. Incubation was performed for 3
to 10 hours.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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). A recent observation that Wnt2b inhibits the expression of
Notch and proneural genes in the retina led to the proposal that Wnt2b keeps
RPCs undifferentiated in the peripheral OC
(Kubo et al., 2005). In order to assay whether canonical Wnt signaling promotes the proliferation of RPCs in an intact tissue, the proliferation of retinal cells in an explant, in the presence or absence of CA-ß-catenin, was assessed. Retinas were electroporated with a control GFP plasmid with the ubiquitous CAG promoter/enhancer (pCAG:GFP) only, or with a mixture of plasmids encoding CA-ß-catenin and GFP (pCAG:CA-ß-catenin+pCAG:GFP). When CA-ß-catenin was expressed in embryonic day 5.5 (E5.5) explants, the fraction of mitotic RPCs decreased compared with that of the GFP-expressing (or non-electroporated) control cells (25.2±2.4% to 10.7±3.5%, n=5; P=0.0001; Fig. 1A).
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)] was used to infect the OV at HH stage 10. Viral particles
were injected or viral DNA was electroporated into the OV, whereupon viral
expression and replication would initiate in infected or electroporated cells,
allowing the virus to spread throughout the eye by further infecting dividing
cells. Viral infection could be tracked by immunohistochemical (IHC) detection
of a viral gag antigen using antibodies such as 3C2 or -p27 gag
(Chen and Cepko, 2002).
Injection of bromodeoxyuridine (BrdU) was then carried out at later stages, 1
hour prior to harvest. The effects on cell proliferation were initially
assayed at E4.5, when patches of infected cells were relatively small,
indicating that viral infection occurred locally, and had not spread
throughout the eye. We did not observe any significant effect on retinal cell
proliferation, except in rare infected patches of cells that showed a partial
inhibition of retinal cell proliferation when compared with uninfected retinas
(see Fig. S1 in the supplementary material). The effects on proliferation were
also assayed later. At E7.5, a significant reduction in the number of
BrdU-labeled cells was observed in extensively infected regions. These
infected areas were thinner and folded, compared with RCAS:GFP-infected
control retinas (Fig. 1D-H,
Fig. 3). The percentage of
cells that incorporated BrdU in severely folded central retinas resulting from
CA-ß-catenin infection was 9.7±3.9% (n=4,
Fig. 1B,H), which was
significantly reduced relative to the central and peripheral parts of the
control retina [19.2±2.9% (n=4, P=0.01) and
30.3±2.8% (n=4, P=0.004), respectively;
Fig. 1B,D,E,G], but similar to
that of ciliary/iris epithelia from control retina (9.8±1.1%,
n=4, P=0.863; Fig.
1B,F,G). The fraction of cells in M-phase of the cell cycle also
was assessed using an -pH3 antibody. A similar reduction of the
fraction of cells labeled with -pH3 was seen in vivo in highly folded
areas of the RCAS:CA-ß-catenin-infected retina. The percentage of
-pH3-stained cells in the RCAS:CA-ß-catenin-infected, thin and
folded epithelium was 0.6±0.7% (n=4,
Fig. 1C,H), which is
significantly less than was observed in the central and peripheral parts of
the control retina [3.1±1.4% (n=4, P=0.017) and
4.5±0.6% (n=4, P=0.001), respectively;
Fig. 1C-E,G], but similar to
that of ciliary/iris epithelia from control retina (0.5±0.3%,
n=4, P=0.309; Fig.
1C,F,G). Consistent with the reduction of BrdU incorporation and
pH3 staining in vivo, the level of cyclin D1 RNA was decreased in
RCAS:CA-ß-catenin-infected retinas at E5.5 (see Fig. S1 in the
supplementary material).
Complementary expression of Wnt signaling and RPGs in the early OV and OC
The expression of Wnt signaling genes was compared with those of RPGs.
