Division of Molecular Neurobiology, National Institute for Basic Biology, and Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki 444-8585, Japan
* Author for correspondence (e-mail: madon{at}nibb.ac.jp)
Accepted 11 July 2003
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SUMMARY |
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Key words: Retina, Chick, Retinotectal projection, CBF1, BMP2
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Introduction |
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The Eph family of receptors are classified into two subfamilies, EphA and
EphB, according to their preference for either glycosyl-phoshpatidyl-inositol
(GPI)-anchored ephrin A ligands or transmembrane ephrin B ligands
(Eph Nomenclature Committee,
1997). In chick, the EphA3 receptor is expressed in a temporal
high/nasal low gradient in the retinal ganglion cells
(Cheng and Flanagan, 1994
), and
ephrin A2 and ephrin A5 are expressed in posterior high/anterior low gradients
in the tectum (Cheng and Flanagan,
1994
; Drescher et al.,
1995
; Feldheim et al.,
1998
; Frisén et al.,
1998
). Several lines of evidence suggest that ephrin A ligands are
repulsive to EphA-expressing axons (Cheng
and Flanagan, 1994
; Drescher
et al., 1995
; Feldheim et al.,
1998
; Frisén et al.,
1998
), and thereby elongation of retinal axons stop at precise
locations along the AP axis on the tectum (SC)
(Frisén et al., 1998
;
Brown et al., 2000
;
Feldheim et al., 2000
).
In addition to EphA3, four EphA receptors (EphA4, EphA5, EphA6 and EphA7)
are uniformly expressed in the chick retina
(Monschau et al., 1997;
Connor et al., 1998
). On the
other hand, ephrin A2 and ephrin A5 are expressed in nasal high/temporal low
gradients in the retina (Marcus et al.,
1996
; Connor et al.,
1998
; Hornberger et al.,
1999
). Overexpression of ephrin As in temporal axons leads to
errors in the topographic targeting of temporal axons
(Dütting et al., 1999
;
Hornberger et al., 1999
),
suggesting a role for retinal ephrin As in the formation of topographic
projections. Thus, EphAs uniformly expressed in the retina are also thought to
be involved in the topographic projection along the AP axis, together with
ephrin As gradiently expressed in the retina.
The achievement of graded distributions of topographic molecules along the
AP and DV axes in the retina and tectum during development is a crucial step
in the formation of the topographic map. From the early developmental stages
of the retina and tectum, a number of molecules that belong to morphogens and
transcription factors show asymmetrical expression patterns along the two
axes. Studies on some of these molecules have demonstrated that they regulate
the graded distributions of topographic molecules, which finally lead to the
topographic projection, along the two axes in the retina and optic tectum. We
previously found that two winged-helix (WH) transcription factors, chick
brain factor 1 and 2 (CBF1 and CBF2), are
expressed specifically in the nasal and temporal regions of the developing
chick retina, respectively (Yuasa et al.,
1996). We further demonstrated that ectopic misexpression of
CBF1 or CBF2 reversed the topographic map in the
retinotectal system along the AP axis
(Yuasa et al., 1996
). Although
CBF1 and CBF2 are supposed to determine the regional
specificity along the NT axis in the retina through the regulation of
expression of their downstream target genes, their modes of action remain
unknown. In addition to CBF1, two homeobox transcription factors,
SOHo1 and GH6, are also expressed specifically in the nasal
region of the developing retina (Schulte
and Cepko, 2000
). Misexpression of these molecules in the retina
results in projection errors of retinal axons along the AP axis, due to the
repression of EphA3 expression
(Schulte and Cepko, 2000
). As
CBF1 and CBF2 are expressed in the retina before the
expression of SOHo1 and GH6 starts, there is the possibility
that these homeobox transcription factors are downstream target genes of
CBF1 or CBF2. However, the relationship between CBFs and
these homeobox proteins has not been fully investigated.
We recently identified a novel secreted molecule, Ventroptin, which is an
antagonist of bone morphogenetic protein 4 (BMP4) in the retina, and
demonstrated that Ventroptin is implicated in the retinotectal topographic
projection along both the DV and AP axes
(Sakuta et al., 2001). At the
early developmental stages of the eye, Ventroptin is specifically
expressed in the ventral retina, in a complementary pattern to that of the
dorsal-specific expression of BMP4. The counteraction between BMP4
and Ventroptin governs the regional specification along the DV axis by
regulating the distributions of downstream target genes, such as Tbx5
and Vax (Sakuta et al.,
2001
). At the later stages (E6 onward), Ventroptin begins
to be expressed in a nasal high/temporal low gradient, in addition to a
ventral high/dorsal low gradient. This is the first demonstration of a
molecule with a double-gradient pattern of expression in the retina.
