1 Program in Developmental Biology, The Research Institute, Hospital for Sick
Children, Toronto, Ontario M5G 1X8, Canada
2 Program in Genetics, The Research Institute, Hospital for Sick Children,
Toronto, Ontario M5G 1X8, Canada
3 Department of Molecular and Medical Genetics, University of Toronto, Toronto,
Ontario, Canada
4 Laboratory of Developmental Neurogenetics, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892,
USA
5 MRC Human Genetics Unit, Western General Hospital, Crewe Road South, Edinburgh
EH4 2XU, UK
6 Ottawa Health Research Institute, Ottawa, Ontario K1H 8L6, Canada
7 Department of Cellular and Molecular Medicine, and University of Ottawa Center
for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario K1H 8M5,
Canada
8 Department of Pediatrics, University of Toronto, Toronto, Ontario,
Canada
Author for correspondence (e-mail:
mcinnes{at}sickkids.ca)
Accepted 2 November 2004
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SUMMARY |
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Key words: Mouse, Mitf, Chx10, FGF, RPE, Neuroretina
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Introduction |
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The majority of studies examining RPE versus neuroretinal cell identity
decisions have focused on the ability of differentiated or developing RPE
cells to `transdifferentiate' into neuroretinal cells in response to external
signals (Reh and Pittack,
1995). The neuroretinal-inducing signal appears to come from the
surface ectoderm (Mikami,
1939
), suggesting a model in which surface ectodermal signals
induce the underlying OV neuroectoderm to become NR rather than RPE. Although
FGF has long been considered the surface ectodermal signal that induces
neuroretinal cell fate, recent evidence suggests that surface ectodermal FGFs
(or factors mimicking FGF signaling) pattern and organize the RPE and NR in
the OV, rather than inducing neuroretinal cells
(Hyer et al., 1998
;
Nguyen and Arnheiter, 2000
).
This model is consistent with the step-wise specification of neuroretinal and
RPE cells that begins early in embryogenesis, before the OV stage.
Of the regulatory molecules implicated in RPE cell specification, the basic
helix-loop-helix leucine-zipper transcription factor Mitf is the best
characterized. Mitf is considered to be crucial for the acquisition
and maintenance of RPE cell identity. In the mouse, Mitf is expressed
throughout the neuroectoderm of the E9.0 OV, and is subsequently downregulated
in the presumptive NR at E9.5 (Nguyen and
Arnheiter, 2000). However, Mitf expression continues in
the RPE and ciliary margin throughout early eye development. Mutations in
mouse Mitf result in microphthalmia, a lack of RPE cell
differentiation and an RPE-to-neuroretinal change in cell identity in the
dorsal region of the eye (Bumstead and
Barnstable, 2000
; Hodgkinson
et al., 1993
; Hughes et al.,
1993
; Nguyen and Arnheiter,
2000
). Other studies suggest that Mitf is involved in OV
organization and pattern formation in response to external signals.
Mitf expression is repressed by surface ectodermal FGF
(Nguyen and Arnheiter, 2000
),
and upregulated by activin signals from the mesenchyme surrounding the RPE
(Fuhrmann et al., 2000
). In
addition, Pax2, Pax6 (Baumer et
al., 2003
), Otx1, Otx2
(Martinez-Morales et al.,
2003
; Martinez-Morales et al.,
2001
), Gas1 (Lee et
al., 2001
) and Ap2a
(West-Mays et al., 1999
) are
necessary for RPE cell identity; Pax2, Pax6 and the Otx genes are
upstream of Mitf.
Apart from the role of FGF in regulating neuroretinal cell identity, little
is known about the mechanisms of neuroretinal specification. In the mouse,
ectopic expression of Ras (Zhao
et al., 2001) or Mek1
(Galy et al., 2002
) results in
RPE-to-NR changes in cell identity, presumably by mimicking the reception of
an FGF signal from the surface ectoderm. In addition, misexpression of the
early eye-specifying transcription factor Six6/Optx2 leads to an RPE-to-NR
transformation in chick (Toy et al.,
1998
). In the mouse, however, no loss-of-function studies have
identified a NR-specifying molecule.
We considered the Chx10 homeodomain transcription factor to be a strong
candidate regulator of neuroretinal specification or maintenance.