Wnt2b, the only identified Wnt in the peripheral OC
(Liu et al., 2003), was
expressed in the SE apposed specifically to the dorsal region of the OV
(Fig. 2A,B). In the early OC,
Wnt2b expression was seen in the presumptive RPE and peripheral rim
(Fig. 2C,D). This pattern was
maintained throughout the OC stages and later expression was confined to the
iris epithelium (see Fig. S1 in the supplementary material)
(Jasoni et al., 1999).
Similarly, Lef1 and Fz4 were weakly expressed in the dorsal region of the
invaginating OV (Fig. 2E; data
not shown). Later, they were expressed in the periphery of the OC, including
in the precursors of ciliary and iris epithelia
(Fig. 2G; Fig. S1 in the
supplementary material). Expression of Chx10
(Chen and Cepko, 2000) was
limited to the central and ventral areas of the invaginating OV, as its
expression was gradually reduced in the dorsal OV
(Fig. 2F) where Wnt signaling
genes, Lef1 and Fz4, were observed. At the OC stage and later, Chx10 was
highly expressed in mitotic RPCs in the more central regions, but weakly in
the periphery (Fig. 2H; Fig. S1
in the supplementary material). Other RPGs, such as Notch1
(Bao and Cepko, 1997) and
Cyclin D1, are expressed in mitotic RPCs at the OC stage (data not shown).
These data reveal complementary, but partially overlapping, expression of RPGs
and Wnt signaling genes at both the OV and OC stages.
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). However,
this study did not address when the Wnt signaling could first be detected. A
Wnt signaling reporter, SuperTopAP (Fig.
2I), was constructed to investigate this point in chicks.
SuperTopAP contains eight tandem TCF/Lef1-binding sites, and has alkaline
phosphatase (AP) as a reporter in place of the original luciferase gene of
SuperTopFlash (Veeman et al.,
2003). SuperTopAP was electroporated into the early OV at HH stage
(st.) 10 and the retinas were examined at a later OV stage (st. 13), or at an
early OC stage (st. 15). When examined at st. 13, strong Wnt reporter activity
was detected in the dorsoposterior OV, which will develop into the RPE and the
ciliary body/iris epithelia (n=9,
Fig. 2J). By contrast, the
portion of the anterior OV that will give rise to the NR, did not exhibit Wnt
signal activity. Similarly, 24 hours post-electroporation, Wnt reporter
activity was high in both the RPE and the tip of the peripheral OC, but was
very low in the adjacent central and inner OC (n=15,
Fig. 2K). However, there was
very weak reporter activity in the central OC, in some cells on the scleral
side of the NR where it abuts the RPE (Fig.
2K'), consistent with a previous report
(Liu et al., 2003). There was
an absence of Wnt activity in the ventral OV and OC, which is likely to be due
to the delayed development of the ventral retina relative to the dorsal
counterpart (Fig. 2J,K). As a
negative control, SuperFopAP (Fig.
2I) was made, in which the TCF/Lef1-binding sites were mutated, as
in SuperFopFlash (Veeman et al.,
2003). SuperFopAP gave no detectable signal in the developing
retina and RPE (n=12, Fig.
2L). In summary, the pattern of Wnt reporter activity is similar to the expression of Wnt2b and Lef1 at the OC stage. Both Wnt activity and Wnt2b expression were evident in the peripheral OC and RPE, whereas they were undetectable or very low in the NR-forming areas of the OV and OC.
Wnt signal activation induces thinning and extensive retinal folding
Reduced proliferation of RPCs upon Wnt signal activation might result from
an inhibition of the cell cycle in RPCs. Alternatively, a cell fate change,
from highly mitotic retina to that of less mitotic peripheral tissue
(Beebe, 1986;
Kubota et al., 2004), could
lead to a reduction in cell proliferation.
To further explore the in vivo role of Wnt signaling, a gain-of-function
study was carried out by expressing CA-ß-catenin or Wnt2b using a
replication-competent retrovirus (RCAS:CA-ß-catenin or RCAS:Wnt2b) in the
developing OV. The efficacy of CA-ß-catenin in the developing retina was
demonstrated through its ability to ectopically activate a SuperTopAP Wnt
reporter in the central retina, where normal Wnt activity is low or absent
(see Fig. S2 in the supplementary material; compare with
Fig. 2K). Eyes infected with
RCAS:CA-ß-catenin did not show any obvious superficial phenotypes at
E4.5. Retinal sections showed patches of infected cells, but the thickness of
the retinal epithelium was not severely affected (see Fig. S1 in the
supplementary material). A rare morphological change was the formation of a
kink at the scleral side of an infected patch. When the tissues were harvested
at E7.5 (after which animals die as a result of the spread of the virus), a
disorganized head with protruding eyes was observed (12/17; 71% of all
infected retinas, Fig. 3A,B).