Asymmetrical expression of Ventroptin along the NT axis regulates the
graded expression of ephrin A2 along this axis, but not of ephrin
A5 or EphA3 in the retina, which is associated with the
retinotectal mapping along the AP axis
(Sakuta et al., 2001
). As
expression of BMP4 declines in the retina from E5, a member of the
TGFß family other than BMP4 is supposed to appear in a temporal
high/nasal low gradient along the NT axis with a complementary pattern to the
Ventroptin expression (Sakuta et
al., 2001
).
In the present study, to gain insight into the downstream target genes of
CBF1, we employed, for the first time, electroporation of a
retroviral vector carrying the CBF1 gene into the optic vesicle, and
examined effects of the misexpression of CBF1 on the expression of
topographic molecules and other asymmetrically distributed molecules. The in
ovo electroporation of retrovirus vector at Hamburger- Hamilton (HH) stage 8
(Hamburger and Hamilton, 1951)
allows the immediate (at stage 10) and sustained expression of a transgene. We
show that ectopic expression of CBF1 in the temporal retina represses
expression of EphA3 and CBF2, and induces that of SOHo1,
GH6, ephrin A2 and ephrin A5. The mode of action of CBF1 on
these molecules has been revealed to be classified into three distinct
categories. In addition, we show that a TGFß family member,
BMP2, begins to be expressed in a pattern complementary to that of
Ventroptin, with a double-gradient along the NT and DV axes from E5
onward. Misexpression of BMP2 in the developing retina represses
expression of Ventroptin and ephrin A2. Moreover, we
demonstrate that CBF1 perturbs BMP signaling through a DNA
binding-independent mechanism, which resultantly leads to the induction of
ephrin A2 expression. Based on these results, we suggest that CBF1
plays pivotal roles in the determination and maintenance of the NT specificity
in the retina by integratively regulating the expression of asymmetrically
distributed molecules through multiple mechanisms.
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Materials and methods |
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To make CBF1-eve/RCAS, the repression domain of the Drosophila
Even-skipped protein (Han and Manley,
1993) from pCS2eve was first fused with CBF1 DNA binding domain
(amino acid residues from 139 to 253). It was inserted into SLAX-NS myc, which
was prepared with a shuttle vector SLAX-NS by inserting double myc-epitope
tags at the NcoI-EcoRI site, and then subcloned into RCAS-NS
retrovirus vector using NotI and SpeI sites. The
CBF1AA mutant was generated by substitutions of asparagine 189 and
histidine 193 with alanines by PCR, and cloned into RCAS-NS or pcDNA3.1(+)
(Invitrogen). To prepare the BMP2/RCAS construct, the coding region of mouse
BMP2 was once inserted into SLAX-NS, and then subcloned into RCAS-NS.
Ventroptin/RCAS was described previously
(Sakuta et al., 2001
).
Mouse Smad1, Smad4 and Alk3 were cloned from a P0 mouse
retina cDNA library by PCR and their sequences were verified by DNA sequence
analysis. Smad1 and Smad4 were fused with double
flag-epitope tags at the N terminus by PCR. A constitutively active mutant
Alk3 (Alk3-CA) was generated by substitution of glutamine
233 with aspartic acid by PCR as described previously
(Hoodless et al., 1996). They
were subcloned into pcDNA3.1(+) vector.
In ovo electroporation
In ovo electroporation was performed as described previously
(Sakuta et al., 2001).
Retrovirus constructs for the electroporation were suspended at a
concentration of 0.1-1.0 µg/µl in 10 mM Tris-HCl, 0.25 mM ethylendiamine
tetraacetic acid (EDTA), pH 8.0 containing 0.05%(w/v) Fast Green (Sigma).
Embryos were electroporated at HH stage 8 and incubated in a humidified
incubator. The embryos of normal size and morphology were used for assays.
In situ hybridization and riboprobes
Section in situ hybridization and whole-mount in situ hybridization were
carried out as described previously
(Suzuki et al., 2000). All the
samples were treated in the same way throughout the study. Image acquisition
and figure processing of the sections and whole-mount samples were performed
as reported (Suzuki et al.,
2000
). We always compared electroporated experimental side with
non-electroporated control side of one and the same embryo or section.
Templates used for digoxigenin-labeled RNA probes were as follows: the 516
bp fragment of chick CBF1 (nucleotide residues 1252-1761; GenBank
Accession Number U47275), the 656 bp fragment of chick CBF2
(1191-1846; U47276), the 724 bp fragment of chick EphA3 (2267-2990;
M68514), the 541 bp fragment of chick ephrin A2 (94-634; L40932), the
681 bp fragment of chick ephrin A5 (25-705; X90377), the 491 bp
fragment of chick ephrin A6 (1-491; AF317286), the 782 bp fragment of
chick SOHo1 (34-815; S69380), the 567 bp fragment of chick
GH6 (4-570; AF227921) and the 1179 bp fragment of chick BMP2
(1-1179; AY237249). The template for the probe of Ventroptin has been
described previously (Sakuta et al.,
2001).
