1 Department of Genetics and Howard Hughes Medical Institute, Harvard Medical
School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
2 Department of Pediatric Oncology, Dana Farber Cancer Institute, 44 Binney
Street, Boston, MA 02115, USA
Author for correspondence (e-mail:
cepko{at}genetics.med.harvard.edu)
Accepted 10 June 2004
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SUMMARY |
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Key words: BAC transgenic, Mitf, orJ, Fate mapping, Microarray, Compartments
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Introduction |
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Chx10, a paired-like homeodomain transcription factor, is expressed in the
presumptive neural retina, probably in response to inductive signals from the
prospective lens ectoderm (Liu et al.,
1994; Nguyen and Arnheiter,
2000
). In this respect, Chx10 is the earliest characterized
specific marker of retinal progenitor cells. Fibroblast growth factor (Fgf)
signals from the surface ectoderm may play a role in patterning the domains of
the optic vesicle and may be required for Chx10 expression in the presumptive
neural retina (Nguyen and Arnheiter,
2000
). In mouse, as well as the chick, Chx10 is expressed in what
appears to be nearly all retinal progenitor cells, but is absent from all of
the postmitotic cell types except for bipolar interneurons
(Belecky-Adams et al., 1997
;
Burmeister et al., 1996
;
Chen and Cepko, 2000
) and a
subset of Müller glia (Rowan and
Cepko, 2004
). Mutations in Chx10 cause the ocular
retardation phenotype in mice, including the orJ mutant,
which has a spontaneous mutation that leads to a premature stop codon and
failure to produce Chx10 protein
(Burmeister et al., 1996
). This
phenotype correlates with a reduction in proliferation of retinal progenitor
cells, especially those in the periphery of the retina
(Bone-Larson et al., 2000
;
Burmeister et al., 1996
). The
central retina does appear to undergo differentiation, but the retina
differentiates into a poorly laminated structure
(Bone-Larson et al., 2000
;
Burmeister et al., 1996
).
Molecular analysis has revealed the absence of bipolar cells in these retinas,
although all other cell types could be found
(Burmeister et al., 1996
).
Several peripheral structures of the eye are abnormal in
orJ mice, including the ciliary body and iris, and the
lens is cataracterous (Bone-Larson et al.,
2000
; Tropepe et al.,
2000
). The ciliary body appears to be expanded at the periphery of
the retina, but does not undergo proper morphogenesis. Chx10
expression has not been analyzed in these peripheral structures in the mouse.
In the chick, however, Chx10 has proven to be a useful marker of
cells in the peripheral retina and nonpigmented cells of the ciliary body
(Fischer and Reh, 2003
;
Kubo et al., 2003
).
It has been known for several decades that ocular retardation mutant mice
can show a range of variability in severity of the phenotype, predicting the
presence of genetic interactors (Osipov
and Vakhrusheva, 1983). In fact, genetic interactions between
ocular retardation mutants and a number of microphthalmia mutants, including
the fidget mouse and two different naturally occurring Mitf mutant
mice, microphthalmia (MitfMi) and white
(MitfWh), have been observed
(Koniukhov and Sazhina, 1985
;
Koniukhov and Ugol'kova, 1978
;
Koniukhov and Sazhina, 1966
).
In each case, the ocular retardation small eye and microphthalmia small eye
phenotypes were ameliorated in the double mutant animals. The genetic
interactions between Mitf and Chx10 mutants indicate a
functional antagonism between Mitf and Chx10, a supposition supported by their
mutually exclusive expression patterns. Genetic modifiers for
orJ mutants have been identified that affect the
proliferation of progenitor cells, as well as the lamination of the
differentiated retina. One set of modifiers was observed by crossing 129/Sv
mice, the original background of orJ mice, to Mus
musculus castaneus. These mice had intermediate-sized, well-laminated
retinas, but the peripheral retina remained undifferentiated and the ciliary
body did not form (Bone-Larson et al.,
2000
). A partially rescued orJ phenotype was
also observed in crosses with p27 nullizygous mice. These retinas
also were intermediate in size and had lamination, although they did not
generate bipolar cells (Green et al.,
2003
).
Mitf is a basic helix-loop-helix (bHLH)-Zip transcription factor that acts
as a master regulator of pigment cell development, and is expressed throughout
neural crest-derived melanocytes and the RPE
(Hodgkinson et al., 1993).
Mitf is initially expressed throughout the optic vesicle, but its
expression is rapidly restricted to the proximal part of the optic vesicle,
the presumptive RPE (Bora et al.,
1998
; Nguyen and Arnheiter,
2000
). The restriction of Mitf expression both spatially
and temporally correlates with induction of Chx10 expression in the
presumptive neural retina. While Mitf is not required for specification of the
RPE, it is essential for its differentiation and maintenance
(Bumsted and Barnstable, 2000
;
Nakayama et al., 1998
;
Nguyen and Arnheiter, 2000
). A
regulatory pathway upstream of Mitf expression has been characterized
involving redundant activities of Pax6 and Pax2 as well as Otx1 and Otx2
(Baumer et al., 2003
;
Martinez-Morales et al., 2003
;
Martinez-Morales et al.,
2001
), but the transcriptional pathway leading to repression of
Mitf within the distal optic vesicle has not been identified. It is possible
that either signaling upstream of Chx10 or Chx10 itself is
responsible for the downregulation of Mitf in this domain.