The overall size of the affected eyes varied: sometimes they were slightly
bigger, but other times, they were similar or smaller than control eyes.
Examination of the infected eyes revealed small folds on the vitreal side of
the retina (Fig. 3C,D). Cross
sections revealed unusually thin areas, and folds, which are similar to the
normal morphology of the ciliary/iris epithelia
(Fig. 3E-I). In severely
affected eyes, the folded area of the retina had only one-cell layer, and
cells had a cuboidal shape, a characteristic of normal ciliary/iris epithelial
cells (Fig. 3J; Fig. S2 in the
supplementary material). Weaker phenotypes were observed when eyes were
harvested at E5.5 (Fig. 4A,B).
Expression of Wnt2b also caused a minor thinning of the retina at E5.5 and
E7.5 [Fig. 4C,D; the average
thickness of the control- and RCAS:Wnt2b-infected thinner retinas at E7.5 were
150 µm (n=7) and 82.4 µm (n=7), respectively;
P=0.007], but both expressivity and penetrance was always much lower
than that induced by CA-ß-catenin.
|
;
Chen and Cepko, 2000), was
examined. Infection with RCAS:CA-ß-catenin or RCAS:Wnt2b caused RPCs to
partially lose retinal progenitor characteristics in that both viruses led to
a partial to complete loss of the RPGs Chx10 (n=10,
Fig. 4A,C and Fig. S2 in the
supplementary material) and Notch1 (n=9,
Fig. 4B,D) at E5.5 and E7.5
(data not shown).
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Wnt signal activation induces peripheral markers
To investigate whether Wnt signaling can switch the NR to peripheral fates,
the expression of genes whose endogenous expression is enriched in the
peripheral OC and ciliary/iris epithelia was investigated. Collagen Type IX is
a marker of both the developing ciliary and iris epithelia at E7.5, although
the expression is weaker in the iris epithelium. Its expression starts around
E4, and is confined to the periphery of the eye at later stages
[Fig. 5A,B; see also Fig. S4 in
the supplementary material (Dhawan and
Beebe, 1994; Kubo et al.,
2003)]. Similarly, Bmp7, an essential factor for ciliary body
formation (Zhao et al., 2002),
is exclusively expressed in the periphery of the OC beginning at E6.5
(Fig. 5C,D).
The expression of peripheral markers in RCAS:CA-ß-catenin-infected
retinas was examined at E5.5 and E7.5. In the E5.5 central retina where
extensive folding had not yet occurred, activation of Wnt signaling led to
ectopic induction of Collagen IX and Bmp7 (n=8 and 5, respectively,
Fig. 5E-H), and a recently
discovered peripheral marker, WFDC1 (n=5,
Fig. 5I,J; J. Trimarchi and
C.L.C., unpublished). Induction of ectopic peripheral markers was confined to
the infected areas, but was not manifest in all infected cells of all infected
areas. At E7.5, when the thinning and folding of the central retina was
apparent, Collagen IX protein and RNA were ectopically induced in the thin and
folded regions of the affected retinas (data not shown). In addition, Wnt2b
and Lef1, genes expressed in precursor cells of the iris and ciliary body,
respectively, were induced in the thin or folded areas (n=3 and 3,
respectively, Fig. 5K,L).
Furthermore, the severely folded, thin central tissue expressed smooth muscle
specific actin (SMA; n=6, Fig.
5M,N), which is normally expressed in invaginating and migrating
iris epithelial cells (Zhao et al.,
2002).
