1 Department of Molecular Cell Biology, Max-Planck-Institute of Biophysical
Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
2 Department of Developmental Biology, Max-Planck-Institute of Biophysical
Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
* Present address: The Salk Institute for Biological Studies, Gene Expression
Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
Present address: Sackler Faculty of Medicine, Department of Human Genetics and
Molecular Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv,
Israel
Author for correspondence (e-mail:
peter.gruss{at}mpg-gv.mpg.de)
Accepted 12 February 2003
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SUMMARY |
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Key words: Eye development, Pigmented retina, Regionalization, Pax2, Pax6, Mitf
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INTRODUCTION |
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Initially, the cells of the early OV are bipotent becausethey can
differentiate into either NR or RPE cells. The first indication for the
determination of different areas within the developing eye is apparent at the
OV stage. Here, the microphthalmia-associated transcription factor Mitf, which
belongs to the family of basic helix-loop-helix (bHLH)/leucine zipper
transcription factors, is first expressed throughout the entire OV
(Bora et al., 1998;
Nguyen and Arnheiter, 2000
).
Subsequently, the Mitf expression is downregulated in the distal
portion of the OV to mark the region of the presumptive RPE in the
proximal-dorsal portion of the OV (Nguyen
and Arnheiter, 2000
). In the adjacent, distal OV, the presumptive
NR region (Burmeister et al.,
1996
; Liu et al.,
1994
), the paired-like homeodomain transcription factor
Chx10 starts to be expressed at the same time. Later on Mitf
and Chx10 continue to be expressed in adjacent regions of the eye,
suggesting a reciprocal regulation between these two factors
(Nguyen and Arnheiter, 2000
).
Interestingly, in Mitf-mutant eyes, portions of the outer RPE layer
transdifferentiate into NR instead of RPE, reflecting the important roles of
Mitf in promoting the RPE differentiation and suppressing NR characteristics
(Mochii et al., 1998
;
Bumsted and Burnstable, 2000
;
Nguyen and Arnheiter, 2000
).
Chx10-mutant NRs are generally hypocellular and lack bipolar cells
(Ferda Perkin et al., 2000; Burmeister et
al., 1996
).
The paired and homeodomain transcription factor Pax6 is assumed to be a
`master regulator' of eye development
(Gehring and Ikeo, 1999;
Ashery-Padan and Gruss, 2001
)
because forced expression of Pax6 alone is sufficient to induce
ectopic eyes in fly and frog embryos
(Halder et al., 1995
;
Chow et al., 1999
). When
Pax6 is lacking no functional eye structures form in organisms as
different as mouse, human, rat, frog and fly (reviewed by
Gehring and Ikeo, 1999
). Mice
heterozygous for Pax6-null mutations exhibit a small eye
phenotype, which is characterized by multiple ocular abnormalities, such as
microphthalmia, lens cataracts and iris defects, and human PAX6
mutations lead to Aniridia, as well as multiple lens and corneal
defects (Hill et al., 1991
;
Glaser et al., 1992
;
Glaser et al., 1994
).
Pax6 is expressed highly throughout the early OV and the surface
ectoderm, and remains expressed in all eye components at the optic-cup stage,
including lens vesicle, outer and inner optic cup layers, and optic stalk
(Walther and Gruss, 1991
).
Later, Pax6 expression becomes restricted to the lens, corneal and
conjunctive epithelia, iris and inner portion of the NR
(Walther and Gruss, 1991
).
Conditional inactivation of Pax6 in the surface ectoderm leads to a
specific ablation of the lens (Ashery-Padan
et al., 2000
). Furthermore, the conditional elimination of
Pax6 in the distal NR causes a complete failure of differentiation of
all NR cell types except amacrine cells
(Marquardt et al., 2001
).
However, the function of Pax6 during the early phase of OV genesis has not
been studied extensively (Grindley et al.,
1995
).
At early stages of eye development, the Pax-family member Pax2 is
co-expressed with Pax6 in the OV, but is absent in the surface
ectoderm (Nornes et al., 1990)
(this study). During optic nerve formation at
E12.5, Pax2
expression becomes restricted to the ventral NR that surrounds the closing
optic fissure and the presumptive glia cells of the optic nerve
(Nornes et al., 1990
;
Torres et al., 1996
). After
E12.5, Pax2-deficient mice exhibit a severe retinal coloboma
a failure to close the choroid fissure. Furthermore, eyes are achiasmatic and
the retinal ganglion cell axons project only ipsilaterally
(Torres et al., 1996
).