A fascinating feature of the developing RPE is its ability to
transdifferentiate into retinal cells under the appropriate circumstances.
This has been observed following the addition of Fgf to RPE in vivo or in
culture (Guillemot and Cepko,
1992; Park and Hollenberg,
1989
; Pittack et al.,
1991
). Most studies have focused on chick RPE, which maintains the
ability to transdifferentiate at later embryonic stages. Rodent RPE has the
ability to transdifferentiate into retina in culture, but this capacity is
lost relatively early in development
(Nguyen and Arnheiter, 2000
;
Zhao et al., 1995
). Transgenic
mice have been generated that overexpress Fgf9 in developing RPE, and these
mice show dramatic RPE to retina transdifferentiation
(Zhao et al., 2001
).
Mitf mutants show spontaneous transdifferentiation of dorsal RPE
(Bumsted and Barnstable, 2000
;
Nguyen and Arnheiter, 2000
),
while mice lacking a cell cycle inhibitor, Gas1, have ventral RPE
transdifferentiation (Lee et al.,
2001
). Mitf function appears to be a key target in
transdifferentiation. In the chick, forced expression of Mitf
prevents RPE transdifferentiation in the presence of Fgf, although on its own,
Mitf does not cause retinal effects
(Mochii et al., 1998
).
Mutation or overexpression of factors thought to be upstream of Mitf,
such as Pax2 and Pax6, have phenotypes consistent with their action being
mediated by Mitf. Thus, Mitf appears to function in part as a safeguard
against transdifferentiation of the RPE into the retina.
A full understanding of the role of Chx10 in retinal development would
require understanding its biochemical functions and the genes it regulates. To
date this has been lacking. Two candidate Chx10 target genes have been
identified. One gene, Foxn4, is co-expressed in progenitor cells with
Chx10, and is highly downregulated in the orJ
mutant (Gouge et al., 2001).
Another candidate gene is cyclin D1, whose RNA levels are
significantly decreased in the orJ mice, possibly leading
to deregulation of p27 (Green et
al., 2003
). In neither case is it known if these effects reflect
direct regulation by Chx10. To further our understanding of the function of
Chx10 and how it might act through downstream targets, we characterized the
fate of cells within the Chx10-expressing domain in
orJ mutant mice. In addition, we present a microarray
analysis of orJ mutant versus wild-type retinas. We show
that several genes are upregulated in the orJ mutant,
including those associated with peripheral retina development and those
controlling pigmentation. Examination of the developing periphery of
orJ retinas revealed the progressive expansion of
pigmented cells towards the center of the retina at the expense of retinal
progenitor cells. Using a multifunctional Chx10 BAC transgenic mouse
reporter, we demonstrate that these pigmented cells transdifferentiated from
retinal cells. Peripheral markers also expand centrally in the
orJ retinas, suggesting a new function for Chx10 in
maintaining boundaries in the peripheral retina. Finally, we show that ectopic
Chx10 in the developing chick RPE causes lack of pigmentation of the
RPE, perhaps through direct repression of Mitf. These studies link Chx10 and
Mitf together as critical determinants in maintenance of the retina and RPE
respectively.
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Materials and methods |
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Immunohistochemistry and X-gal histochemistry
For antibody staining, cryosections were prepared and stained as described
previously (Chen and Cepko,
2002). Primary antibodies used were 1:1000 rabbit anti-GFP
(Molecular Probes) and 1:500 rabbit anti-ß-gal (5'3').
Secondary antibodies used were 1:250 Cy2- or Cy3-conjugated goat anti-rabbit
(Jackson Immunologicals). Following antibody staining,
4',6-diamidino-2-phenylindole (DAPI) was applied to stain nuclei
(Sigma), and the sections were coverslipped and mounted in Gel/Mount
(Biomeda). Dissociated cells were prepared by removing the lens and cornea
from E17.5 orJ eyes and dissociating the remaining tissue
using papain (Worthington) as described previously
(Chen and Cepko, 2002
). Tissue
sections or dissociated cells were stained for ß-galactosidase using
standard methods (Kwan et al.,
2001
). Tissue was stained overnight at 37°C, washed in PBS,
coverslipped, and mounted in Gelvatol (Air Products).
Microarray analysis
Retinal RNA samples were isolated from E12.5 or E13.5
orJ mutant embryos using the Trizol reagent (Gibco).