Chx10 is the earliest known gene to be expressed specifically in the
presumptive NR, beginning at E9.5 (Liu et
al., 1994). Mutations in mouse Chx10 result in
microphthalmia and a pronounced defect in the proliferation of neuroretinal
progenitor cells, but all the cell types of the mature retina are present,
except bipolar cells (Burmeister et al.,
1996
). A comparable phenotype is seen in humans
(Percin et al., 2000
).
Although Chx10 does not appear to be necessary for neuroretinal
specification, its early neuroretinal expression suggests that Chx10
may contribute to the acquisition or maintenance of neuroretinal identity.
To investigate the role of Chx10 in eye development, we examined
the expression of candidate Chx10 target genes in the developing eye of the
homozygous recessive Chx10or-J/or-J mouse
(Burmeister et al., 1996). One
potential target was the key eye development transcription factor gene
Mitf, because mutations in this gene, like mutations in
Chx10, are also associated with a small eye phenotype
(Hodgkinson et al., 1993
;
Hughes et al., 1993
). A
possible regulatory relationship between Chx10 and Mitf in
early stages of mouse eye development is also suggested by the fact that
Mitf expression in the presumptive NR (in the distal OV) is normally
extinguished at E9.5 (Nguyen and
Arnheiter, 2000
), at the time that Chx10 expression in
the distal OV is first observed (Liu et
al., 1994
).
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Materials and methods |
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Chx10or-J/+ mice were genotyped as previously described
(Burmeister et al., 1996).
Mitfmi/+ mice were genotyped by PCR amplification with
mouse Mitf intron 6 forward (5'-GGTGTGCCTCAGTCACTAATG-3')
and exon 7 reverse (5'-CTGGATCATTTGACTTGGGG-3') primers. Genotypes
were determined by heteroduplex analysis on a 9% acrylamide gel. Heterozygous
individuals yielded multiple bands instead of a single 200 bp band that arises
from homozygous normal or mutant mice.
In situ hybridization and immunofluorescence
Embryos were processed and sectioned, and in situ hybridization and
immunofluorescene were performed as previously described
(Chow et al., 2001).
Mitf, Tyr, Tyrp1, Dct, Math5 (Atoh7 - Mouse Genome
Informatics) and human MITF (see below) expression was identified
using mRNA from full-length cDNAs; Chx10 expression was examined
using a 3' UTR probe (Liu et al.,
1994). The localization of Mitf protein was determined using
rabbit anti-Mitf antibodies (Nguyen and
Arnheiter, 2000
) pre-adsorbed to mouse embryo powder (see below),
at a dilution of 1:25. Chx10, Pax6 and NCAM proteins were identified with
affinity-purified anti-Chx10 (Liu et al.,
1994
), anti-Pax6 (a gift from G. Mastick) and anti-NCAM (Santa
Cruz Biotechnology, Santa Cruz CA, H-300) rabbit polyclonal antibodies. The
final dilutions of these primary antibodies were 1:500, 1:500 and 1:100,
respectively. Islet1 protein was identified with an anti-Islet1 (Developmental
Studies Hybridoma Bank, University of Iowa; 39.4D5) mouse monoclonal antibody
at a dilution of 1:100. Notch1 was identified using a goat anti-Notch1 (C-20;
1:25; Santa Cruz Biotechnology, Santa Cruz, CA; sc-6014) polyclonal antibody.
Secondary AlexaFluor488-conjugated F(ab')2 fragment goat
anti-mouse IgG (Molecular Probes, Eugene, OR) and secondary Cy3-conjugated
goat anti-rabbit and anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA)
and donkey anti-goat antibodies (Jackson ImmunoResearch, West Grove, PA) were
used at a dilution of 1:100. Stained sections were examined on a LSM 510 Zeiss
confocal microscope and on a Nikon Eclipse E-1000 conventional upright
fluorescence microscope.
Anti-Mitf antibody was adsorbed on acetone-extracted E15.5 Mitfmi/+ embryo powder in phosphate-buffered saline (PBS) and stored in 10% goat serum, 50% glycerol, 0.05% sodium azide/PBS.