Previously, it has been proposed that Pax2 and Pax6 might be engaged in
reciprocal negative regulation, thereby resulting in the delineation of the
optic-cup versus the optic-stalk domains
(Schwarz et al., 2000). The
co-expression of Pax2 and Pax6 at high levels throughout the entire OV prior
to and concomitant with the establishment of the distinct progenitor domains
of the NR, RPE and optic stalk (i.e. Martinez-Molares et al., 2001) (this
study) prompted us to examine whether Pax2 and Pax6 do synergize during early
retinal development. Despite the early arrest of OV development in
Pax6-null mutants, we show that the establishment of distinct NR, RPE
and optic-stalk-progenitor domains is independent of Pax6 activity. Similarly,
Pax2 is dispensable for the formation of the distinct progenitor domains in
the OV. However, Pax2; Pax6 compound mutants displayed a
dose-dependent reduction in the expression of the RPE determinant Mitf,
accompanied by transdifferentiation of RPE into NR in
Pax2-/-; Pax6+/- embryos. This
resembles the phenotype of Mitf-null mutants. In
Pax2-/-; Pax6-/- OVs, Mitf
fails to be expressed, with NR markers occupying the area usually representing
the Mitf+ RPE domain. Furthermore, we show that Pax2 and Pax6 both
bind to and activate a MITF-RPE promoter element in vitro. Moreover,
the prolonged expression of Pax6 in the Pax2-positive optic
stalk in transgenic mice leads to the ectopic expression of Mitf and
RPE differentiation. Together, these results demonstrate that the redundant
activities of Pax2 and Pax6 are required and sufficient to direct the
determination of RPE, and that this might be achieved by directly controlling
the expression of RPE determinants such as Mitf.
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MATERIALS AND METHODS |
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DNA from either the yolk sac or tail of Pax6lac embryos
was genotyped by PCR as described
(Bäumer et al. 2002).
DNA from either the yolk sac or tail of Pax2-/- embryos was genotyped using two PCRs. The following primers were used to identify the mutant allele: Neo-f4, 5'-CTTCTATCGCCTTCTTGACG-3'; Pax2-r3, 5'-TCCCAGCCATTACTTGAACG-3'. A band of 600 bp indicated the existence of a Pax2-mutant allele. Each PCR assay contained 1 µg of DNA, 1/10 vol of HotStarTag buffer (Qiagen), 200 µM dNTP mix, 10 µM of Neo-f4 primer, 23 µM of Pax2-r3 primer and 1 U HotStarTaq polymerase (Qiagen). Cycling conditions were 95°C for 15 minutes, followed by 34 cycles of 94°C for 45 seconds, 58°C for 30 seconds, 72°C for 1 minute, and a final extension of 72°C for 5 minutes.
For the identification of the wild-type allele, the following primers were used: Pax2-f, 5'-CGGGGCTGCGTTGCTGACTG-3'; Pax2-r, 5'-GCTTTGCAGTGCATATCCATCG-3'. A band of 300 bp indicated the existence of a Pax2 wild-type allele. Each PCR assay contained 1 µg of DNA, 1/10 vol of PCR buffer (Biotherm), 200 µM dNTP mix, 0.33 pmol µl-1 of each primer and 1.5 U Taq polymerase (Biotherm). Cycling conditions were 94°C for 2 minutes, 80°C for 2 minutes, followed by30 cycles of 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 30 seconds, then 72°C for 5 minutes.
Immunohistochemistry
The embryos were fixed for 30 minutes in 4% PFA/PBS (pH 7.8), washed with
PBS, incubated in cold 30% Sucrose/PBS over night and frozen in Tissue
Freezing Medium (Jung). Sections of 6-10 µm were air-dried and stored at
-80°C.
For antibody staining, the sections were washed in PBS (3 X 5 min), blocked with 1% BSA (IgG-free, Sigma), 0.05% Tween-20 in PBS for 30 minutes at room temperature. Primary antibodies were diluted in blocking solution and incubated at 4°C overnight. Primary antibodies: 1:300 rabbit anti-ß-Gal (Cappel); 1:20 monoclonal mouse anti-Pax6 (DSHB); 1:200 rabbit anti-Pax2 (Babco); 1:150 rabbit anti-Mitf (gift of H. Arnheiter); 1:500 rabbit anti-Chx10 (gift of R. McInnes); 1:2000 rabbit anti-Otx2 (gift of F. Vaccarino); 1:75 goat anti-Brn3b (Santa Cruz). After three, 5-10 minute washes in PBS, the secondary antibody was applied in blocking solution for 1 hour [1:500 Alexa 568 goat anti rabbit (Molecular Probes); 1:60 FITC goat anti mouse (Southern Biotechnology)]. After three washes with PBS, counterstaining was performed with Dapi and the sections were embedded with Mowiol.
Hematoxylin-Eosin (HE) stainings were performed using standard protocols.
Bandshift assays
Pax6 and Pax2 proteins were overexpressed using SP6 promoter-coupled
Pax2 and Pax6 cDNA in the TNT in vitro transcription and
translation system (Promega) according to the manufacturer's protocol.
Double-stranded oligonucleotides (see
Fig. 5B for sense sequences)
were end-labeled using polynucleotide kinase and -[32P]-ATP.
The binding reaction was performed for 1 hour on ice in retard buffer (40 mM
HEPES-NaOH, pH 7.6, 8% Ficoll, 10 mM MgCl2, 80 mM NaCl, 0.2 mM
EDTA, 1 mM DTT) with 5 µg poly-dI-dC, 25,000 cpm of the double-stranded
oligonucleotide and 2-10 µl of either Pax2 or Pax6 TNT protein. To test the
binding specificity, either Pax6 or Pax2 polyclonal rabbit antibodies (Babco)
were preincubated 1:10 in retard buffer with poly-dI-dC and protein for one
hour on ice. The probes were run over an 8% polyacrylamide gel. The gel was
exposed to a Kodak Biomax film over night.