Retinal RNA samples of E13.5 wild-type 129/Sv or E12.5 Swiss Webster were used
as comparisons. Each RNA sample was pooled from an entire litter. Care was
taken to avoid contamination from the RPE and lens. 0.3-0.7 µg of total RNA
was RT-PCR amplified for 14 to 18 cycles using the SMART amplification kit
according to the manufacturer's instructions (Clontech). PCR-amplified cDNA
from each experiment and control pair was Klenow labeled with Cy5 and Cy3,
respectively, and color swapped with Cy3 and Cy5, respectively as described
(Livesey et al., 2000).
Labeled probe was then hybridized to microarray slides spotted with 11,500
cDNA clones from the brain molecular anatomy project (BMAP) library (kind gift
of B. Soares, University of Iowa, see
http://trans.nih.gov/bmap/index.htm
for details) and 500 cDNA clones of known identity from our lab collection
(list available by request). Slides were printed and hybridized as described
(Dyer et al., 2003
;
Livesey et al., 2004
). Slides
were then scanned on an Axon Instruments GenePix 4000 scanner and images were
analyzed using the GenePix Pro software package. Gene expression data were
uploaded to the AMAD database for data management and filtering. Gene
expression ratios were normalized after filtering the data to remove
low-intensity and poor-quality spots.
In situ hybridization
Section in situ hybridization was performed as previously described
(Murtaugh et al., 1999) using
20 µm cryosections from OCT-embedded tissue. Whole-mount in situ
hybridization was performed as previously described
(Chen and Cepko, 2000
).
Riboprobes labeled with DIG were detected with NBT/BCIP (Sigma). Riboprobes,
gene nomenclature, and relevant references are described in Table S1
(supplementary material).
Cloning of a partial chick Tfec cDNA
Total RNA from E4 chicks was isolated using the Trizol reagent according to
the manufacturer's instructions (Life Technologies) and cDNA was generated
using superscript II reverse transcriptase (Life Technologies). cDNA was
amplified using degenerate primers to the Mitf family bHLH domain as
described elsewhere (Rehli et al.,
1999). PCR products were subcloned and sequenced to determine
their identity; 3/16 colonies coded for a bHLH domain that shared higher
homology to Tfec orthologs than Mitf. The remaining clones
encoded Mitf. 5' RACE was performed using the Marathon cDNA
Amplification Kit (Clontech) with the following gene-specific primer:
5'-TCACAGCAGATACGCGGAGCAATGG-3' to obtain a partial chick
Tfec cDNA, which was subcloned into pCR2.1 (Invitrogen) and
sequenced. This sequence has been submitted to Genbank as accession number
AY502941. Using available EST data, a full-length chick Tfec cDNA was
compiled and the nucleotide sequence data are available in the Third Party
Annotation Section of the DDBJ/EMBL/GenBank databases under the accession
number TPA: BK004078
In ovo electroporation
Electroporations were performed by injecting 0.5 µg of
pMiw-Chx10 (1.3 Kb EcoRI/KpnI coding sequence
fragment) along with 0.25 µg pMiw-GFP expression vector
(Schulte et al., 1999) into
the right optic vesicle of HH stage 9-10 chick embryos as described previously
(Chen and Cepko, 2002
). Embryos
were harvested at stage 15/16. Electroporated embryos with normal eye sizes
and strong GFP fluorescence (visualized on a Leica MZFLIII dissecting
microscope) were further analyzed by whole-mount in situ hybridization
analysis.
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Results |
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To better illustrate the pattern of neopigmentation, orJ mutant Chx10 BAC transgenic animals were immunofluorescently stained for GFP at P0 (Fig. 2K) or P17 (Fig. 2M). In both cases, regions of mostly non-pigmented neuroepithelium included some lightly pigmented cells (Fig. 2J,L arrows). These regions expressed high amounts of GFP (Fig. 2K,M), demonstrating that they expressed Chx10. Furthermore, GFP-positive nuclei could be observed surrounded by pigment granules. At P1, the newly transdifferentiating region was still peripherally located within the eye, whereas by P17 it was more central, illustrating the progressive feature of transdifferentiation in the orJ eye.
In order to unambiguously determine whether ß-gal- or GFP-positive cells were pigmented, as well as quantify the extent of transdifferentiation, E17.5 Chx10 BAC/R26R double transgenic orJ eyes were dissociated and immunofluorescently or histochemically stained for ß-gal (Fig. 2N,R,S) or GFP (Fig. 2P). Examination of ß-gal-positive cells under Nomarksi illumination revealed that many of these cells (59/100) were pigmented, with some of them heavily pigmented (Fig. 2N, arrowheads). Similar analysis of GFP-positive cells also indicated that a significant, but smaller number of cells (18/100), contained pigment granules. These GFP-positive pigmented cells were more lightly pigmented than typical pigmented cells (Fig. 2Q arrowhead and insert) and were probably cells in the process of transdifferentiation. Histochemical staining of dissociated cells for ß-gal also revealed cells that were clearly pigmented and expressing ß-gal (Fig. 2R,S).
