Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
* Author for correspondence (e-mail: Walter.Gehring{at}unibas.ch)
Accepted 12 April 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: sine oculis, so, hedgehog, hh, eyes absent, eya, eyeless, ey, Ocelli, Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determination of the eye primordium requires several nuclear proteins that
are known to act as transcriptional regulators. The Drosophila Pax6
gene eyeless (ey) was the first gene shown to display the
capacity to induce ectopic eye morphogenesis upon ectopic expression
(Halder et al., 1995). A
second Drosophila Pax6 gene, twin of eyeless (toy),
like ey encodes a protein with two DNA-binding domains
(Czerny et al., 1999
). Further
genes involved in early eye determination are eyegone (eyg),
which also shows some similarity to Pax6
(Jun et al., 1998
;
Chao et al., 2004
;
Dominguez et al., 2004
),
eyes absent (eya) and dachshund (dac),
both encoding nuclear proteins (Bonini et
al., 1993
; Mardon et al.,
1994
), and sine oculis (so)
(Cheyette et al., 1994
).
Analyses of the expression patterns of these genes combined with genetic
approaches have revealed a complex genetic regulation network during compound
eye development. toy is the first of the mentioned genes to be
expressed during embryogenesis and activates ey in the eye primordium
(Czerny et al., 1999
).
so is required later for the development of the entire visual system,
including the compound eyes, the ocelli, the optic lobe of the brain and the
larval photoreceptors designated as Bolwig's organ
(Cheyette et al., 1994
;
Serikaku and O'Tousa, 1994
;
Pignoni et al., 1997
).
eya expression comes up later in the compound eyes and can be found
in the ocelli-specifying region in third instar eye imaginal discs. Recently,
eya has been shown to have protein phosphatase activity
(Li et al., 2003
;
Tootle et al., 2003
).
so and eya are both required for compound eye and ocellus
formation, as the respective mutants lack both visual systems
(Cheyette et al., 1994
;
Zimmerman et al., 2000
).
so, eya and dac have been shown to be regulated by
ey (Halder et al.,
1998
; Niimi et al.,
1999
; Zimmerman et al.,
2000
). SO and DAC have been proposed to function as co-factors for
EYA, and genetic studies in Drosophila have demonstrated synergistic
interactions between so, eya and dac during eye development
(Chen et al., 1997
;
Pignoni et al., 1997
). The
respective protein complexes feed back on ey expression and
eya and dac, like ey and toy, are capable
of inducing ectopic eye morphogenesis
(Bonini et al., 1993
;
Bonini et al., 1997
;
Pignoni et al., 1997
).
Although much knowledge has been gathered during the last years about the complex genetic network that orchestrates eye development, only a small number of observed regulatory interactions have been analysed down to the level of DNA-protein interactions. Analysis of further components controlling expression patterns of genes involved in early eye development should therefore provide important details on the genetic hierarchy that mediates eye specification and may help to identify direct targets of the known eye specification genes.
Among the already described direct interactions, toy has been
shown to induce ey expression by an eye-specific enhancer in
embryonic eye precursor cells, but not during larval stages in the later
emerging eye imaginal disc (Czerny et al.,
1999). However, ey, together with toy, directly
regulates so expression by an eye-specific enhancer that is deleted
in the so1 mutant allele
(Niimi et al., 1999
;
Punzo et al., 2002
).
Furthermore, ey-regulated, eye-specific enhancers have been
identified using deletions within the eya gene locus
(Zimmerman et al., 2000
).
In this study, we address the regulatory potential of a previously
described so7 enhancer fragment during ocellar morphogenesis. So7
represents the DNA fragment that is deleted in the so1
mutation and contains the ey- and toy-regulated enhancer
element so10 (Punzo et al.,
2002). We show that a 27 bp fragment within so7, soAE, is
sufficient to expand expression of a reporter gene to the ocellar region when
fused to the so10 enhancer and, consequently, this so10-soAE enhancer fragment
is sufficient to rescue the eyeless and the ocelliless phenotype of
so1/so1 flies when used as a driver
for so. Furthermore, we show that soAE is a direct target of
so in compound eye and ocellar development and that the
autoregulatory feedback of so on its own expression is required for
the ocellus-specific expression of so.