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RESULTS |
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The activity of Pax2 or Pax6 is dispensable for proximo-distal
patterning of the early OV
To elucidate the early function of Pax2 and Pax6 during OV formation, we
examined the patterning of Pax6-/- and
Pax2-/- OVs. At E12.5 the RPE determinant Mitf is
completely restricted to the RPE in the wild-type eye
(Fig. 2A), whereas Chx10
remained confined to the NR (Fig.
2C). Remarkably, expression of both Mitf and Chx10 is initiated in
the Pax6-/- OV (Fig.
2E,G), although neither retinal neurogenesis nor RPE
differentiation is initiated in Pax6-null mutants
(Grindley et al., 1995; data
not shown). At E12.5, the Mitf-positive domain was at the very distal tip of
the Pax6-/- OV (Fig.
2E), just adjacent to a more proximal Chx10-positive domain
(Fig. 2G). Similar to the wild
type, in the mutant eye virtually no overlap could be detected between Chx10
and Mitf-positive areas (Fig.
2A,C). At this stage, Otx2 was expressed at a high level in the
RPE and the distal-most NR, and decreased sharply towards the proximal half of
the NR (Fig. 2B) (see also
Martinez-Morales et al.,
2001
). In addition Otx2 was expressed in the surface ectoderm
(Fig. 2B). The immunoactivity
in the dorsal optic stalk might be due to an, as yet, unpublished
cross-reactivity with Otx1 (Baas et al., 2000) because optic-stalk expression
has been reported for Otx1, but not for Otx2
(Fig. 2B)
(Martinez-Morales et al.,
2001
). Surface ectoderm of Pax6 mutant failed to express
Otx2 (Fig. 2F), possibly
reflecting the failure of the lens ectoderm specification in
Pax6-null embryos (Grindley et
al., 1995
). However, the distal, Mitf-positive domain in
Pax6-/- OV co-expressed high levels of Otx2
(Fig. 2F), indicating that this
region corresponds to the wild-type Mitf-positive, Otx2-positive, RPE domain.
In addition, in the Pax6-/- Ovs, a distal (high)-proximal
(low) gradient of Otx2 activity was observed in the Chx10-positive NR domain
(Fig. 2F-G), which matched the
Otx2 expression characteristics in wild-type NR
(Fig. 2B).
By E12.5, the expression of Pax2 in the wild-type eye was confined to the optic stalk and the ventral NR (Fig. 1J; Fig. 2D; see also Fig. 4C). In the Pax6-/- OV at E12.5, Pax2 expression was detected in the medial Chx10-positive, Otx2 (low), NR domain (Fig. 2F-H, `m'). Furthermore, higher levels of Pax2 activity were localized in the proximal region of the OV, the presumptive optic stalk (Fig. 2H, `p'). A similar distribution of Mitf, Chx10, Otx2 and Pax2 in wild-type and Pax6-/- embryos was also observed in E9.5 and E10.5 OVs (data not shown). Together these results indicate that in the mutant OV, the domains `d' and `m' represent the anlagen of the RPE and the NR, respectively.
We next examined whether the distribution of Pax6 expression itself was
affected in the OV of Pax6-null mutants. Recently, we found that the
ß-galactosidase (ß-gal) activity in the transgenic
Pax6lacZ/+ knock-in line mainly reflects the endogeneous
expression pattern of Pax6
(St-Onge et al., 1997;
Bäumer et al., 2002
). At
E12.5 in Pax6lacZ/+ eyes, ß-gal was expressed in the
NR, anterior RPE, lens and corneal ectoderm, but was largely absent from the
optic stalk (Fig. 2B, inset).
The E12.5 Pax6lacZ/lacZ (Pax6-/-) OV
had high levels of ß-gal expression in the distal and the medial (`RPE'
and `NR'), but much lower levels in the proximal `optic-stalk' domain
(Fig. 2F, inset), which is
comparable to the ß-gal expression in Pax6lacZ/+ eyes
(Fig. 2B, inset). In situ
hybridization with a Pax6 riboprobe revealed essentially the same
localization of Pax6 transcripts in the distal OV of
Pax6Sey/Sey embryos
(Grindley et al., 1995
; data
not shown). Therefore, the distribution of Pax6 transcripts detected
in Pax6-null mutants reflects the largely undisturbed proximo-distal
patterning of the OV.
Pax2-/- eyes display retinal and optic nerve coloboma,
which are visible by E12.5 (Torres et al.,
1996) (data not shown). Morphologically, these eyes can be
identified after this stage by elongation of the NR towards the optic stalk
(Torres et al., 1996
;
Schwarz et al., 2000
)
(Fig. 3G; data not shown). We
further studied the putative function of early Pax2 expression during OV
regionalization of Pax2-/- OVs and eyes from E9.5 to E12.5
(Fig. 2I-P; data not shown).
Interestingly, the expression domain of Mitf in the RPE
(Fig. 2I,M), Otx2 expression in
the RPE, NR and a subpopulation of optic-stalk cells
(Fig. 2J,N), and Chx10 in the
NR (Fig. 2K,O) were identical
in wild-type and Pax2-/- OVs. Pax6 expression in mutant
eyes was comparable to wild type up to stage E11.5
(Fig. 2L,P; data not shown).