Identification of genes affected in the orJ mutant
To identify genes affected in the orJ mutant, a
microarray analysis was performed comparing wild type to
orJ retinas. RNA samples were compared at E12.5 or E13.5
in development, very early time points following the onset of Chx10
expression. At this time, the difference in eye size is small and
transdifferentiation is not observed (data not shown).
Table 1 shows the genes that
were upregulated greater than 1.7-fold in the orJ versus
wild-type retinas and Table 2
shows the genes that were downregulated 0.5-fold compared to wild-type
retinas. These cutoffs were arbitrarily determined, and a number of genes with
ratios below the cutoffs showed expression differences as analyzed by in situ
hybridization (see below). A more complete list of putatively affected genes
is shown in Tables S2 and S3 (supplementary material).
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Table 1 also shows a number
of genes evaluated by in situ hybridization (in bold). The genes that were
tested by in situ hybridization from the array list were verified to be
differentially expressed in the orJ mutant (see below).
One particular gene, Crhbp, evaluated because of a known expression
pattern in the peripheral retina (Blackshaw
et al., 2004), was verified to be upregulated in the
orJ mutant, even though the fold upregulation by
microarray analysis was 1.29. This suggested that a high percentage of the
genes identified by microarray analysis might be significantly differentially
expressed in the orJ mutant.
Downregulated genes (Table
2) with known functions fitted into a more definite pattern than
upregulated genes. The bHLH transcription factors, Neurod1 and
Math3, are required for neuronal differentiation and were highly
downregulated. Several other genes found to be downregulated corresponded to
structural genes and known markers (e.g. Gap43 and Snap25)
of neurons. In situ hybridization analysis of a number of genes identified in
Table 2 showed significant
downregulation of these genes (see below). Two markers of cell cycle
progression, cyclin D1 and E2f1, were downregulated
according to the microarray analysis, although less so than the cutoff
assigned to Table 2. Cyclin D1
has previously been shown to be downregulated in orJ
retinas (Green et al.,
2003).
In situ hybridization analysis of affected genes
In order to verify that some genes identified as differentially regulated
by microarray analysis were changed in expression level, and to better
identify genes that may play a role in transdifferentiation, in situ
hybridization analysis was performed (Fig.
3; see Fig. S1 in the supplementary material). Several additional
genes not present on the microarray were analyzed because of their previously
characterized expression patterns. In situ hybridizations were performed at
E14.5, when early phenotypes were apparent, and at E17.5, when
transdifferentiation was at an intermediate stage (see
Fig. 1).
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Most genes analyzed with ONBL expression patterns were not expressed in the zones of the peripheral eye in orJ/+ mice, or were only expressed transiently in the peripheral retina (E2f1, cyclin D1 and cyclin E1) with the exception of Chx10. To determine whether Chx10 was differentially regulated in orJ mutants from other ONBL-expressed genes because of its peripheral expression, Hes1, a gene expressed similarly to Chx10 was analyzed (see Fig. S1I in the supplementary material). Hes1 expression in the orJ mutant behaved like that of Chx10 rather than other ONBL-expressed genes (see Fig. S1I in the supplementary material), suggesting that peripheral gene expression was differentially affected in the orJ mutant.
Class II: INBL- and INBL+ONBL-expressed genes
The loss of expression of genes enriched in retinal progenitor cells, as
well as genes that play a role in neurogenesis, suggested that the genesis of
retinal neurons might be affected in orJ mutant retinas.
Since a number of genes found by microarray analysis to be downregulated in
orJ mutants were markers of early-born neurons, we used in
situ hybridization to examine genes expressed in the INBL. Gap43,
Brn3b (Pou4f2 Mouse Genome Informatics) and
Snap25 were expressed in the INBL at E14.5 and E17.5 in
orJ/+ heterozygotes
(Fig. 3C and Fig. S1J,K in the
supplementary material). Gap43 was restricted to only a few cells in
the INBL in the central retina in orJ mutant retinas
(Fig. 3C). Similar results were
obtained for Brn3b and Snap25 (see Fig. S1J,K in the
supplementary material). This decrease in the number of cells expressing
markers of retinal ganglion cells and amacrine cells correlated with the
restricted location and reduced number of cells expressing ONBL markers, and
was further indicated by the finding of a hypocellular INBL in
orJ mutant retinas.
Stathmin 1, one of three stathmin family member genes observed by microarray analysis to be downregulated, was evaluated by in situ hybridization. In orJ/+ eyes, stathmin 1 was expressed throughout the INBL and in many ONBL cells at E14.5 and E17.5 (see Fig. S1L in the supplementary material). In orJ mutant retinas, stathmin 1 was expressed in a similar fashion to the combination of Gap43/Brn3b and Neurod1, and in a much-reduced number of cells, which was especially apparent at E17.5 (see Fig. S1L in the supplementary material). The paired box transcription factor Pax6 was expressed throughout the ONBL and INBL, and at high levels in zones 1, 2 and 3 (see Fig. S1M in the supplementary material). Unlike stathmin 1, Pax6 appeared slightly upregulated in orJ mutant retinas and persisted even in pigmented regions. These data, like the comparison of Chx10 and Hes1 with other ONBL-expressed genes, suggest that the peripheral component of Pax6 may have contributed to the high expression level of Pax6 in the orJ mutant retina.