Northern blot analysis
Total RNA was prepared from one-third of the nasal or temporal E8 chick
retina electroporated with CBF1/RCAS, with Trizol Reagent (Invitrogen)
following the manufacturer's protocol. Control RNA were prepared from the left
eye of the same embryos. Northern blot analysis with 20 µg of total RNA was
performed as described previously (Suzuki
et al., 2000). Templates used for probe preparation were as
follows: the same fragments of chick EphA3, ephrin A2 and ephrin
A5 used for in situ hybridization, the 300 bp fragment of chick
CBF2 (nucleotide residues 1418-1717; GenBank Accession Number
U47276), and the 477 bp fragment of chick glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (218-794; K01458).
DNA pull-down assay
Chick embryonic fibroblasts were transfected with retrovirus vectors for
myc-tagged CBF1 or myc-tagged CBF1AA mutant using Lipofectamine
plus (Invitrogen), and cultured for a week. The nuclear extract preparation
and DNA pull-down assay were performed as described previously
(Mukai et al., 2002). For DNA
pull-down assays, biotininylated double-stranded oligonucleotides derived from
the sequence named B2 in the HNF1
promoter were synthesized by
Qiagen: B2 is known as the BF-1 binding sequence
(Li et al., 1995
). The eluates
of precipitates were subjected to SDS-PAGE, followed by immunoblot analyses
with the anti-myc primary antibody 9E10 (Santa Cruz Biotechnology) and
horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences).
CBF1 or its mutant proteins were visualized by chemiluminescence using ECL
plus (Amersham Biosciences) and the lumino-image analyzer LAS-1000plus
(Fujifilm).
Luciferase assay
A minimal promoter construct (pGL3ti) was first made from pGL3-basic
reporter vector (Promega) by inserting oligonucleotides carrying the
adenovirus major-late promoter TATA box and the mouse terminal
deoxynucleotidyl transferase gene initiator in the BglII site as
described previously (Jonk et al.,
1998). The BMP responsive reporter (pGL3ti-12GCCG) was constructed
by further inserting four oligonucleotides, each containing three copies of
the GCCG motif (Kusanagi et al.,
2000
), into the XhoI site of pGL3ti. Luciferase assays
were carried out using human embryonic kidney 293 cells. Cells grown in
96-well microplates at a density of 4x104 cells per well were
transiently transfected with expression plasmids in a combination of the
reporter (50 ng), receptor (10 ng) and Smads (10 ng each), together with or
without the expression plasmid for CBF1 (20 ng) or CBF1AA (20 ng).
Luciferase activity was measured with a Dual-Glo Luciferase Assay System
(Promega) using Fluoroskan Ascent FL (Labsystems). Total amounts of the
transfected DNAs were kept the same throughout the experiments by addition of
the empty vector, and firefly luciferase activities were normalized using the
Renilla luciferase activity of phRL-SV40 (Promega)
co-transfected.
Isolation of cDNA fragments encoding TGF-ß family molecules
A set of degenerative oligonucleotide primers were synthesized based on the
regions conserved among TGFß family molecules except for the growth
differentiation factor (GDF) subfamily; the 5' primer sequence was
5'-TGGVANGAYTGGATHRTNGC-3' and the 3' primer sequence was
5'-ARNGTYTGNACDATNGCRTG-3'. Another set of primers was also
synthesized based on the regions conserved among GDF molecules: the 5'
primer sequence was 5'-TGGGAYGAYTGGATHRTNGC-3' and the 3'
primer sequence was 5'-TAYAARCARTAYGARGAYATGGT-3'. The
first-strand cDNA was synthesized using total RNA isolated from E8 chick
retina with oligo dT primers. PCR was carried out for 30 cycles each
consisting of denaturation at 95°C for 60 seconds, annealing at 46°C
for 60 seconds and extension at 72°C for 60 seconds. The PCR products were
subcloned into pGEM-T Easy vector (Novagen) and sequenced.