However, at stage E12.5, unlike wild type, the Pax6 activity was maintained in
the optic stalk region (Schwarz et al.,
2000
) (data not shown), suggesting that the late Pax2
expression is required for the downregulation of Pax6 in the optic
nerve. Together, these results indicate that the general subdivision of the OV
along the distal-proximal axis into distinct RPE, NR and
optic-stalk-progenitor domains is independent of Pax6 and of Pax2 activity,
suggesting a redundant function of Pax2 and Pax6 during these early events of
OV development.
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Similar defects have been described following loss of function of
Mitf in eyes of the mi/mi mice
(Scholtz and Chan, 1987;
Mochii et al., 1998
;
Bumsted and Burnstable, 2000
).
Therefore, we examined the expression of Mitf in
Pax2-/-; Pax6+/- embryos. At E9.5,
immunoreactivity of Mitf in the Pax2-/-;
Pax6+/- and wild-type OV was comparable. Notably, at E10.5 and
E11.5, the outer layer of the wild-type optic cup was Mitf-positive
(Fig. 3K-L), but Mitf
expression was reduced in Pax2-/-;
Pax6+/- optic cups (Fig.
3N-O).
To determine whether the RPE transdifferentiation observed in the
Pax2-/-; Pax6+/- mutants reflected a
secondary effect of either loss or reduction of the lens
(West-Mays et al., 1999;
Ashery-Padan et al., 2000
), we
studied Mitf expression in lens-CRE/Pax6flx/flx
mutants, in which the lens is genetically ablated by specific inactivation of
Pax6 in the surface ectoderm via the Cre/lox-approach
(Ashery-Padan et al., 2000
).
However, although both lens-CRE/Pax6flx/flx and
Pax2-/-; Pax6+/- mutant eyes show
multiple NR domains (Ashery-Padan et al.,
2000
), the position of the RPE and the level of Mitf
expression at E11.5 were comparable to wild type (compare
Fig. 3L with
Fig. 3P). Together, these
results indicate that Mitf expression is initiated but not maintained
at a sufficient level in the optic cups of the
Pax2-/-; Pax6+/- embryos, which leads
to a transdifferentiation of a second NR at the expense of RPE.
Mitf expression and OV regionalization depend on cooperative
Pax2 and Pax6 activity
The reduced levels of Mitf expression in Pax2-/-;
Pax6+/- eyes could be caused by remaining Pax6 activity.
Likewise, the initiation of Mitf expression in Pax6-null
mutants could result from functional compensation by Pax2.
To examine these two possibilities, we studied Mitf expression in the complete absence of Pax2 and Pax6. Remarkably, although expression of the early OV markers Lhx2 and Six3 was still detectable in the Pax2-/-; Pax6-/- OVs (data not shown), Mitf expression was not initiated (Fig. 4F,H). Expression of Chx10 and Otx2 were also initiated in mutant eyes (Fig. 4J,N,L,P). Therefore, we conclude, that the lack of Mitf expression in the OV of the double-null mutants is a specific defect rather than a general failure of OV development. Similar to the wild type (Fig. 4I), the expression of Chx10 at E9.5 was mainly confined to the distal portion of the Pax2-/-; Pax6-/- OV (Fig. 4J). Some additional Chx10-positive cells were also detected in the more proximal part of the Pax2-/-; Pax6-/- OV that is usually occupied by the Mitf-positive RPE domain (Fig. 4J, arrow). Although in the absence of Pax6, Chx10 expression was regionalized mostly within the `m' domain of the OV remnant (Fig. 2G), in Pax2-/-; Pax6-/- OV, Chx10 immunoreactivity was confined to the very distal tip (region `d') of the vesicle, occupying the normally Mitf-positive progenitor domain of the RPE. By contrast, expression of Otx2 was detected first at E9.5 in both wild-type and Pax2-/-; Pax6-/- OVs (Fig. 4M,N) possibly slightly expanded proximally in the mutant OV (Fig. 4N, arrow). At E11.5, wild-type RPE expressed high levels and NR low levels of Otx2 (Fig. 4O). At this stage, the distal-most region of the Pax2-/-; Pax6-/- OV was Otx2 positive (Fig. 4P), which indicates that the distal tip of the Pax2-/-; Pax6-/- OV co-expressed the NR marker Chx10 and the RPE marker Otx2.
Accordingly, we conclude that, in absence of both Pax2 and Pax6 function, the bipotent, early OV cells could not be further specified into the determined NR and RPE domains, a process that possibly involves a direct control on Mitf-gene activity.
Pax2 and Pax6 can bind and activate a MITF RPE promoter in
vitro
The specific loss of Mitf expression in Pax2/Pax6
double-null mutants raised the question of whether Mitf was a direct
target gene of Pax2 and Pax6. The Mitf gene produces at least four
different splice variants (see Fig.