Class III: Peripherally-expressed genes
Gas1 and p57 were both found to be significantly
upregulated in the orJ mutant microarray analysis. In situ
detection of Gas1 at E14.5 in orJ/+ eyes showed
expression exclusively in the peripheral retina in zones 1 and 2, but not in
pigmented cells (Fig. 3D). At
E17.5, it remained highly expressed in these zones and was also expressed at
low levels in the ONBL in orJ/+ eyes. In
orJ eyes, Gas1 was highly upregulated in the
peripheral retina and expressed at lower levels in more central parts
(Fig. 3D). At E17.5,
Gas1 was highly upregulated throughout the entire
orJ retina. Another gene also functioning as a cell cycle
inhibitor, p57, was highly expressed in the peripheral retina and
showed a similar expression pattern to Gas1 in
orJ/+ and orJ eyes (see Fig. S1N in
the supplementary material). A gene not represented on the microarrays, but
with known important roles in ciliary body development, Otx1, was
analyzed by in situ hybridization, and it too showed an expression pattern in
orJ/+ and orJ eyes highly similar to
Gas1 and p57 (see Fig. S1O in the supplementary
material).
To address the role of gene expression specific to zone 1, Wfdc1, a potential tumor suppressor gene and a peripheral retinal marker (J. Trimarchi and C.L.C., unpublished), was analyzed by in situ hybridization (Fig. 3E). The limits of Wfdc1 expression defined zone 1 (Fig. 3E, arrows). In the orJ retina at E14.5, Wfdc1 expression levels were not significantly altered, but its expression was expanded centrally. At E17.5, Wfdc1 expression was observed in some cells in the center of the orJ retina and throughout peripheral parts of the retina that had not yet undergone transdifferentiation (Fig. 3E, inset). Another peripherally-expressed gene, Crhbp, showed essentially the same expression pattern (see Fig. S1P in the supplementary material). Crhbp showed a small but significant upregulation by microarray analysis. This upregulation was prominent at E13.5 but not at E12.5, and like the in situ hybridization analysis, indicated a slow expansion of zone 1. A third marker of zone 1, Igf2 (J. Trimarchi and C.L.C., unpublished), was altered in the orJ retina like Wfdc1 and Crhbp (see Fig. S1Q in the supplementary material).
Class IV: RPE transcription factors
To examine changes in genes expressed in zones 2 and 3 and evaluate factors
involved in controlling pigmentation, Mitf, Tfec and Otx2
were examined by in situ hybridization. In orJ/+ eyes,
Mitf was expressed throughout the RPE at E14.5 and at higher levels
in peripheral RPE in zone 3 and zone 2 as well
(Fig. 3F). Mitf was
also expressed in migrating melanoblasts and in zone 4 at E17.5. Mitf
expression abutted Chx10 in the peripheral retina at E14.5 and E17.5,
and was never detected in wild-type retinas. In the orJ
retina, Mitf was ectopically expressed at high levels in the retina,
in a manner similar to Gas1, while there was a lessened expression in
the center. However, by E17.5, Mitf was highly upregulated throughout
the entire orJ retina
(Fig. 3F). Tfec was
the most upregulated gene as evaluated by microarray analysis. At E14.5, in
orJ/+ eyes, Tfec was barely detectable in zone 2
and was not detected at E17.5 (apparent staining is likely to be background)
(Fig. 3G). In the
orJ retina, Tfec was ectopically expressed in the
retina, but more peripherally than Mitf and at lower levels. At
E17.5, Tfec was moderately expressed throughout the entire retina in
orJ eyes. In orJ/+ eyes
(Fig. 3G), Otx2 was
expressed in both the ONBL and RPE, including zone 3, at E14.5, but not the
peripheral retina (see Fig. S1R in the supplementary material). The
Otx2 expression pattern mimicked that of ONBL-specific genes in the
orJ retina, except that Otx2 was detected in the
periphery in lightly pigmented cells in the peripheral retina only at
E14.5.
Activation of Mitf target genes in transdifferentiation
To address whether ectopic Mitf and Tfec in the
orJ retina was sufficient to activate their cognate target
genes, Mitf target genes were analyzed by in situ hybridization at E17.5,
during an intermediate stage of transdifferentiation
(Fig. 5). Trpm1 is a
direct target of Mitf and depends on Mitf for expression in the developing
RPE. Like Mitf, Trpm1 was expressed in zone 2 in
orJ/+ heterozygotes and was ectopically expressed
throughout the retina in orJ mutants, including
unpigmented cells (Fig. 5A). In
contrast, Dct was highly expressed in pigmented cells in
orJ retinas as well as in regions of non-pigmented cells
in the central retina. Its expression correlated with regions that had lost
neural retinal identity, but had not yet undergone pigmentation, and
complemented the expression of ONBL and INBL markers
(Fig. 5B). Tyrosinase,
the rate-limiting enzyme in melanin synthesis, was only expressed in pigmented
cells in both orJ/+ heterozygotes and
orJ mutant retinas
(Fig. 5C). Therefore, different
classes of Mitf target genes define a progressive program of
transdifferentiation beginning with direct Mitf target genes like
Trpm1, then proceeding to express Dct, and finally
tyrosinase, coinciding with the appearance of pigment. This ordered
gene expression progression was also observed at E14.5 in
orJ mutant retinas (see Fig. S2A-C in the supplementary
material).