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Results |
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Although ephrin A5 and ephrin A2 are also known to be expressed in nasal
high/temporal low gradients (Cheng and
Flanagan, 1994), their spatial and temporal expression patterns in
the retina had not been fully investigated. Thus, we examined their expression
during development of the chick retina by whole-mount and section in situ
hybridization. Expression of ephrin A5 was first detected in the
optic vesicle at HH stage 12, while asymmetrical distributions were not
detected (Fig. 1A). At stage
18, expression of ephrin A5 was absent in the dorsal and ventral
regions, but observed in the nasal and temporal retina
(Fig. 1B, part a). At this
stage, the expression was significantly stronger on the nasal than temporal
side (Fig. 1B, part a). It was
evident also by section in situ hybridization. In a section along the NT axis,
ephrin A5 expression was observed in the nasal and temporal third of
the retina, although the expression was stronger on the nasal than temporal
side (Fig. 1B, part b). Thus,
ephrin A5 was not expressed in a nasal-specific fashion at stage 18
(E3). At E6, a nasal high/temporal low gradient was clearly visible for the
first time, although the expression was also observed in the periphery of the
temporal retina (data not shown). At E8, a similar nasal high/temporal low
expression gradient and expression in the temporal periphery was observed
(Fig. 1C, parts a-c). At this
stage, ephrin A5 transcripts in the central retina were mainly
distributed in the ganglion cell layer (GCL), while those in the peripheral
regions were observed in whole cell layers like the retina at early stages
(Fig. 1C, parts a-c).
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Misexpression of CBF1 affects expression patterns of asymmetrically
distributed molecules along the NT axis in the retina
We examined the expression of the above-mentioned molecules when a
replication-competent avian retrovirus RCAS-BP vector carrying the
CBF1 gene (CBF1/RCAS) was introduced into the optic vesicle of HH
stage 8 embryos by in ovo electroporation. The transgene expression could be
detected as early as HH stage 10, prior to the stage when the polarity along
the NT axis appears to be determined, and persisted (data not shown).
At first, we examined the expression of SOHo1, GH6, EphA3 and CBF2 at HH stage 18-20 (E3) in the electroporated embryos, because these molecules clearly show graded distributions along the NT axis in the native embryos at this stage. At E3, SOHo1 and GH6 were expressed mainly in the nasal retina and the lens ectoderm but not in the temporal retina (Fig. 2A,C), while EphA3 expression was observed in the temporal retina (Fig. 2E). Here, the EphA3 expression was more faithfully complementary to that of SOHo1 than that of GH6.
|
We next examined the expression of EphA3, ephrin A5, ephrin A2 and
CBF2 at E8. At this stage, these molecules distribute asymmetrically
along the NT axis in the retina. Misexpression of CBF1 continuously
repressed the expression of EphA3 in the GCL in the temporal retina
(Fig. 3B,E; four out of five
embryos). In some embryos, a complete loss of EphA3 expression was
observed (two out of five embryos). Conversely, CBF1 misexpression
induced ectopic expression of ephrin A5
(Fig. 3G,J; five out of five
embryos) and ephrin A2 (Fig.
3L,O; five out of seven embryos) in the GCL in the temporal
retina: In the control retina, the expression of these ephrins is weak in the
GCL in the temporal retina (Fig.
3I,N). The expression of CBF2 in the temporal retina was
completely repressed by the misexpression of CBF1
(Fig. 3Q,T; five out of six
embryos). We further examined the effects of misexpressed CBF1 on
expression of the EphA4 receptor, which is uniformly expressed in the
developing retina (Holash and Pasquale,
1995; Dütting et al.,
1999
; Hornberger et al.,
1999
). When CBF1 was misexpressed in the developing
retina, the expression of EphA4 was not affected (data not
shown).
|
Repressing construct of CBF1 similarly regulates expression of SOHo1,
GH6, EphA3, CBF2, and ephrin A5 as the wild-type, but not expression of ephrin
A2
We next analyzed the mode of action of CBF1. As BF-1 is suggested to act as
a transcriptional repressor (Li et al.,
1995; Bourguignon et al.,
1998
), we examined whether this function is essential to regulate
the expression of SOHo1, GH6, EphA3, CBF2, ephrin A5 and ephrin
A2 in the developing retina. We designed a RCAS vector (CBF1-eve/RCAS)
expressing a chimeric protein that consists of an even-skipped repression
domain (Han and Manley, 1993
)
and a winged-helix DNA binding domain (WH domain) of CBF1 as shown in
Fig. 4A. It has been
demonstrated that nuclear localization of CBF1 is determined by a sequence
within the WH domain (Chang et al.,
1996
). Consistent with this report, the chimeric protein was found
to be concentrated in the nucleus when expressed in the chick embryonic
fibroblasts (Fig. 4B), and in
the chick retinal cells (data not shown).
|
Mutant CBF1 deficient in DNA binding ability exerts similar effects
on expression of SOHo1, GH6, EphA3, CBF2 and ephrin A2 as the wild-type CBF1,
but not on expression of ephrin A5
We next tested whether DNA binding of CBF1 is essential for the activity to
regulate the expression of asymmetrically distributed molecules. Two amino
acid residues in the WH domain of murine BF-1, Asn219 and His223, are
suggested to be important for binding to DNA, and it has been shown that the
mutant generated by changing these two residues to alanine (AA mutant) is
devoid of DNA binding ability (Dou et al.,
2000). We prepared CBF1AA/RCAS to express the mutant
CBF1 protein in which Asn189 and His193 were changed to alanine: These two are
equivalent to Asn219 and His223 in murine BF-1, respectively
(Fig. 5A). We examined the
ability of the wild-type CBF1 and CBF1AA mutant proteins to bind to
the BF-1 binding sequence by DNA pull-down assay beforehand. The wild-type
protein was co-precipitated with the double-stranded DNA fragment containing
the BF-1 binding sequence (Fig.