5A). Three of them, Mitf-A, Mitf-H and Mitf-D are expressed in RPE
cells (Hallsson et al., 2000;
Udono et al., 2000
;
Takeda et al., 2002
). The
recently identified MITF-A promoter drives expression in RPE and
melanocyte cell lines (as expected by the expression pattern of this splice
variant) as well as in HeLa cells, which might indicate a more widespread
activity caused by a missing repressor element in this promoter fragment
(Udono et al., 2000
). However,
our results suggested a role of Pax2 and Pax6 in activation, rather than in
repression of Mitf. Therefore, we studied the effect of the binding
of Pax2 and Pax6 proteins on the human MITF-A promoter. The 2.2 kb
MITF-A promoter sequence is 66.3% identical to the homologous mouse
sequence, in some regions 79-91%, compared with sequence information by Celera
(data not shown). The MITF-A promoter, which is located upstream of
the first exon, was fused to the luciferase reporter gene
(Fig. 5A) (Udono et al., 2000
).
Using the consensus sequence of optimal Pax2 and Pax6 binding to DNA
established by Epstein and co-workers
(1994)
(Fig. 5B), five potential
binding sites, A1-A5, were identified (Fig.
5B). Bandshift assays using in vitro overexpressed Pax2 and Pax6
proteins and 32P-labelled oligonucleotides (for the sequences see
Fig. 5B) revealed that A5
represented a MITF-A promoter sequence that can bind both Pax2 and
Pax6 (Fig. 5C, red arrow). The
other four oligonucleotides only bound unspecific components of the assay, as
shown by controls without overexpressed proteins
(Fig. 5C, -Pax). The binding
specificity of the A5 site was verified by preincubation of Pax2 and the Pax6
proteins with specific antibodies, which impaired formation of the binding
complex (Fig. 5D, lanes 2 and
5). By contrast, addition of the Pax6 antibody to the Pax2 protein, and vice
versa, did not purturb the binding (Fig.
5D, lanes 3 and 6). Therefore, we concluded that the A5 sequence
in the MITF-A promoter represents a specific binding site for Pax2
and Pax6.
To examine the potential transactivation of this promoter element by Pax2
and/or Pax6, we co-transfected Cos-7 cells with the luciferase-coupled
MITF-A promoter, CMV-Pax2-cDNA and/or
CMV-Pax6-cDNA. CMV-Pax1-cDNA was used as a control
(Fig. 5E). These
co-transfections showed that the basal level of luciferase activity driven by
the MITF-A promoter was increased 13-fold by the addition of
Pax6,
40-fold by addition of Pax2, and
12-fold by an equimolar
mixture of Pax2 and Pax6 (Fig.
5E). Pax1 increased the luciferase activity only fivefold
(Fig. 5E).
These results demonstrate that both Pax2 and Pax6 can bind to and activate the MITF-A promoter. The fact that the mixture of Pax2 and Pax6 did not surpass the activation obtained by either Pax2 and Pax6 alone accords with the single binding site for both factors identified in this promoter sequence (Fig. 5D). It is likely that both factors compete for this binding site and that the double-mutant phenotypes result from the requirement of a specific concentration of either protein for sufficient activation of Mitf.
A similar competitive relationship has been reported for the segmentation
genes kreisler (Mafb Mouse Genome Informatics) and
Krox20, which control the expression of the Hoxb3 gene in
rhombomere 5 (Manzanares et al.,
2002).
Ectopic expression of Pax6 in the Pax2-positive optic nerve results
in the development of ectopic, Mitf-expressing RPE
To test if Pax2 and Pax6 can direct the expression Mitf in vivo,
we took advantage of a previously generated transgenic mouse line
(pPax2Pax6) that expresses Pax6 under the control
of a Pax2-upstream promoter fragment. This drives expression of
Pax6 in the optic stalk (Schwarz
et al., 2000) (Fig.
6A). At E13.5, Mitf expression was confined to the
developing iris (Fig. 6C,G, ir)
and to some cells in the RPE (Fig.
6E, arrows).
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In summary, these results indicate that (1) the expression of Mitf is not initiated in the absence of both Pax2 and Pax6, (2) both Pax2 and Pax6 can bind to and activate the MITF-A promoter in vitro, and (3) the forced co-expression of Pax2 and Pax6 in vivo leads to RPE development and ectopic expression of Mitf.
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DISCUSSION |
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Probably because of these signaling events, the neuroepithelium of the
distal OV becomes patterned into distinct NR-progenitor and RPE-progenitor
domains, as indicated by the segregation of the expression domains of Chx10
and Mitf (see Fig. 2).
Cross-repressive interactions between Chx10 and Mitf are thought to mediate
the sharpening and stabilization of the boundary between NR-progenitor and
RPE-progenitor domains (Nguyen and
Arnheiter, 2000), which might, therefore, mark a switch from
exogenous to OV-autonomous patterning mechanisms.
It was unclear how the expression of progenitor factors such as
Chx10 and Mitf is initiated in the OV neuroepithelium
because both factors are activated, even in the absence of the surface
ectoderm (see below) (Nguygen and Arnheiter, 2000). Recently, we demonstrated
that the expression of the naso-temporal axis markers BF1
(Foxg1 Mouse Genome Informatics) and BF2
(Foxd1 Mouse Genome Informatics) is dependent on Pax6
activity (Bäumer et al.,
2002). This provides a link between broadly expressed retinal
determinants and retinal-axial patterning. In Pax6-null mutants,
however, proximo-distal patterning of the OV into distinct NR, RPE and
optic-stalk-progenitor domains appears to be unaffected
(Fig. 2E-H). This rules out a
`master' function of Pax6 during the specification of murine retinal identity.