|
Chx10 is sufficient to repress the expression of RPE genes
Microarray and in situ hybridization analysis pointed to Mitf
family members as possible key target genes for Chx10 regulation. To determine
if the expression of Chx10 could lead to repression of these genes,
Chx10 was misexpressed in the developing RPE in chick by in ovo
electroporation. A plasmid encoding chick Chx10, along with another plasmid
encoding GFP as a cotransfection reporter, were transduced into early chick
optic vesicles by electroporation, and embryos were allowed to develop for 1
day in ovo (Fig. 6A).
Non-transfected optic vesicles from the same embryo
(Fig. 6B) or electroporations
with GFP alone (Fig. 6C) were
used as controls and showed identical marker expression (compare
Fig. 6F,I,L,O,R,U to
Fig. 6E,H,K,N,Q,T).
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Discussion |
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Several lines of evidence support the notion that the neural retina directly transdifferentiates into pigmented cells. However, the additional expression domain of Chx10 in the nonpigmented ciliary body epithelium raises the possibility that the transdifferentiation observed is one of nonpigmented ciliary body cells into pigmented cells. In this model, the neural retina would die and be replaced with an expanding population of pigment-fated cells. The fate mapping analysis and extensive expression studies presented here do not support this alternative model. Firstly, in situ hybridizations and microarray data do not indicate that pigment-fated cells undergo the rapid proliferation required to populate the orJ retina, nor do they indicate abnormally high rates of cell death in the neural retina. Specifically, markers of proliferation, including E2f1, cyclin E1, and cyclin D1, were downregulated in regions of the orJ retina undergoing pigmentation, while cell cycle inhibitors, including p57 and Gas1, were concomitantly upregulated in these regions. Secondly, analysis of markers from the peripheral retina, particularly in zone 2, showed their expansion into parts of the retina that also expressed bona fide neural retina markers well before transdifferentiation was observed. The overall temporal pattern of gene expression and high number of fate-mapped pigmented cells even at E17.5 support the direct transdifferentiation of neural retinal cell into pigmented cells.
A molecular program of transdifferentiation
Genes expressed in either the RPE or different peripheral regions of the
retina were upregulated in the orJ mutant retina and were
identifiable by microarray analysis. By analyzing candidate genes and other
genes with known expression patterns by in situ hybridization, we have formed
a model for transdifferentiation that also possibly explains why it occurs
progressively (Fig. 4A). The
model designates zones of gene expression for purposes of description,
although as discussed below, these zones may represent functional compartments
(except zone 4, which is not part of the neuroepithelium).
Fig. 4B is a tabular summary of
some of the genes presented in this study
(Fig. 3,
Fig. 5 and Fig. S1,S2 in the
supplementary material) and their inclusion in the different zones.
The expansion and ectopic expression of genes normally restricted to zone 2 of the peripheral retina appears to be an early event in the orJ retina. The upregulation, however, was not uniform and was stronger in the periphery than the center. While Mitf, and some of its target genes, showed broad upregulation, other melanogenic genes were restricted to peripheral regions, and thus pigmentation did not occur in all Mitf-expressing cells. Where zone 1 genes and zone 2 genes then began to overlap (shown as yellow in Fig. 4A), Dct expression became pronounced. Shortly thereafter, tyrosinase was expressed and the tissue became pigmented and adopted a zone 3-like appearance. Concurrent with pigmentation was a downregulation of genes expressed in zone 1, as well as Gas1 and p57. The next progression occurred within zone 1 which began to expand centrally. As new parts of the retina began coexpressing zone 1 and zone 2 genes, Dct expression expanded centrally and marked non-pigmented cells programmed to initiate pigmentation. The cells did not initiate pigmentation until tyrosinase, the rate-limiting enzyme in melanin production, became expressed.
It is striking how the progression of transdifferentiation occurred so slowly. Even as late as P17, there were still regions of the retina just beginning to undergo pigmentation (see Fig. 2L). The rate-limiting step appears related to the expansion of zone 1, as zone 2 completely expanded throughout the retina well before pigmentation reached this area. The central boundary of zone 1 was defined by the expression of progenitor cell genes in the ONBL. The downregulation of ONBL-expressed genes was observed in the peripheral retina prior to those regions acquiring pigmentation, and by E17.5, the entire retina appeared to lose neural identity. These events may precipitate loss of the central boundary of zone 1 and lead to the expansion of zone 1 into central parts of the retina. Determination of the ONBL-expressed genes that are responsible for defining the boundary with zone 1 will yield interesting insights into the mechanism of compartmentalization of the retina.