5B, part a). However, CBF1AA was not co-precipitated
(Fig. 5B, part a), although
sufficient amounts of mutant proteins were expressed
(Fig. 5B, part b). Thus, the
mutation at Asn189 and His193 abolishes the ability of CBF1 to bind the BF-1
binding sequence.
|
CBF1 inhibits the BMP signaling in the retina
We previously demonstrated that a BMP antagonist, Ventroptin, promotes
expression of ephrin A2 but not ephrin A5 in the developing
retina (Sakuta et al., 2001).
In addition, Noggin, a structurally unrelated BMP antagonist, has the same
activity as Ventroptin in the promotion of ephrin A2 expression
(Sakuta et al., 2001
). These
results indicate that BMP signaling regulates the expression of ephrin
A2, which prompted us to examine the possibility that CBF1 modulates BMP
signaling. BMPs transduce their signals into the cell through a family of
mediator proteins known as Smads. Upon phosphorylation by the BMP receptors,
Smad1 associates with Smad4 and translocates into the nucleus where the
complex recruits DNA-binding proteins to activate specific gene
transcription.
Indeed, the reporter activity was stimulated when a constitutively active BMP receptor, Smad1 and Smad4 expression plasmids were co-transfected into HEK293 cells with a reporter construct that consists of a BMP-responsive promoter and the luciferase gene (Fig. 6A). Using this reporter assay system, we examined whether CBF1 interferes with BMP signaling. We found that CBF1 completely inhibited the activation of the reporter gene by the Smad complex (Fig. 6A). In addition, the CBF1AA mutant also blocked the activation of the reporter gene (Fig. 6A). These results indicate that CBF1 inhibits BMP signaling by a DNA binding-independent mechanism.
|
Weak expression of BMP2 was first observed in the dorsotemporal retina at E5 (data not shown). From E6 onwards, a temporal high/nasal low expression gradient as well as a dorsal high/ventral low gradient of BMP2 expression became evident (Fig. 6B, parts a,b). The BMP2 expression was positive in the GCL and inner nuclear layer in the central retina, and in the whole layers in the periphery (Fig. 6B, parts a,b). This is somewhat different from that of Ventroptin: Expression of Ventroptin is not observed in the GCL. The BMP2 expression was restricted to the dorsotemporal one third of the retina, while the Ventroptin expression expanded into the dorsotemporal retina (Fig. 6B, parts a-e).
BMP2 controls ephrin A2 expression along the NT axis
The complementary expression patterns of Ventroptin and
BMP2 suggest that these molecules repress the expression of their
counterpart. To test this possibility, we examined effects of misexpression of
these molecules. When Ventroptin was misexpressed in the retina by in
ovo electroporation, BMP2 expression in the temporal regions was
completely repressed (Fig. 6C,
part b; seven out of seven embryos). Similarly, misexpression of BMP2
inhibited expression of Ventroptin in the nasal retina
(Fig. 6C, part d; six out of
six embryos). We previously demonstrated that misexpression of
Ventroptin induces ephrin A2 expression in the temporal
retina (Sakuta et al., 2001).
Therefore, we next tested effects of BMP2 misexpression on ephrin
A2 expression. Misexpression of BMP2 repressed expression of
ephrin A2 in the nasal retina
(Fig. 6D, part b; seven out of
nine embryos), suggesting that the counteraction between Ventroptin and BMP2
controls the graded distributions of ephrin A2 along the NT axis.
Finally, to address whether CBF1 is involved in the regulation of BMP
signaling in the developing retina, we examined the expression of
Ventroptin and BMP2 when CBF1 was misexpressed in
the retina. Expression of BMP2 in the GCL was repressed by
misexpression of CBF1, while expression in the inner nuclear layer
was not reduced (Fig. 6E, part
b; five out of nine). On the other hand, expression of Ventroptin
appeared not to be altered by misexpression of CBF1 (data not shown;
six out of six embryos). These phenotypes were recapitulated when the
CBF1AA mutant was misexpressed
(Fig. 6E, part c; four out of
nine embryos). These results suggest that CBF1 represses the expression of
BMP2 in the ganglion cells by inhibiting BMP signaling. In
conclusion, these results are consistent with our view that CBF1 regulates the
ephrin A2 expression through inhibition of BMP signaling
(Sakuta et al., 2001).