The remarkable inverted orientation of the Pax6-mutant OV, in which
the presumptive RPE domain faces the surface, might contribute to the failure
of specification of the mutant surface ectoderm, and strongly resembles the
results of ablation experiments (Nguyen
and Arnheiter, 2000
). Therefore, the surface ectoderm in
Pax6 mutants might lack signaling factors that are required for the
activation of either RPE-repressing or NR-activating factors.
In the present study we provide evidence that the combined action of Pax2
and Pax6 directly mediates the initiation of Mitf expression in the
OV and, thereby, determines the RPE-progenitor domain. Subsequently, the
extraocular and OV-autonomous patterning events mentioned above
(Araki et al., 2002) restrict
the initial, broad Mitf-expression domain to the future
RPE-progenitor domain, thereby assuring that RPE differentiation is confined
to the future outer layer of the optic cup.
Redundant and distinct functions of Pax2 and Pax6 in regionalization
of the OV
In contrast to the distinct regionalization of Pax2-/-
and Pax6-/- OVs into NR and RPE-progenitor domains, the
Pax2-/-; Pax6-/- OV is incorrectly
patterned in this respect. The RPE marker Mitf fails to be expressed in the
Pax2-/-; Pax6-/- OV and a second RPE
marker, Otx2, largely colocalizes with Chx10, a NR marker
(Fig. 4). The expansion of
Chx10 into the region usually occupied by the RPE-progenitor domain probably
reflects the failure to express the NR repressor Mitf in this region. However,
co-localization of Otx2 and Chx10 indicates that the normal determination of
the NR domain is also affected, which indicates additional, redundant roles of
Pax2 and Pax6 in establishing this progenitor domain. Therefore, the
determination of both NR and RPE from bipotent OV cells appears to be
dependent on redundant Pax2 and Pax6 function.
Consequently, in Pax6 mutants Pax2 activity is sufficient to
direct the formation of the NR-progenitor and RPE-progenitor domains, but Pax2
activity alone is not sufficient to induce optic-cup formation
(Fig. 2)
(Grindley et al., 1995).
Conversely, Pax6 activity is sufficient to direct optic-cup formation in
Pax2-/- mutants, but not to maintain the sharp border
between optic cup and optic nerve after E12.5, and the differentiation of the
optic nerve itself (Fig. 3G)
(Torres et al., 1996
). These
observations imply distinct, nonredundant functions of these two Pax proteins
at later stages of OV development. Interestingly,
Pax2-/-; Pax6+/- mutants have reduced
RPE differentiation as well as more severe optic-nerve defects than
Pax2-/- mutants (Fig.
3D), which might indicate additional functions of Pax6 in
optic-nerve formation.
The involvement of Pax2 and Pax6 in the regionalization of other tissues
has already been implied. For example, Pax6 can restrict the expression of
Pax2 at the boundary between the diencephalon and mesencephalon in
chick embryos (Matsunaga et al.,
2000) and this border is affected in absence of Pax6 in
mice (Stoykova et al., 1996
;
Mastick et al., 1997
;
Pratt et al., 2000
). Pax6 is,
furthermore, required for the specification of ventral-progenitor-cell
identity in the spinal cord and hindbrain
(Ericson et al., 1997
;
Takahashi and Osumi, 2002
),
and the lack of Pax6 function causes a prominent ventralization of the
molecular patterning and morphogenesis of the embryonic forebrain
(Stoykova et al., 2000
;
Yun et al., 2001
).
In the OV, after E10, the expression of Pax6 and Pax2 increasingly
segregates and becomes mutually exclusive after E12.5. During these later
stages both factors might acquire distinct functions in the further
specification of the optic cup and the optic nerve domains, where they might
now repress each other (Macdonald et al.,
1995; Schwarz et al.,
2000
). Taken together, these observations suggest that, during eye
development, Pax2 and Pax6 initially function redundantly in the OV during the
determination and patterning of the RPE, NR and, possibly,
optic-stalk-progenitor domains. During later stages, a switch appears to occur
that brings out the distinct functions of both factors, so that they now
mediate the differentiation of discrete tissue compartments of the eye in a
mutually exclusive manner (Macdonald et
al., 1995
; Schwarz et al.,
2000
). Interestingly, redundant as well as distinct functions have
been implied for other Pax-family members, such as Pax3 and Pax7 in spinal
cord and somite development (Borycki et
al., 1999
; Mansouri and Gruss,
1998
), Pax1 and Pax9 in sclerotome development
(Neubüser et al., 1996
;
Peters et al., 1999
), and Pax2
and Pax5 in different developing organs
(Schwarz et al., 1997
;
Urbanek et al., 1997
;
Bouchard et al., 2000
). The
recruitment of the same factor to drive distinct processes during sequential
stages in the development of the same tissue or organ is starting to become a
recurring theme in developmental biology (reviewed by
Marquardt and Pfaff,
2001
).