Genetic modifiers of the orJ phenotype
Modifiers of the orJ phenotype have been described that
partially rescue eye size and lamination. In neither case was the peripheral
orJ phenotype rescued
(Bone-Larson et al., 2000;
Green et al., 2003
). This
raised the possibility that another factor could compensate for some Chx10
functions in the central but not peripheral retina. One would then predict
that in the genetic background employed here, the compensating factor was
detrimentally affected, leading to a more severe phenotype. A candidate gene
for this function is the Chx10 homolog, Vsx-1. Vsx-1 in
other organisms including chick and zebrafish is co-expressed with
Chx10 in retinal progenitor cells, but has not been detected in
mammalian progenitor cells (Chen and Cepko,
2000
; Chow et al.,
2001
; Passini et al.,
1997
). We performed in situ analysis for Vsx-1 and did
not see it expressed in either orJ/+ mice or
orJ mice on any background, probably ruling out
Vsx-1 as a modifying allele (data not shown).
An alternative possibility for a genetic modifier relates to the extent of
upregulation of Mitf and other zone 2 genes. Mitf is
normally not expressed at any level in the retina, thus Mitf
upregulation in the orJ mutant may be influenced by
genetic modifiers in terms of the spatial limits of Mitf expression
or the absolute quantity of RNA. One might then predict that Mitf in
rescued orJ mice might only be ectopically expressed in
the periphery and/or may be expressed at lower levels. A further prediction is
that broad and strong overexpression of Mitf in a mildly affected
orJ strain would lead to a more dramatic phenotype.
Interestingly, Mitf was one of the candidate genes preliminarily
identified by Bone-Larson and colleagues as a modifier of the
orJ phenotype
(Bone-Larson et al., 2000).
Another candidate gene that might control the degrees of severity of the
orJ transdifferentiation phenotype is the
Mitf-related transcription factor Tfec. Normally,
Tfec is only transiently expressed in the RPE and zone 2, and its
ectopic expression in the retina along with Mitf might have distinct
consequences. It is also notable that Tfec appeared to be upregulated
less centrally than Mitf at E14.5, possibly marking the region of the
retina actively undergoing transdifferentiation. Gas1 is also an interesting
candidate, not only because of its characterized role as a cell cycle
inhibitor, but also its functions in the RPE. Mice lacking Gas1 show
transdifferentiation of the ventral RPE to neural retina, suggesting a role
for Gas1 in ventral RPE identity (Lee et
al., 2001). Mitf mutant mice also show
transdifferentiation of RPE to neural retina, but this occurs only dorsally
(Bumsted and Barnstable, 2000
).
Thus, the concurrent expression of Gas1 and Mitf in the
retina of orJ mice may have a distinct effect on cell
fate, functioning in an opposite direction to their loss-of-function
phenotypes. Finally, there remains the possibility that mesenchymal cells play
a role in controlling the spatial and temporal features of
transdifferentiation, and these functions could be altered by genetic
background.
Transcriptional targets of Chx10
One of the goals of orJ microarray analysis was to
identify genes that may be directly controlled by Chx10. If Chx10 functioned
as a transcriptional activator, one would predict that direct target genes
would be downregulated in the orJ retina. A large number
of downregulated transcripts were observed by microarray analysis, but the
ones studied in greater detail by in situ hybridization were not necessarily
downregulated on a per cell transcript level, but rather through a reduction
in the number of cells expressing them. Many of these downregulated genes were
markers of neurogenesis and early differentiation. Further characterization of
genes downregulated in the orJ retina may give new
insights into this molecular program.
Genes upregulated in the orJ retina were the focus of
this study and are candidate targets for repression by Chx10, e.g. Mitf,
Tfec, Gas1, p57 and Otx1. However, some upregulated genes are
likely targets of Mitf-dependent activation rather than repression by Chx10.
Genes like Trpm1 and Cd63 are probably upregulated through
Mitf transcription. Trpm1, in particular, is
transcriptionally activated in a concentration-dependent fashion by Mitf and
is not expressed in Mitf mutant RPE
(Miller et al., 2004). In this
respect, analysis of orJ mice by microarray analysis would
be an ideal way to identify new target genes of Mitf and Tfec, provided the
array had a large set of RPE genes. Neither Gas1, p57 nor
Tfec require Mitf for their expression and therefore their
misexpression in orJ mice is probably secondary to, or
independent of, Mitf expression (see Fig. S3 in the supplementary
material). However, p57 is expressed elsewhere in the retina in
subsets of progenitor cells, and Gas1 is expressed in the ONBL at
E17.5 in cells that potentially express Chx10, so Gas1 and
p57 may not be ideal candidate target genes for repression by
Chx10.