Inhibition of Wnt signaling interferes with the development of the peripheral eye
In order to determine whether Wnt signaling is necessary for the
development of the peripheral eye, loss-of-function experiments were carried
out in vivo. Reduction of Wnt signaling was achieved by expression of
inhibitory molecules, Lef1-En, a fusion of Lef1 with the En repressor (G.
Kardon and C. Tabin, unpublished) or a dominant-negative allele of Lef1,
DN-Lef1, a deletion of the ß-catenin-binding domain
(Kengaku et al., 1998) (data
not shown). First, the effects of RCAS:Lef1-En on peripheral marker expression
were examined after in vivo electroporation. Collagen IX expression was
occasionally reduced at the periphery of the E4.5 retina when Lef1-En or
DN-Lef1 was expressed (2/8; 25%, Fig.
6A,B). However, the expression of Collagen IX was only
significantly reduced when viral infection was maximal, as judged by the
signal intensity of IHC for a viral gag protein
(Fig. 6A',B').
The effects of viral expression of Lef1-En on the maturing peripheral
tissues were examined at E14-E16, when the structures of the ciliary body and
iris are morphologically distinguishable. The majority of the
electroporated/infected eyes (13/17; 76% affected) exhibited a partial loss of
ithe ris (Fig. 6C). Sections of
the affected eyes revealed a localized hypoplasia of the iris and, to a lesser
extent, the ciliary body, compared with unaffected regions in the same eye
(Fig. 6D-F), which are
indistinguishable from RCAS:GFP-infected retinas (0/26; 0% affected, data not
shown). To further examine this phenotype, the development of iris-derived
muscles in the severely affected eyes was examined using an -SMA
antibody. Consistent with the morphological defects seen in
RCAS:Lef1-En-infected eyes at E14, a severe reduction of -SMA-positive
cells in the stromal layer of the developing iris was observed in severely
affected eyes (4/10; 40%, Fig.
6G) compared with the unaffected side (0/10; 0%,
Fig. 6H). RCAS:Lef1-En-infected
animals also had defects in RPE pigmentation. As shown in
Fig. 6I,J,
RCAS:Lef1-En-infected areas of the RPE exhibited hypopigmentation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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) also
reported a loss of retinal differentiation markers and a loss of Notch1
following the introduction of Wnt2b, but observed the induction of Chx10, Pax6
and Rx1 at E3.5.
The second line of evidence is the observation of expression of Collagen
IX, Bmp7 and WFDC1 following exposure to Wnt2b/CA-ß-catenin, as was also
seen by Kubo et al. (Kubo et al.,
2003). It is clear that Collagen IX is a marker of the ciliary
body, as it is expressed in the peripheral OC, in the ciliary epithelium
during mid-embryonic stages, and in the pars plana at later embryonic stages
(see Figs S4, S5 in the supplementary material). It is not clear whether
Collagen IX is also a marker of retinal CMZ stem cells, which is the
interpretation favored by Kubo et al.
(Kubo et al., 2003). Induction
of Bmp7 is consistent with a fate change, as it has been shown that Bmp
signaling is essential for ciliary body formation
(Zhao et al., 2002). We also
found induction of Wnt2b and Lef1, two additional markers of the periphery,
following the introduction of CA-ß-catenin. We also demonstrated that the
thin and folded tissue infected with CA-ß-catenin expresses smooth muscle
actin, which is normally expressed in invaginating iris epithelial cells that
transdifferentiate into smooth muscle cells in the stroma of the iris
(Fig. 5M). The expression of
all of these peripheral markers, including a distinctive iris marker, suggests
that the cells exposed to a high level of Wnt signal are induced to form the
ciliary body (expressing Collagen IX and Bmp7) and iris (expressing Wnt2b and
SMA). The third line of evidence is the morphology of retinal tissue infected
with CA-ß-catenin. It is thin and highly folded, resembling the
morphology of normal ciliary body/iris tissue. Although folding could be
secondary to overproliferation, we did not find evidence of overproliferation,
as discussed below. The fourth line of evidence is that abrogation of
Lef1-mediated transcription led to a loss of iris tissue in vivo, some
disorganization of the ciliary body, and a reduction of peripheral marker
expression. The partial decrease of Collagen IX expression by Lef1-En or
DN-Lef1 (Fig. 6A) is likely to
be due to the partial, not complete, loss of Lef1 activity in the peripheral
retina. Alternatively, Lef1-En-mediated partial inhibition of the
transcription of Collagen IX might only occur within the area most dependent
upon high Lef1 activity. This is the area where Wnt2b is maximal, at the tip
of the OC, in the region containing precursors of iris cells. The fact that
the loss-of-function experiments yielded data that are complementary to the
gain-of-function data strongly supports a model in which high Wnt signaling
induces the most peripheral eye fate(s).