Hematoxylin and Eosin staining
To perform Hematoxylin and Eosin staining, paraffin wax-embedded sections
were deparaffinized in xylene and rehydrated in a graded ethanol series.
Sections were then immersed in Hematoxylin for 10 minutes, in tap water for 30
seconds and acid alcohol (0.5% HCl/70% ethanol) for 10 seconds. The slides
were then incubated in 0.5% Eosin/70% ethanol for 3 minutes, dehydrated in a
graded ethanol series, immersed in xylene and mounted in Permount (Fisher
Scientific).
NR-MITF transgenic mice
The NR-MITF construct consisted of the human PAX6
neuroretinal enhancer and minimal ß-globin promoter driving the
expression of the human M-MITF cDNA (without exon 6) followed by an
SV40 polyadenylation signal (see Fig. S1 in the supplementary material).
A human M isoform MITF (M-MITF) cDNA was obtained by
generating a hybrid EST clone (due to an EST mutation: 5' EST Accession
Number N36632, 3' EST Accession Number N34462). The 5' and
3' sequences were linked using a ClaI site at nucleotide 478 of
the M-MITF cDNA. The sequence starting at nucleotide 68 of the
M-MITF cDNA and extending to the poly-A tail (nucleotide 1900)
was cloned into pT3T7D (Pharmacia), thus creating the plasmid
M-MITF-pT3T7D. This M-MITF cDNA does not contain the
alternatively spliced exon 6. This plasmid was digested with AccI,
blunted and digested with HindIII.
As the putative neural retinal enhancer (NRE) of the human PAX6
gene is located in intron 4 (Kammandel et
al., 1999), a 6 kb EcoRI fragment encompassing exons 3, 4
and part of exon 5 of the PAX6 gene was identified and subcloned into
the pBluescript-SK (pBS-SK) cloning vector (Stratagene).
This fragment was then excised as a NotI fragment for subsequent
ligation into the NotI site of the p1229 reporter plasmid
that contains the minimal ß-globin promoter, the lacZ
gene and the polyadenylation sequence from SV40. Transgenic studies
demonstrated that this PAX6 enhancer is sufficient to direct
expression in the developing mouse neuroretinal epithelium as detected by
staining for lacZ expression (G.C.S., unpublished). The
ß-globin promoter from
PAX6-NRE-ßglobin-lacZ-SV40 was amplified from this
plasmid with the primers T7 (5'-GTAATACGACTCACTATAGGGC-3') and
lacZ5'Rev (5'-AAGTTGGGTAACGCCAGGG-3'). This PCR
product was digested with NcoI, blunted and digested with
SacII. The M-MITF cDNA and ß-globin fragments
were ligated into pBS-SK digested with SacII and
HindIII, to generate the plasmid BMpBS-SK. The SV40
polyadenylation sequence was amplified from
PAX6-NRE-ßglobin-lacZ-SV40 with the primers SV40Fwd
(5'-CCATCGATCCGGGCAGGCCATGTCTGC-3') and T3
(5'-AATTAACCCTCACTAAAGGG-3'). This PCR product was digested with
ClaI and HindIII and subcloned into pBSSK to
generate the plasmid SV40pBS-SK. SV40pBS-SK was digested with
HindIII and the liberated fragment was ligated into the
HindIII site of BMpBS-SK to generate BMSpBS-SK. The
PAX6-NRE-ßglobinlacZ-SV40 construct was digested with
NotI and SalI to remove all inserted sequences, which were
replaced with the NotI-SalI fragment from
BMSpBS-SK, thus generating the plasmid BMS. The 6 kb
PAX6-NRE was ligated into the NotI site of BMS to
generate the NR-MITF construct (see Fig. S1 in the supplementary
material). The NR-MITF insert was released with SalI prior
to microinjection.
The purified NR-MITF insert was injected into both C57BL/6;SJL (Hospital for Sick Children Transgenic Facility) and C57BL/6;C3H (Ottawa Health Research Institute) donors; germline-transmitting NR-MITF mice were identified using Southern blot analysis. EcoRI-digested genomic DNA was probed with radiolabeled full-length M-MITF cDNA; NR-MITF-positive individuals yielded 1 kb and 5 kb bands. Transgenic mice were also identified by PCR using primers in exon 3 (5'-CCCAGGCATGAACACACATTCAC-3') and exon 9 (5'-GTGCTCCGTCTCTTCCATGC-3'), resulting in a 1 kb product.