Based on observations that members of the same subgroup of the Hox-gene
family can, to a large extent, substitute for each other, it has been proposed
recently that the quantity rather than the quality of a required factor might
be decisive for some developmental mechanisms
(Duboule, 2000;
Greer et al., 2000
). The
dose-dependence of the eye phenotype on the Pax6 concentration in
Pax2-/-; Pax6+/- compound mice, as
shown in this study, corroborates this idea. Moreover, the Pax2 dose
dependence of Mitf expression can be observed in OVs of
Pax6-/-; Pax2+/- embryos that are
phenotypically identical to Pax6-/- OVs. These
Pax6-/-; Pax2+/- OVs express less Mitf in the
distal presumptive RPE region than the Pax6-/- OVs,
although the medial presumptive NR region is highly Chx10 positive in both
genotypes (data not shown; Fig.
2).
Mitf as a putative target gene of Pax2 and Pax6
Mitf was the only OV determinant identified that failed to be expressed in
Pax2-/-; Pax6-/- OV
(Fig. 4). Maintained expression
of another RPE marker, Otx2, in mutant OVs implied a specific loss of Mitf
activity rather than a complete failure in the specification of RPE
characteristics. As Otx2 is co-expressed with the NR marker Chx10, we assume
that the bipotent character of the OV cells is maintained, possibly due to the
absence of Mitf.
Transdifferentiation of the RPE in Pax2-/-;
Pax6+/- eyes closely resembles the ocular phenotype of
Mitf-deficient mice (Scholtz and
Chan, 1987; Bumsted and
Burnstable, 2000
; Nguyen and
Arnheiter, 2000
). Because RPE differentiation appears to be normal
in the lens-ablated lens-CRE; Pax6flx/flx mutants
(Ashery-Padan et al., 2000
)
(this study) this defect is unlikely to be due to the partial loss of lens
tissue in these mutants.
Interestingly, our results indicate that both Pax2 and Pax6 can
specifically bind to and activate the MITF-A promoter in vitro (see
Fig. 5). This might indicate
that one aspect of the complicated regulation of the Mitf gene might
involve direct binding of Pax2 and Pax6, although the in vitro results are
indirect because a transgenic approach to study the in vivo function of the
binding has not yet been examined. However, the fact that the pan-specific
Mitf-antibody did not detect Mitf activity in Pax2-/-;
Pax6-/- OV might indicate a general requirement of Pax2 and
Pax6 function for the expression of all Mitf isoforms in the RPE. To follow
this hypothesis, it will be necessary to further characterize Mitf
regulatory elements, such as the MITF-H, MITF-D and MITF-A
promoters. Although the H-form of Mitf also occurs at low level in the RPE, we
were unable to identify binding sites for Pax2 and Pax6 in the MITF-H
promoter (Udono et al., 2000),
and did not detect activation of this promoter in cell-culture experiments
(data not shown). Because the promoter region of MITF-H is less well
conserved than the MITF-A promoter between humans and mice (data not
shown), it is also possible that the MITF-H promoter did not
adequately represent the mouse promoter in the in vitro analysis.
Alternatively, the MITF-A promoter region could act as an enhancer to
control the expression of the other isoforms. Furthermore, because expression
driven by the MITF-A promoter is more widespread than wild-type
Mitf expression (Udono et al.,
2000
) (N.B. and D.S., unpublished), the existence of other
regulatory elements that restrict Mitf expression, is likely. This
issue requires further intensive studies, including mutational analysis of
potential transcription-factor binding sites.
To date, more detailed information is available concerning the regulation
of Mitf activity during melanocyte development (reviewed by
Tachibana, 2000). Mutations in
the MITF gene in humans cause type II Waardenburg syndrome, a severe
disease that specifically affects melanocyte function
(Tassabehji et al., 1994
;
Hodgkinson et al., 1998
;
Lee et al., 2000
).
Intriguingly, in melanocyte cell lines the human MITF-M promoter is
activated by another Pax-family member, PAX3, which is also involved
in type II Waardenburg syndrome (Watanabe
et al., 1998
; Potterf et al.,
2000
).
An essential role of Pax6 in RPE differentiation has been suggested
previously. Quinn and coworkers established chimeric mice from
Pax6-deficient and wild-type ES cells to examine the influence of
Pax6 deficiency in different compartments of the eye. When these
mutants had incorporated a high percentage of Pax6-deficient cells in
the outer layer of the optic cup, the eyes had a disorganized optic cup with a
folded NR and reduced RPE differentiation
(Quinn et al., 1996). This
resembles the phenotype of Pax2-/-;
Pax6+/- eyes. Further studies, possibly involving
tissue-specific inactivation of Pax6 in the RPE, should specifically
address the direct function of Pax6 in RPE differentiation.
Another hint for the possible function of Pax6 in inducing RPE
differentiation came from experiments in which Pax6 was ectopically
expressed. In frog embryos, ectopic expression of Pax6 leads to the
formation of complete ectopic eye structures outside the optic system, but
after overexpression within the optic system it induces only RPE, not NR,
along the (Pax2-postive) optic nerve (Chow
et al., 1999). In transgenic mice that express Pax6 under the
control of a Pax2-regulatory element, differentiation of ectopic RPE
(again without NR differentiation) was observed in the distal optic nerve
(Schwarz et al., 2000
) (this
study). Although, initially, we interpreted this phenotype as an indication of
the retinal differentiation potential of Pax6 in a region of the optic stalk
that expresses lower concentrations of Pax2
(Schwarz et al., 2000
), we had
to refine this interpretation following our more recent results showing the
important role of the redundant function of Pax2 and Pax6 in RPE development
(see below). The region of ectopic RPE formation in these mice appears to
correlate with the region that endogenously expresses Otx1 in the
dorsal optic stalk (see Fig.