|
; Rowan and Cepko,
2004).
Not all infected areas showed a robust phenotype when assayed for gene
expression changes and/or morphological changes. Instead, or in addition to
the slow kinetics noted above, the lack of a phenotype might be due to the
fact that there are Wnt antagonists, Sfrp1 and Sfrp2, in the retina
(Blackshaw et al., 2004;
Esteve et al., 2003). A high
level of Wnt signaling early when patterning is likely to occur might be
needed to cause a fate switch and this might not have been achieved in all
infected areas.
Kubo et al. (Kubo et al.,
2003; Kubo et al.,
2005) performed two studies in which they investigated the role of
Wnt2b signaling in the early chick retina. Cultures of dissociated and
reaggregated cells treated with Wnt2b displayed an increase in proliferation.
Similarly, using a Wnt2b-expressing retrovirus and in vitro cultures of
infected tissue, the authors found a dramatic increase in proliferation in the
presence of EGF and/or bFGF. As mentioned above, they also found a gain of
retinal progenitor markers, a loss of neuronal markers, and a gain of Collagen
IX expression. Although some of the results reported here confirm and extend
their findings, they also provide an alternative interpretation for some of
their observations. Kubo et al. (Kubo et
al., 2003) argue that expression of Collagen IX and
hyperproliferation are due to the acquisition of stem cell behavior. Cells at
the far periphery of the retina of amphibians and fish exhibit stem cell
characteristics in vivo, and cells isolated from the PCM of mammals can
generate neurospheres in vitro (Ahmad et
al., 2000; Moshiri et al.,
2004; Tropepe et al.,
2000). As Wnt2b-expressing cultured retinas demonstrate a high
degree of proliferation in vitro, it is possible that retinal cells exposed to
a high level of Wnt2b normally in the periphery in vivo are induced to form
stem cells. However, Kubo et al. (Kubo et
al., 2003; Kubo et al.,
2005) did not demonstrate the induction of proliferation in vivo,
so it could be that additional factors are needed for such an induction of
proliferation in vivo, and perhaps for an induction of stem cell properties.
We also examined the effect on proliferation at E4.5 and E7.5 after infection
with retroviruses expressing CA-ß-catenin and Wnt2b at the OV stage, and
did not find an increase in proliferation. The level of proliferation in vivo
in the infected central retinal areas was almost identical to that of the
normal ciliary/iris epithelia, which is lower than that normally found in
uninfected, central retina (Beebe,
1986; Kubota et al.,
2004). As Kubo et al. (Kubo et
al., 2005) clearly demonstrated a dramatic hyperproliferation in
vitro in response to Wnt2b, there might be an inhibition of this role of Wnt
in vivo, perhaps provided by surrounding ocular tissues. The Wnt antagonists
Sfrp1and Sfrp2 are expressed in the central retina
(Blackshaw et al., 2004;
Esteve et al., 2003).
Alternatively, it is possible that some other aspect of the in vitro culture
environment enhances proliferation in response to Wnt signaling.
|
;
Kubo et al., 2005) that may
account for our observations of additional phenotypes is our use of
CA-ß-catenin. We were able to produce phenotypes when Wnt2b was
overexpressed, and these were similar to, although weaker and less penetrant
than, those we obtained with CA-ß-catenin. This was expected for several
reasons. First, ectopic CA-ß-catenin does not require intracellular
signal transducers, as it functions at the last step of the signal
transduction cascade. Second, CA-ß-catenin can bypass the presence of Wnt
antagonists in the central retina. Third, because Wnt2b would take time to be
produced, secreted, received and transduced in order to affect transcriptional
targets, it is possible that high enough levels of Wnt signaling were not
achieved quickly enough to change fate following Wnt misexpression. By
contrast, ß-catenin would act immediately on its targets.