By analysing the DNA-binding specificity of SO in more detail, we were able to identify those nucleotides that are essential for SO-soAE interaction. Using the emerging cis-regulatory signature for so-dependent regulation, we performed a genome-wide search for additional putative so-target genes. Sequences that fit our selection criteria were identified in the ey and hedgehog (hh) loci. We show that both these genes contain eye-specific enhancers that are directly regulated by so. Our results emphasize the importance of autoregulatory feedback loops in morphogenesis and development.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specific genotypes generated: (1) eyFLP; FRT42D, ubiquitinGFP, (2) so2/so2; so10-soAE-lacZ, (3) so2/so2; so7-lacZ, (4) UAS-so/UAS-so;UAS-eya/UAS-eya and (5) so1/so1; so10-soAE-so.
lacZ reporter plasmids and rescue constructs were introduced into w1118 by standard P-element transformation procedures. Three to 10 independent transgenic lines were established for each construct and tested for expression or rescue potential.
Antibody staining on discs was performed according to Halder et al.
(Halder et al., 1998). Primary
antibodies were anti-EyaMab10H6, 1:10
(Bonini et al., 1997
), Rabbit
anti-ß-galactosidase, 1:500 (Promega). Secondary antibodies used were
from Jackson ImmunoResearch Laboratories: Cy5
-rabbit (1:400), Alexa586
-mouse (1:400).
To detect ß-galactosidase activity, third instar larval imaginal discs were fixed and subjected to a standard X-gal colour reaction for 2 hours at 37°C.
Reporter transgenes and rescue construct
Inserts of the reporter constructs were obtained by PCR, using so7 as a
template, and subcloned into the lacZ pCß vector
(Niimi et al., 1999). For the
rescue construct a modified pUAST vector
(Brand and Perrimon, 1993
) was
used. The 5 x UAS sequence was replaced by so10-soAE. so cDNA
was placed downstream of hsp70 within the polylinker resulting in
so10-soAEhsp70so in the pUAST backbone
(so10-soAE-so).
For the constructs: ey enhancer, B4M and B4M SOmut, the sequences given in Fig. 5 were used. A BamHI and a KpnI site was added at the 5' and 3'-end, respectively, and used for subcloning into the lacZ pCß vector.
The hh1 (bar-3) sequence was obtained by PCR
on genomic DNA of wild-type (wt) flies by using the following primer set:
5'-CTGTGCGCTCGAGTGGGCCACACAGGGTGGG-3'; rightward orientation,
5'-CGGCCCGTCTCAGATCTCGGATCTGAGATC-3' leftward orientation.
Mutations were introduced by PCR. For the deletion construct
hh1 5',
5'-GGGGTACCCAAGACAAGTAATCCCCCACCCTCGC-3' was used as rightward
oriented primer (the SO site is mutated by changing GAG to CCC).
so2 mutant
Genomic DNA was amplified by PCR from
so2/so2 flies and sequenced. The
sequences were confirmed on independent amplification events. Genomic DNA
isolation was performed according to Bui et al.
(Bui et al., 2000a). Primers
used for mapping the so2 deletion were:
5'-GAAGGGCACTGCTTACTGAGAGCTCG-3',
5'-GCCCATCGAATCCGCATCTCCCCCAG-3' rightward orientation;
5'-GCGCACACTCGACAAATTTGCGATCTGGC-3' leftward orientation. Primers
are located at positions 2355, 3116 and 6218, respectively, within the last
intron. Nucleotides 3983-5181 are deleted in so2 (the
first nucleotide of the last intron is set as 1).
Southern blotting was performed according to Sambrook and Russel
(Sambrook and Russel, 2001).
Genomic DNA was digested using ClaI, EcoRV and
XhoI. As probes, DIG-labelled PCR products of so10 and so9 were used.
so10 and so9 are described previously
(Punzo et al., 2002
).
Transfections and reporter gene assays
Drosophila S2 cells were maintained in Schneider's insect medium
(Invitrogen) supplemented with 10% fetal calf serum and were transfected with
the Effectene Transfection Reagent (Qiagen). For reporter gene assays 2
x106 cells were transfected with a total of 200 ng plasmid
DNA (20 ng reporter plasmid, 5 ng of a plasmid constitutively expressing
firefly luciferase, the indicated amounts of expression plasmids and the
parental vector pAc5.1B/V5His, to bring the total amount of DNA to 200 ng).
Cells were lysated 48 hours after transfection and lysates were assayed for
ß-galactosidase and luciferase activity as described previously
(Muller et al., 2003).