The finding that misexpression of Chx10 in developing chick RPE
led to a depigmentation and downregulation of Mitf, Tfec and other
pigment markers further supports the notion that Mitf and
Tfec are direct targets of Chx10. Overexpression of a number of
transcription factors in the RPE (e.g. Six3, Six6 and
Rax/Rx) can lead to depigmentation, but this usually occurs via
transdifferentiation of the RPE to retina (C-M.A.C. and C.L.C., unpublished)
(Toy et al., 1998). RPE made
to express Chx10 seemed to maintain proper RPE identity, at least as judged by
the lack of conversion to a retinal identity as viewed through expression of
Six3 and Chx10. These data further suggest that the aberrant
expression of Mitf, Tfec and their target genes was not secondary to
alterations in the center-periphery pattern also observed in the
orJ mutant. Mitf has been implicated as a Chx10
target gene previously, based on its temporal and spatial expression in early
eye development. The boundary between Chx10 and Mitf
persists throughout eye development (discussed further below). An attractive
model then is that Chx10 functions in the optic vesicle to repress
Mitf and permanently keep it repressed in the retina. This repression
does not require continuous Chx10 expression, as mature retinal cell
types that lose Chx10 expression do not ever express Mitf.
Possible derepression of Mitf in the optic vesicle in
orJ mice may help explain the broad ectopic expression of
Mitf in the retina of these mutants. Identification of a
physiologically meaningful Chx10 binding site in Mitf regulatory
regions is a necessary next step in testing this hypothesis of direct
regulation by Chx10.
Insights into other Chx10 mutant phenotypes
The results of this study provide new insights into a number of phenotypes
observed in mice and humans lacking Chx10 function. Human patients with
mutations in Chx10 present with microphthalmia, cataracts and iris
abnormalities (Percin et al.,
2000). While microphthalmia is expected, based upon the earlier
characterization of the orJ phenotype, the anterior eye
phenotypes were unexplained. Conversion of nonpigmented ciliary body
epithelium into pigmented tissue might disrupt formation and function of the
ciliary body and iris. Even in the orJ animals rescued by
a Mus musculus castaneus genetic background, the ciliary body did not
form properly, although the iris partially developed
(Bone-Larson et al., 2000
).
Overt hyperpigmentation was not observed in these eyes, suggesting that Chx10
may play a direct role in differentiation of the ciliary body where it is
expressed in the presumptive nonpigmented ciliary body epithelium.
An intriguing observation made in orJ mice was the
presence of a five-fold increase in the number of retinal stem cells compared
to wild-type counterparts (Tropepe et al.,
2000). Retinal stem cells derive from cells in the pigmented
ciliary body in mammals, which may be a homolog of the ciliary marginal zone
of fish and amphibians (Perron and Harris,
2000
). It was proposed that retinal progenitor cells might
negatively regulate the number of stem cells in the retina, and as a
consequence of fewer progenitor cells in the orJ retina,
more stem cells would be produced. Another possibility might be that the
retinal cells that transdifferentiated into pigment cells form a larger niche
for retinal stem cells, or themselves become retinal stem cells. The
possibility that retinal progenitor cells could become retinal stem cells via
transdifferentiation opens up new experimental possibilities in studying
retinal stem cells.
Compartment boundaries in the peripheral retina
The study of compartment boundaries has been elegantly conducted in
Drosophila using classical genetics and molecular markers (see
Dahmann and Basler, 1999;
Wolpert, 2003
). Much less is
known in vertebrate systems about where compartments are found and how they
are regulated. Several aspects of the data presented here provide evidence for
compartment boundaries in the peripheral retina. First, early gene expression
patterns in the peripheral retina were largely maintained. For example,
Notch1, Fgf15, and several bHLH factors examined were never expressed
in the peripheral zones in the retina. Wfdc1 also was never expressed
outside of zone 1, although Crhbp and Igf2 showed some zone
2 expression. Not only was the spatial organization maintained, but most
markers of the periphery remained expressed in the mature ciliary body.
Second, a combination of expression and fate-mapping analysis of
Chx10 at the periphery revealed it to be expressed in zone 1
throughout development. Fate mapping analysis using R26R in wild type or
orJ/+ heterozygotes showed that labeled cells stayed
within their boundaries and labeled cells were never observed to be pigment
cells. This finding strongly implicates a boundary between future pigmented
and nonpigmented cells within an initially nonpigmented population, and
suggests that even if some cell mixing occurs, the eventual fate of a cell is
still lineage-fixed relative to its initial specification. Third, loss of
function of Chx10 in the orJ mutant caused normally
peripherally restricted genes to become expanded to the center of the retina.
Therefore, Chx10 function appears linked to maintaining at least one important
boundary in the peripheral retina. Strikingly, the most peripherally expressed
genes, in zone 2, expanded more dramatically and temporally before zone 1
cells in the orJ mutant. This finding implies that
formation and/or maintenance of the boundary between zone 1 and zone 2
requires Chx10.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/20/5139/DC1
* Present address: Hydra Biosciences, 790 Memorial Drive, Suite 203,
Cambridge, MA, 02139, USA
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