CA-ß-catenin is known to have effects on cell-cell adhesion. Although
we cannot completely exclude the possible involvement of CA-ß-catenin in
producing thin and folded phenotypes by affecting the adhesion
(Ouchi et al., 2005), several
lines of evidence support the interpretation that the observed phenotypes are
mainly caused by Wnt signal activation. First, we observed similar phenotypes
with both CA-ß-catenin and Wnt2b, as discussed above. Second,
gain-of-function phenotypes obtained with CA-ß-catenin and Wnt2b were
complementary to the loss-of-function phenotypes obtained with Lef1-En or
DN-Lef1. Third, the expression of CA-ß-catenin was sufficient to activate
a canonical Wnt reporter (see Fig. S2 in the supplementary material).
A recent study of the Hdac1 mutant of zebrafish found that
introduction of CA-ß-catenin in vivo did lead to hyperproliferation in
the central retina (Yamaguchi et al.,
2005). This finding might indicate that zebrafish, which have a
continuously proliferating ciliary marginal zone (CMZ), use Wnt signaling to
drive normal CMZ proliferation. Similarly, Xenopus frizzled 5 is
expressed in the domain-flanking stem cells in the CMZ, and is involved in
regulating proliferation and differentiation
(Van Raay et al., 2005). The
fact that there is rapid and ongoing conversion of CMZ cells to RPCs in fish
and amphibians throughout their lives might call for a regulation of this
conversion that is not needed in animals with only a transient, or no, CMZ,
i.e. in chick and mouse. If Wnt signaling is the regulator of this conversion
in fish and amphibians, perhaps some aspect of this role of Wnt is also
present in chicks, and it is this role that was revealed by the in vitro
behavior of chick retinal cells exposed to high Wnt2b levels
(Kubo et al., 2003;
Kubo et al., 2005).
Wnt signaling at the OV stage
Previous models of the patterning of the eye recognized the early division
of the OV into domains that would give rise to the outer OC and inner OC, and
thus the RPE and retina, respectively
(Chow and Lang, 2001;
Graw, 2003;
Martinez-Morales et al.,
2004). We are now suggesting that there is a third domain of the
OV, determined by high Wnt signaling, which will give rise to the peripheral
OC, and thus the ciliary body and iris. Expression of Wnt2b/Lef1/Fz4 and
canonical Wnt reporter activity had been previously observed at the OC stages,
but not at the OV stage (Jasoni et al.,
1999; Kubo et al.,
2003; Liu et al.,
2003). The sensitive reporter SuperTopAP allowed the observation
of Wnt activity at the OV stage. Both Wnt reporter activity and Lef1
expression were observed as early as the stage when the OV invaginates, when
Wnt2b is not expressed in the OV (Fig.
2A,J). Furthermore, within the invaginating OV, the expression
pattern of Lef1 is complementary to that of Chx10, an early retinal marker
(Fig. 2E,F). This observation
indicates that the initial blueprint for the patterning of the ciliary
body/iris via Wnt2b/ß-catenin signaling is present as early as the OV
stage. In addition to the source of Wnt2b in the SE and periphery of the OC,
Wnt2b is expressed in the early lens [Fig.
2D; Fig. S1 in the supplementary material
(Jasoni et al., 1999)].
Induction of the peripheral fates of the OC had been proposed to be dependent
upon signals from the lens (Thut et al.,
2001). Thus this previous model, and data presented here, are
consistent with the model that Wnt2b produced by the lens, the SE and the
peripheral OC coordinate to produce high Wnt signaling, and thus induce the
peripheral fates of the OC. Other factors, including unidentified Wnts
expressed in overlying mesoderm or neuroectoderm, might also contribute to the
Wnt signal activation in the OV. The latter possibility is supported by the
observation that extraocular mesenchyme possesses an activity that induces
Wnt2b in the RPE and represses RPGs in the NR
(Fuhrmann et al., 2000).