2). This indicates that Otx1 is required to allow Pax6;
Pax2-mediated RPE differentiation. Because Otx1 and Otx2 are thought to be
essential for RPE development
(Martinez-Morales et al.,
2001
), this would reflect the situation during normal RPE
determination. In this case, Otx2 is co-expressed with Pax2 and Pax6 in the
RPE-progenitor domain (see Fig.
2) (Martinez-Morales et al.,
2001
), and Otx1, which can take over various functions of Otx2 in
different tissues (Acampora et al.,
1999
; Martinez-Morales et al.,
2001
) in the ectoptic situation of the transgenic optic stalk.
In summary, Pax6 activity appears to be sufficient to induce RPE development, whereas both Pax2 and Pax6 are necessary and sufficient to activate the RPE determinant Mitf in a competent tissue.
Redundant and divergent roles of Pax2 and Pax6 in eye
development
To summarize, initially Pax2 and Pax6 carry out redundant functions in
setting up the RPE progenitor domain in the OV neuroepithelium. This shared
role is demonstrated clearly by their entirely overlapping expression domains
in the OV neuroepithlium and by their redundant function in mediating Mitf
expression. Later, during optic-cup stages, the distribution of Pax2 and Pax6
segregate to give their well-documented, mutually exclusive patterns in optic
stalk and optic cup, respectively. At these stages Pax2 is necessary for
oligodendrocyte differentiation in the optic stalk and for the closure of the
choroid fissure (Torres et al., 1997), whereas Pax6 is required for normal
retinal neurogenesis (Marquardt et al.,
2001) and iris morphogensis
(Glaser et al., 1992
).
Therefore, the later segregation of Pax2 and Pax6 activities reflects their
divergent functions during later eye development and might be necessary for
them to carry out their functions in different tissues of the eye.
In this respect it might be significant that the late retinal enhancer of
Pax6 (`alpha') (Kammandel et al.,
1999; Bäumer et al.,
2002
), which is repressed by Pax2
(Schwarz et al., 2000
), is
excluded from the Pax2-positive choroid fissure (see
Bäumer et al., 2002
). In
this light, the original model in which the mutual repression of Pax2 and Pax6
was assumed to direct the spatial segregation of territories in the early eye
(Schwarz et al., 2000
) might
only apply to later aspects of eye development, such as optic-cup
morphogenesis. Such mutual repression might assure the spatial exclusion of
their diverging functions in optic nerve/choroid fissure and optic cup/retina.
It remains to be shown how the switch from coordinate expression and function
to divergent activities and mutual exclusion is achieved at the level of gene
regulation.
Pax transcription factors as regulators of bHLH transcription factors
in cellular determination
Mitf is not the first bHLH transcription factor that is known to be
regulated by Pax-family transcription factors. During muscle development, Pax3
is involved in the activation of MyoD, a myogenic bHLH transcription factor
(Maroto et al., 1997;
Tajbakhsh et al., 1997
),
although it is unclear whether activation is direct or indirect
(Borycki et al., 1999
).
Similarly, direct activation of the bHLH factors Myf5 and
MyoD by Pax7 during the differentiation of pluripotent muscle-stem
cells into satellite cells was assumed
(Rudnicki et al., 1993
;
Megeney et al., 1996
;
Seale et al., 2000
) (reviewed
by Tajbakhsh and Buckingham,
2000
). Recently, a direct requirement of Pax6 activity for the
activation of another bHLH factor, Ngn2, in the spinal cord
(Scardigli et al., 2001
) and
the developing neuroretina (Marquardt et
al., 2001
) was demonstrated. We previously found that Pax6
normally binds to and activates Ngn2-specific enhancers
(Marquardt et al., 2001
;
Scardigli et al., 2003
) that
are not activated in Pax6-deficient embryos
(Scardigli et al., 2001
).
Furthermore, after specific inactivation of Pax6 in the distal NR in
-Cre/Pax6flx/flx mice, bHLH factors Mash1
and Math5 are not expressed
(Marquardt et al., 2001
).
Another bHLH transcription factor, Neurod1 (previously
NeuroD), is still expressed in the distal NR of this Pax6
mutant (Marquardt et al.,
2001
), but is absent in Pax6-/- OV, which
might indicate its dependence on early Pax6 expression (data not
shown).
Pax transcription factors are often required for the determination of a
specific cell fate from multipotent cells
(Nutt et al., 1999;
Marquardt et al., 2001
;
Marquedt and Gruss, 2001; Seale et al.,
2000
; Borycki et al.,
1999
) and bHLH transcription factors function in the
differentiation of determined progenitor cells
(Cepko, 1999
;
Kageyama et al., 1997
). Taken
together, several lines of evidence indicate that the presence of particular
Pax transcription factors in different progenitor cells might be a general
requirement for the initiation of a number of specific differentiation
pathways.
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ACKNOWLEDGMENTS |
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