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Discussion |
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This strategy allowed the immediate and sustained expression of the
transgene, because the mRNA is directly transcribed from the incorporated RCAS
vector DNA shortly after the gene transfer, which is followed by the
production of retroviral particles and widespread infection in the retina.
When CBF1/RCAS was introduced by in ovo electroporation into the optic vesicle
of HH stage 8 embryos, the transgene expression was detected at 6 hours after
the electroporation, as early as HH stage 10, just before the stage at which
the polarity along the NT axis appears to be determined by the endogenous CBF1
expression (Dütting and Meyer,
1995; Dütting and Thanos,
1995
; Thanos et al.,
1996
), indicating that this in ovo electroporation method fulfills
the requirements for the functional study of CBF1.
CBF1 controls all of the asymmetrically distributed molecules along
the NT axis in the developing retina
We show that misexpression of CBF1 in the temporal retina results
in induction of SOHo1, GH6, ephrin A2 and ephrin A5, and
down-regulation of EphA3 and CBF2 expression
(Fig. 7). These results
indicate that all of the asymmetric molecules so far identified to be involved
in the formation of the topographic map along the AP axis
(Yuasa et al., 1996;
Drescher et al., 1995
;
Nakamoto et al., 1996
;
Schulte and Cepko, 2000
;
Dütting et al., 1999
;
Hornberger et al., 1999
) are
downstream genes of CBF1. Endogenous expression of CBF1
begins around HH stage 8-9 in the retina, and is confined to the nasal retina
prior to HH stage 11 (Yuasa et al.,
1996
; Yamagata et al.,
1999
), preceding all other genes that are asymmetrically expressed
along the NT axis during development. CBF2 is first asymmetrically
expressed in the temporal retina at HH stage 11
(Yamagata et al., 1999
).
Expression of SOHo1 and GH6 is restricted in the nasal
retina from HH stage 12-14 (Yamagata et
al., 1999
; Schulte and Cepko,
2000
). EphA3 expression is confined to the temporal
retina at HH stage 15 (Yamagata et al.,
1999
; Schulte and Cepko,
2000
). In addition, as shown in this report, graded distributions
of ephrin A5 and ephrin A2 are observed after HH stage 18
(E3) and E5, respectively. The order of onset of these genes thus supports the
view that CBF1 determines the NT specificity, thereby controlling the
expression of a series of asymmetrically distributed molecules in the
developing retina. It is likely that misexpression of CBF1 endues the
temporal retinal ganglion cells with the character of nasal ganglion cells by
inducing the expression of SOHo1, GH6, ephrin A5 and ephrin
A2, and inhibiting the expression of CBF2 and
EphA3.
|
CBF1 regulates the expression of asymmetrically distributed molecules
through multiple mechanisms
BF-1 has been shown to act primarily as a transcriptional repressor
(Li et al., 1995;
Bourguignon et al., 1998
). In
addition to the functions which are dependent on their ability to bind to DNA,
several transcription factors have been reported to function even when their
DNA binding-ability is deficient (Beato et
al., 1995
; Um et al.,
1995
; Plaza et al.,
1997
; Schuur et al.,
2001
; Zhao et al.,
2001
). This DNA binding-independent function is also observed in
murine BF-1 (Dou et al., 2000
;
Rodriguez et al., 2001
). To
clarify the modes of action of CBF1, we examined effects of misexpression of
CBF1-eve and CBF1AA mutants. As a result, we found that the
downstream asymmetric molecules can be classified into three distinct
categories by the mode of action of CBF1
(Fig. 7A): (1) those whose
expression is affected only by the CBF1-eve mutant (written in red), (2) those
whose expression is affected only by the CBF1AA mutant (in green),
and (3) those whose expression is affected by both the CBF1-eve and
CBF1AA mutants (in blue).
Ephrin A5 belongs to the first category. Consistent with the reports that CBF1 acts as a transcriptional repressor, misexpression of CBF1-eve shows similar activity for the expression of ephrin A5 as the wild-type CBF1. Because misexpression of CBF1AA mutant protein has no effect on the expression of ephrin A5, the expression of this molecule is regulated only by the function of CBF1 as a transcriptional repressor (Fig. 7A). How does CBF1, a transcriptional repressor, induce the expression of ephrin A5? The simplest explanation is that CBF1 functions as a transcriptional repressor of some other putative intermediate repressor(s) which inhibits the expression of ephrin A5 (indicated by `X' in Fig. 7B). The onset of ephrin A5 expression appears to be triggered first by a transcription activator(s) at stage 12 to yield the homogeneous expression in the retina (Fig. 1A). Subsequently, the factor X may begin to be expressed in the temporal retina where CBF1 is absent, and represses expression of ephrin A5 in the temporal retina. In misexpressed embryos, ectopic CBF1 probably abrogates the repression by factor X from the temporal retina, and then expression of ephrin A5 is re-induced by the transcriptional activator(s) which is ubiquitously expressed in the retina.