RPE-CHX10 transgenic mice
The RPE-CHX10 construct consisted of the mouse Dct
enhancer/promoter driving the expression of the human CHX10 cDNA (see
Fig. S1 in the supplementary material).
The Dct enhancer/promoter construct (pPdct) was a
generous gift from T. Hornyak. This construct contains 3.3 kb of sequence
upstream of the open reading frame of the mouse Dct gene in a
pcDNA vector backbone (Hornyak et
al., 2001
). To generate the human CHX10 cDNA construct, a
second XhoI site was inserted 3' to a 3.1 kb human
CHX10 cDNA in pBS-KS to generate CHX10-2Xho.
CHX10-2Xho was digested with XhoI to release the CHX10
cDNA, which was then ligated to XhoI-digested pPdct to
generate RPE-CHX10 (see Fig. S1 in the supplementary material). The
RPE-CHX10 insert was released with SalI prior to
microinjection.
The purified RPE-CHX10 insert was injected into C57BL/6;C3H donors (Ottawa Health Research Institute) and germline-transmitting mice were identified. RPE-CHX10 mice were genotyped using EcoRI-digested genomic Southern blots probed with a radiolabeled full-length human CHX10 insert. RPE-CHX10-positive individuals yielded a major 3 kb band, and minor undigested bands that varied with the individual transgenic mouse lines. RPE-CHX10 mice were also identified by PCR using primers located in the homeodomain (5'-GAAGAAGCGGCGACACAGG-3') and CVC domain (5'-CCTCCAGCGACTTTTTGTG-3'), resulting in a 350 bp product.
OV cultures
OV cultures and associated immunofluorescence were performed as previously
described (Nguyen and Arnheiter,
2000).
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Results |
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The eyes of mice either hemizygous (see Fig. S1 in the supplementary material) or homozygous (data not shown) for the NR-MITF transgene were normal. Thus, in a wild-type background, the transgenic expression of MITF in the developing NR failed to alter retinal cell identity. We then considered that this failure resulted from the activity of the wild-type Chx10 gene. We therefore increased the gene dosage of neuroretinal MITF:Chx10 by crossing mice carrying the NR-MITF/+ transgene to animals heterozygous or homozygous for mutant Chx10, and examined the resulting retinal phenotypes at P0. In animals hemi-or homozygous for the human MITF transgene and carrying one Chx10 wild-type allele (NR-MITF/+;Chx10or-J/+ and NR-MITF/NR-MITF;Chx10or-J/+), no changes were observed in the NR (data not shown). By contrast, the NR in NR-MITF/+;Chx10or-J/or-J individuals in two independent transgenic mouse lines changed dramatically, to a pigmented monolayer or PML (Fig. 4A-D). The PML resembled a mature RPE, not only in being a highly pigmented monolayer but also in not expressing the neuroretinal marker protein, Notch1 (see Fig. S2 in the supplementary material). By contrast, the cells of the PML were still partially neuroretinal in identity, because they expressed both the neural marker NCAM (Fig. 4E,F) and the neuroretinal protein Pax6 (Fig. 4G,H). In addition, the PML has not begun to differentiate into the earliest retinal cell type, the ganglion cell, as it did not express Islet1 (see Fig. S2 in the supplementary material).
|
We similarly found that the eyes and retinas of animals either hemizygous
(see Fig. S1 in the supplementary material) or homozygous (data not shown) for
the RPE-CHX10 transgene in a wild-type background were also normal.
Thus, either the absolute level of CHX10 expression achieved in the
developing RPE of wild-type mice, or the ratio of CHX10:Mitf gene
products, was insufficient to alter cell identity. To evaluate the latter
possibility, we increased the ratio of RPE-CHX10 transgene:wild-type
Mitf by crossing mice carrying the RPE-CHX10 transgene onto
mice heterozygous for a semi-dominant mutant allele of Mitf, the
mi allele. At P0, the retinas of
RPE-CHX10/+;Mitfmi/+ mice had a significant
decrease in the overall level of RPE pigmentation compared with
Mitfmi/+ mice (Fig.