However, because Wnt2b has an expression pattern that is almost identical to
the Wnt reporter activity, it is likely to plays a major role in canonical Wnt
signaling at the OC stage (Fig.
2C,D,K).
A role for Wnt2b in RPE and extraocular development is suggested by the
Wnt2b expression pattern, which expands to the dorsal RPE and the tip of the
developing OC (Fig. 2C,D)
(Jasoni et al., 1999;
Kubo et al., 2003). A recent
study found that Wnt signal in periocular SE was required to suppress lens
formation in these cells, providing a mechanism for the division of the lens
from non-lens in the early SE (Smith et
al., 2005). Wnt2b may be the Wnt that is upstream of
ß-catenin for this pattern formation. In support of a role for Wnt
signaling in RPE development was our finding that RCAS:Lef1-En and
RCAS:DN-Lef1 led to a lack of pigmentation in the RPE at later stages of
development (Fig. 6I,J and data
not shown). This was of interest because retinal tissue was observed to
convert into RPE in a mouse mutant lacking Chx10
(Horsford et al., 2005;
Rowan and Cepko, 2004), and we
found that CA-ß-catenin introduction into the retina led to loss of Chx10
in the chick. In addition, it is known that ectopic expression of Chx10 in the
developing RPE causes a lack of pigmentation, and thus the hypopigmentation we
observed following the introduction of RCAS:Lef1-En and RCAS:DN-Lef1 might
have been caused by a de-repression of Chx10 in the RPE. It will be worth
examining RPE cells expressing DN-Lef1 at later stages of development to see
whether they express other retinal markers and eventually differentiate into
retinal cells.
Wnt/wg signaling in vertebrate and invertebrate eye development
Our findings provide an additional link between the development of the
vertebrate and invertebrate eye. In Drosophila, photoreceptor cells
are surrounded at the periphery with a non-neural cuticular structure.
wg, the Drosophila homolog of the Wnt genes, is expressed in
the margin of the eye imaginal disc, which is the anlage of peripheral eye
tissues. Activation of wg, or armadillo, the
Drosophila ß-catenin, in the eye imaginal disc promotes head
cuticle formation at the expense of ommatidia
(Baonza and Freeman, 2002), and
has been proposed to act as a morphogen to pattern the peripheral structures
(Tomlinson, 2003;
Treisman and Rubin, 1995).
Wnt signaling thus promotes the development of the non-neural, peripheral
support structures in both Drosophila and chicks. The similarity of
wg/Wnt expression and function in eye development provides an
additional line of evidence that strengthens the proposed evolutionary
conservation of the vertebrate and invertebrate eyes
(Fig. 7). The modern version of
this model originated with the observation of a conserved expression and
activity for the eyeless/Pax6 gene
(Gehring, 2002;
Gehring, 2004;
Halder et al., 1995;
Onuma et al., 2002). The fact
that wg/Wnt appears to play a role in patterning the central and
peripheral eye structures suggests that the visual structure of the last
common ancestor of flies and vertebrates had not only a photoreceptive
component, but a support structure as well. A conserved unit of neural and
non-neural eye tissues has also been suggested by the observation of a
single-celled dinoflagellate that has several of the support
structures of an eye, including pigment, a lens, a cornea and a photoreceptor
(Gehring, 2004). The fact that
Pax6 plays a role in the development of not only the NR, but also the
supporting tissues, such as the lens, cornea, iris and RPE, might also be seen
as being in keeping with this model.
An alternative interpretation of the use of Wnt/Wg for both vertebrate and
invertebrate eye patterning needs to be considered. Because Wnt pathway genes
are commonly used for many developmental processes, Wnt signaling in eye
development may only reflect the utility of Wnts in development, rather than a
homologous process (Fernald,
2004). The identification of additional genes that play a key role
in the patterning of these early eye domains, and establishment of a conserved
role for them, will be required to strengthen the model of a homologous
process for the establishment of neural and non-neural division of the early
eye.
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Supplementary material |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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