Ephrin A2 belongs to the second category. Surprisingly, expression
of ephrin A2 is not affected by misexpression of the CBF1-eve fusion
protein. By contrast, CBF1AA exerted the same effect on the
expression of ephrin A2 as the wild-type protein. Therefore,
expression of ephrin A2 is regulated only by a DNA
binding-independent mechanism, which clearly contrasts with ephrin A5
whose expression is regulated only by a DNA binding-dependent mechanism
(Fig. 7A). In the retina, both
ephrin A2 and ephrin A5 are expressed in a nasal
high/temporal low gradient. Although these ephrins commonly function as
ligands for EphA receptors, their expression patterns are not identical. The
gradient of ephrin A2 expression in the GCL is gentle and wide along
the NT axis, when compared with that of ephrin A5 expression. In
addition, the onsets of their expression are also distinct: ephrin A5
begins to be expressed at HH stage 12 (E2), but ephrin A2 does not
appear until E5. These differences may reflect the distinct regulatory
mechanisms for their expression. We previously demonstrated that expression of
ephrin A2 is induced by misexpression of Ventroptin, a BMP4
antagonist (Sakuta et al.,
2001). We show here that BMP2 begins to be expressed in a
temporal high/nasal low gradient along the NT axis from E5. Furthermore,
Ventroptin counteracts BMP2 as well as BMP4, and BMP2 represses
ephrin A2 expression. Therefore, it is likely that the DNA
binding-independent control mechanism used by CBF1 on ephrin A2 is
relevant to BMP2 signaling (Fig.
7B). This view is consistent with the expression patterns of
BMP2 and ephrin A2 in the retina: BMP2 expression
is restricted to the temporal third of the retina, while the expression of
ephrin A2 expands into the temporal side, making a gentle gradient
along the NT axis. In the present study, we demonstrated by in vitro reporter
assay that CBF1 indeed interferes with BMP signaling through a DNA
binding-independent mechanism. In addition, misexpression of CBF1
represses the expression of BMP2 in the GCL. This notion is supported
by a recent study describing the interaction between BF-1 and Smad1
(Rodriguez et al., 2001
):
Smad1 is an intracellular signaling intermediate for the receptors for BMP2
and BMP4 (Piek et al., 1999
),
and it is reported that BF-1 interferes with BMP signaling via interaction
with Smad molecules (Rodriguez et al.,
2001
). The loss of the BF-1 gene in mice leads to ectopic
expression of BMP4 in the dorsal telencephalic neuroepithelium
(Dou et al., 1999
). All these
results indicate that CBF1 interferes with the BMP signaling.
SOHo1, GH6, EphA3 and CBF2 belong to the third category.
The expression of these molecules is affected by misexpression of both the
CBF1-eve fusion protein and CBF1AA mutant protein, indicating that
the expression of these molecules is regulated by CBF1 through not only a DNA
binding-dependent but also a DNA binding-independent mechanism
(Fig. 7A). Regulation of these
four molecules, especially the induction of SOHo1 and GH6 in
the temporal retina, by the misexpressed CBF1-eve fusion protein or
CBF1AA mutant protein appeared to be milder than that by the
wild-type CBF1 (compare Fig. 2B and
2D with Fig. 4C,
parts a,b and Fig. 5C, parts
a,b). Thus, cooperation of DNA binding-dependent and DNA binding-independent
mechanisms of CBF1 should be necessary for the full regulation of
SOHo1 and GH6 by CBF1. As SOHo1 and GH6
repress the expression of EphA3 in the retina
(Schulte and Cepko, 2000),
repression of EphA3 expression by CBF1 likely resulted from
induction of SOHo1 and GH6 by CBF1. It might also
be possible that CBF1 directly downregulates the expression of
EphA3 not through SOHo1 or GH6. This is, however,
unlikely because EphA3 is homogenously expressed in the optic vesicle
at HH stage 12, when CBF1 is topographically expressed
(Yamagata et al., 1999
). In
addition, CBF1 and EphA3 are co-expressed in the same region
of the chick forebrain (H.T., T.S., H.S. and M.N., unpublished). Thus,
CBF1 probably represses EphA3 expression through induction
of SOHo1 and GH6 expression. To explain the induction of
SOHo1 and GH6 expression by CBF1 through a DNA
binding-dependent mechanism, the existence of a putative intermediate
repressor(s), which inhibits expression of SOHo1 and GH6,
must be postulated (indicated by `Y' in
Fig. 7B). As in the regulation
of ephrin A5 expression, misexpressed CBF1 abrogates the
repression by factor Y, and then the expression of SOHo1 and GH6 is
induced by a transcriptional activator(s) which is ubiquitously expressed in
the retina.