4M,N). Most notable, however, was the transformation of the dorsal
RPE in RPE-CHX10/+;Mitfmi/+ animals to a
thickened multicellular layer (Fig.
4O-R). This thickened structure was a neuroretinal-like layer
(NRLL), as it expressed the progenitor markers Pax6, Rax, mouse
Chx10 (Fig. 4S-V) and
Notch1 (see Fig. S2 in the supplementary material) and had begun to
differentiate, as shown by the presence of Islet1-expressing cells (see Fig.
S2 in the supplementary material). The similarity of the NRLL to the
transdifferentiated neuroretinal phenotype found in the dorsal RPE of mice
with a severe loss of Mitf function is striking
(Nguyen and Arnheiter, 2000;
Packer, 1967
). All of the
above findings suggest that Mitf and Chx10 act
antagonistically in the developing eye, and that the levels of
Mitf:Chx10 gene products in the optic cup are major determinants of
retinal development.
Chx10 is necessary for neuroretinal cell identity
The modification by Mitf of the ability of Chx10 to
define the phenotype of the NR, together with the importance of genetic
background in the phenotype of the Chx10 mutant eye
(Bone-Larson et al., 2000;
Green et al., 2003
), suggested
that in some genetic backgrounds the failure to maintain neuroretinal identity
may result from the loss of Chx10 function alone. To evaluate this
possibility, we examined the eyes of P0 mice with a mixed
129/SvJ;C57Bl/6;SJL (
75%;20%;5%) mouse background that resulted
from crossing NR-MITF/+ and Chx10or-J/or-J
animals. The majority of Chx10or-J/or-J mice in this mixed
background had neuroretinas with a mixture of pigmented and non-pigmented
cells (a `salt and pepper' phenotype) (Fig.
5A,B), suggesting that the RPE and neuroretinal cells are properly
specified, but are incorrectly organized within the OV. Remarkably, however,
in a small fraction (
8%) of Chx10or-J/or-J mice with
this mixed background, the NR was replaced by a PML phenotype comparable with
the PML of NR-MITF/+;Chx10or-J/or-J animals
(Fig. 5C,D). Altogether, these
results establish, first, that Chx10 is required, at least in some
genetic contexts, not for the specification of neuroretinal cell identity, but
for neuroretinal organization and maintenance; and, second, our work
demonstrates that the level of expression of the MITF protein is critical in
transforming the NR to an RPE-like structure, as this phenotype is seen
infrequently in Chx10or-J/or-J mice, but in most
Chx10or-J/or-J mice carrying a MITF
transgene.
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Discussion |
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The absence of ectopic neuroretinal expression of the Mitf-regulated pigmentation enzyme transcripts in the Chx10or-J/or-J mouse may reflect low levels of Mitf expression, or to the absence of essential co-factors. The lack of ectopic Otx1 and Otx2 expression in the Chx10 mutant NR argues that Chx10 may specifically repress Mitf, rather than downregulating an entire RPE developmental program. However, the expansion of the expression domains of Mitf, Tyr, Tyrp1 and Dct in the ciliary margin of the Chx10or-J/or-J eye indicates that Chx10 is required not only to repress the neuroretinal expression of Mitf, but also to constrain the size of the presumptive ciliary margin. Thus, Chx10 and Mitf may regulate ciliary margin size in an antagonistic manner.
We confirmed and extended earlier findings
(Konyukhov and Sazhina, 1966)
that the expression of Mitf modifies the Chx10 mutant
phenotype and contributes to the failure of lamination and the hypocellularity
of the Chx10 mutant. We propose that the proliferation defects in the
Chx10or-J/or-J NR may result from the shift towards an RPE
cell identity, an identity normally associated with decreased proliferation
(at least compared to the NR) (Green et
al., 2003
; Packer,
1967
). The Chx10-mediated regulation of proliferation is mediated,
at least partially, through p27kip1
(Green et al., 2003
), and may
also be Mitf dependent. Consistent with a possible Mitf-dependent regulation
of p27kip1, p27kip1 is expressed in
the developing RPE (Defoe and Levine,
2003
) and Mitf directly stimulates p21 (a p27kip1
family member) (Goding et al.,
2004
).