Expression of SOHo1, GH6 and CBF2 is also regulated by
CBF1 through a DNA binding-independent mechanism
(Fig. 7A). As expression of
SOHo1, GH6 and CBF2 is not affected by misexpression of
Ventroptin or BMP2
(Sakuta et al., 2001) (H.T.,
T.S., H.S. and M.N., unpublished), these molecules are not regulated by BMP
signaling. Several transcription factors are known to regulate the expression
of their downstream target genes by interacting with other transcription
factors (Beato et al., 1995
;
Um et al., 1995
;
Plaza et al., 1997
;
Schuur et al., 2001
;
Zhao et al., 2001
). Thus, it
is probable that CBF1 regulates expression of SOHo1, GH6 and
CBF2 via interaction with other transcription factors which directly
regulate the expression of SOHo1, GH6 and CBF2.
What is the meaning of using two distinct mechanisms, DNA binding-dependent
and DNA binding-independent (Fig.
7A), to regulate the expression of topographic molecules? It may
be for security to maintain regulation of the EphA/ephrin A system: For
example, if the DNA binding-dependent mechanism is lost by a mutation in the
WH domain, CBF1 can still regulate the asymmetrical distribution of
EphA3 and ephrin A2 through the remaining DNA
binding-independent mechanism. However, if the DNA binding-independent
mechanism is lost, CBF1 can still regulate the asymmetrical distribution of
EphA3 and ephrin A5 through the DNA binding-dependent
mechanism. Mice deficient in ephrin A2 and ephrin A5
suggests the importance of this redundancy: In the single knockout mice, a
substantial part of the retinal axons normally projected onto the SC
(Frisén et al., 1998;
Feldheim et al., 2000
). As the
total EphA/ephrin A system plays an essential role in the formation of the
retinotectal map, this dual regulatory system by CBF1 might have evolved
during the evolution of the visual system.
BMP signaling plays pivotal roles in the topographic mapping along
both axes
BMP2 and BMP4 are implicated in many different processes of vertebrate
development (for a review, see Balemans and
Hul, 2002). The extent of BMP action is controlled in part by the
influence of antagonists such as Noggin, Chordin and Follistatin.
Counteractions between BMPs and their antagonists, which appear to make a
gradient of BMP signaling, are suggested to play important roles in vertebrate
development.
At the early developmental stages of the eye (stage 11 to E5), the
counteraction between Ventroptin and BMP4 governs the regional specification
in the retina along the DV axis (Sakuta et
al., 2001). In the embryos misexpressing Ventroptin in
the retina, projections of almost all the dorsal retinal axons shifted
dorsally on the tectum (Sakuta et al.,
2001
). At the later stages (E6 onward), in proportion to the
disappearance of BMP4 expression from the dorsal retina, Ventroptin
begins to be expressed in both a nasal high/temporal low and a ventral
high/dorsal low gradient in the retina. In the present study, we found that
BMP2 begins to be expressed from E5 in a pattern complementary to
that of Ventroptin along both the NT and DV axes. In addition,
BMP2 and Ventroptin repressed the expression of each other
in the developing retina. Therefore, at the later stages when retinal axons
actively begin to invade the tectum, the counteraction between Ventroptin and
BMP2 appears to play an important role in the topographic projection along the
AP axis in addition to the DV axis. This view is consistent with our previous
results that almost all temporal axons labeled with DiI extended to the
posterior end of the tectum, when Ventroptin was misexpressed, owing
to the induction of ephrin A2 expression in the temporal retina
(Sakuta et al., 2001
).
In the present study, we showed that misexpression of CBF1 moderately inhibits expression of BMP2, while misexpression of Ventroptin does it completely. These results suggest that BMP signaling in the nasal retina is mainly repressed by Ventroptin. Thus, CBF1 may fine-tune BMP signaling. From this point of view, CBF1 presumably triggers the shift in the expression pattern of Ventroptin to the double gradient. Around E5, BMP4 expression in the dorsal retina begins to disappear. Concomitantly, the expression of Ventroptin would be enhanced more in the nasal retina because of the inhibition of BMP signaling by CBF1, which leads to the nasal high/temporal low expression gradient of Ventroptin. Then, BMP2 begins to be expressed with a double-gradient, in a fashion counter to Ventroptin, and this counter gradient would be fixed afterwards. Our study thus demonstrated that two distinct BMP signals sequentially play pivotal roles in the topographic projection along the two axes.
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ACKNOWLEDGMENTS |
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