We also demonstrated that Chx10 is required for the
RPE-hyperproliferation phenotype of the Mitfmi/mi mutant,
suggesting that Mitf may normally repress Chx10 activity in the
developing RPE. However, Chx10 is not ectopically expressed
throughout the entire RPE in Mitf mutants early in eye development
(data not shown), arguing that the repression of Chx10 activity in the
Mitfmi/mi RPE is not mediated by the repression of
Chx10 expression. Mitf may therefore repress neuroretinal cell
identity in the developing RPE by some other mechanisms that repress both
Chx10 activity indirectly and the function and/or expression of other
neuroretinal specification genes also expressed in the
Mitfmi/mi RPE (Nguyen
and Arnheiter, 2000). Finally, the relatively normal eye that
results from the Chx10:Mitf double mutant emphasizes that although
Chx10 and Mitf are essential for the maintenance of retinal
identity, other genes must also be involved in these processes and can at
least partially compensate for loss of Chx10 and Mitf.
Chx10 and Mitf function antagonistically in a pathway with FGF to regulate retinal cell maintenance
Through the use of transgene expression in Chx10 and Mitf
mutant mice, we established that Chx10 and Mitf act
antagonistically during development to regulate the acquisition and/or
maintenance of retinal cell identity. However, neither transgenic RPE
CHX10 nor neuroretinal MITF were sufficient to alter retinal
cell identity in a wild-type context. The levels of the NR-MITF and
RPE-CHX10 transgenes, their spatiotemporal expression patterns, or
the cellular environment may have limited the effects of the transgenes.
Although high levels of Mitf expression are sufficient to establish
pigment cell identity in some contexts
(Lister et al., 1999;
Planque et al., 2004
;
Planque et al., 1999
;
Tachibana et al., 1996
), the
levels of ectopic Mitf expression achieved in those studies (using
strong promoters) are likely to have been higher than those obtained in the NR
from the human PAX6 enhancer we used
(Kammandel et al., 1999
).
Similarly, the Dct promoter-enhancer RPE-CHX10 construct we
used was unable to change RPE identity in wild-type mice. This result
contrasts with the finding that a Dct promoter-enhancer construct
driving RPE Fgf9 or Ras conferred an RPE-to-NR change in a
wild-type background (Zhao and Overbeek,
1999
). The differences between these results may be due to
variations in transgene expression levels or to differences in the roles of
Chx10 versus Fgf9 and Ras in neuroretinal
development. For example, ectopic RPE Fgf9/Ras may mimic the combined
role of various FGFs or other ligands activating receptor tyrosine kinases
[e.g. epidermal growth factor (EGF), see below] that confer neuroretinal cell
identity.
We established that Chx10 is required for neuroretinal
maintenance, at least in some genetic backgrounds, as a PML instead of a
differentiated NR formed in Chx10or-J/or-J mutants. A
similar PML was observed in the great majority (74-100%) of
NR-MITF/+;Chx10or-J/or-J animals in which the dose of
MITF:Chx10 genes was high. In these transgenic mice the NR was
properly specified and initially normal in appearance, but after E11.5 it
transdifferentiated into a PML. We suggest that the transdifferentiation to a
PML of most of the NR-MITF/+;Chx10or-J/or-J neuroretinas
is likely to reflect the higher level of Mitf expression from the combined
neuroretinal expression of the endogenous Mitf gene and the
MITF transgene, in contrast to the formation of a PML in only a small
fraction (8%) of Chx10or-J/or-J mutants lacking the
NR-MITF transgene. Furthermore, the formation of a PML in the
majority of NR-MITF/+;Chx10or-J/or-J animals suggests that
the levels and/or activity of the Mitf pathway may be the major or sole
variable in the Chx10or-J/or-J genetic background that led
to the formation of a PML in some animals. The failure to obtain a fully
differentiated RPE from the transgenic expression of MITF may be due
to the fact that the Chx10or-J/or-J NR has been specified
before the onset of MITF expression from the transgene at E9.5
(Kammandel et al., 1999
). The
mixed RPE/neuroretinal cellular phenotype of the PML may be that of an OV-type
cell. Alternatively, it may indicate an additional role for Mitf and Chx10 in
the development of the ciliary margin, which forms the boundary of the NR and
RPE. In any case, these results provide strong evidence that Chx10
and Mitf act antagonistically in the regulation of retinal cell
identity.
The inability of FGF to change the RPE to a NR in
Chx10or-J/or-J optic vesicles indicates that Chx10
transduces the surface ectodermal FGF signal and represses Mitf in
the OV. Our proposal that FGF and Chx10 act within a single pathway
is supported by the similar neuroretinal phenotypes - a `salt and pepper'
mixture of pigmented (presumed RPE) cells and un-pigmented (presumed NR) cells
- observed in both the majority of Chx10or-J/or-J mutants
in a mixed genetic background and in developing chick eyes from which the
proposed FGF source (the surface ectoderm) was removed
(Hyer et al., 1998). That
cells of the OV lacking either Chx10 or an FGF signal appear to
become either NR or RPE cells further argues that the
FGF
|Chx10|Mitf pathway we identified is necessary for
neuroretinal maintenance rather than specification. The disorganization of the
neuroretinal and RPE cells in optic vesicles without FGF or Chx10
also highlights the need for this pathway in organizing and patterning the
already-specified NR and RPE in the developing OV.
The role of FGF in patterning and organizing the OV was defined by in vitro
loss-of-function studies (Hyer et al.,
1998; Nguyen and Arnheiter,
2000
). By contrast, FGF over-expression induces the
transdifferentiation of RPE into NR (Fig.
6B) (Hyer et al.,
1998
; Nguyen and Arnheiter,
2000
; Park and Hollenberg,
1989
). This contradiction may be resolved by a model in which FGFs
function redundantly in eye development, consistent with the lack of
phenotypes in Fgf1 and/or Fgf 2 mutants
(Dono et al., 1998
;
Miller et al., 2000
;
Ortega et al., 1998
). In this
model, some FGFs, including at least FGF1 and FGF2 from the surface ectoderm
(Nguyen and Arnheiter, 2000
)
organize and pattern the OV by activating Chx10. Subsequently,
Chx10 or other FGF-induced genes in the NR may then activate
Fgf8 (Vogel-Hopker et al.,
2000
), Fgf9 (Zhao et
al., 2001
) and/or Fgf15
(McWhirter et al., 1997
;
Thut et al., 2001
) to further
develop and maintain the NR. It is entirely conceivable, however, that FGF8,
FGF9 or FGF15 also act upstream of Chx10, as they will activate the same
signaling cascades as FGF1 or FGF2. Indeed, FGFs themselves may not be the
surface ectodermal signals that organize the OV. Rather, the FGFs may mimic
another surface ectodermal ligand by activating the FGF receptor tyrosine
kinase signaling pathway. However, no other known ligand is capable of causing
RPE-to-NR transdifferentiation (including EGF, insulin, NGFß, IGF1, IGFII
and TGFß1) (Nguyen and Arnheiter,
2000
; Park and Hollenberg,
1991
; Pittack et al.,
1991
), although EGF can substitute for FGF or surface ectoderm to
organize the OV (Nguyen and Arnheiter,
2000
). We suggest, therefore, that FGFs or other unidentified
molecule(s) organize the OV by activating Chx10, which then represses
Mitf.
The mechanisms regulating retinal cell identity
On the basis of our work, we propose that major function of Chx10 is to
prevent Mitf from imposing an RPE cell identity on the developing NR
(Fig. 7). Alternatively, Chx10
may also have pro-neuroretinal functions apart from the repression of
Mitf, and the balance of the Chx10-dependent neuroretinal and
Mitf-dependent RPE regulatory activities is a key determinant of retinal cell
identity. Although Chx10 and Mitf are essential for the
maintenance of retinal cell identity, it has been established that other genes
participate in regulating cell identity, including the early eye transcription
factor Six6/Optix (Toy et al.,
1998), FGF family members, Gas1
(Lee et al., 2001
) and
Ap2a (West-Mays et al.,
1999
).
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Cancer Research UK Edinburgh Oncology Unit and University
of Edinburgh Cancer Research Centre, Western General Hospital, Crewe Road
South, Edinburgh EH4 2XR, UK
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