Electrophoretic mobility shift assays
Radioactively labelled probes were generated by annealing and filling in
partially overlapping oligonucleotides in the presence of
(-32P)ATP. Binding reactions were carried out in 20 µl of
100 mmol/l KCl, 20 mmol/l HEPES pH 7.9, 20% glycerol, 1 mmol/l DTT, 0.3% BSA,
0.01% NP40 containing 10,000 cpm probe and 1 µg dIdC. As a protein source,
full-length SO protein was synthesized in reticulocyte lysates using the T7
promotor according to the manufacturer's specification (Promega). For the
binding reaction, 1 µl of a standard 50 µl reaction was used. After
incubation for 30 minutes at 4°C, the reactions were analysed by
non-denaturing 6% polyacrylamide gel electrophoresis followed by
autoradiography. For the cold competition experiments, the proteins were first
incubated with a 100 x molar excess of unlabeled double-strand
oligonucleotides for 10 minutes at RT, followed by incubation with the
radiolabelled probe at 4°C for 30 minutes.
|
Alignments of different Drosophila species were obtained from http://hanuman.math.berkeley.edu/genomes/drosophila.html.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
So10 (400 bp) and so9 (1.2 kb) (Fig.
1A) are subfragments of so7. so10 mediates expression in the
compound eye part of third instar eye-antennal imaginal discs and contains the
previously described ey- and toy-specific binding sites
(Fig. 1D). These include five
binding sites bound by toy. Three of these are also binding sites for
ey and are important for compound eye development, whereas the two
toy-specific sites are required for ocellar development
(Niimi et al., 1999;
Punzo et al., 2002
).
Consistent with its expression pattern, so10 is able to rescue the eyeless
phenotype but not the ocelliless phenotype of so1 mutant
flies (Punzo et al.,
2002
).
so9-mediated expression appears at the posterior margin of the eye disc (similar to Fig. 1C). When combined with so10, so9 provides additional transcriptional input to expand the expression to the ocellar region.
Trans-acting factors that bind the cis-regulatory so9 element and cooperate with toy to confer expression in the ocellar region were unknown when this work was started. In order to locate the binding sites of such additional transcription factors we first aimed at the isolation of the smallest version of so9 that still would be able to drive expression of a lacZ reporter to the ocellar region of eye imaginal discs when combined with so10 (Fig. 1A). Our search resulted in the identification of a fragment as small as 27 bp (Fig. 1A, number 21), which in the following text will be referred to as soAE (sine oculis autoregulatory element).
|
sine oculis is able to recognize its own enhancer
In soAE three sequence motifs can be found that are reminiscent of
well-known transcription-factor-binding sites. These are a motif related to
the Pax6-consensus-binding site
(Epstein et al., 1994), a
TAAT-motif that is a hallmark of most homeodomain recognition sequences and a
GATA-motif. We mutated these sites, and tested the respective fragments
(so10-mutPAX, so10-mutHD, so10-mutGATA) for the resulting expression
patterns.
so10-mutPAX-mediated expression was indistinguishable from the so10-soAE expression pattern (Fig. 2C). Conversely, mutating the putative homeodomain-binding site (so10-mutHD) or the GATA sequence (so10-mutGATA) resulted in loss of reporter gene expression in the ocellar region (Fig. 2A,B).
We then oligomerized soAE four times, to boost its expression. As a result, an expression signal became apparent posterior and slightly in front of the MF (Fig. 2E) as well as in the optic lobe (data not shown). However, 4xsoAE was not able to drive expression in the ocellar region. Additional copies of soAE did not lead to a further strengthened expression. Expression of 10xsoAE, for example, appears blotchy and weaker in the eye disc than expression of 4xsoAE (Fig. 2F).
As the expression pattern of 4xsoAE is reminiscent of so-expression in the eye disc, we hypothesized that so itself might be the soAE regulating factor. Both expression patterns show a signal in the optic lobe as well as posterior to, within and in a few cells in front of the MF. The only difference is the ocellar expression of so, which cannot be seen using the 4xsoAE reporter construct.
The idea that so itself is the soAE-binding factor was further
supported by previous work in which Hazbun et al. showed that SO binds in
vitro to (C/T)GATA (Hazbun et al.,
1997), a motif that is present in soAE
(Fig. 4C nt. 7-11).
To determine experimentally whether the expression pattern of the mutated fragments correlates with the ability of these fragments to bind SO in vitro we performed electrophoretic mobility shift assays (EMSAs). SO protein was able to shift radiolabelled mutPAX but failed to bind to mutHD and mutGATA DNA fragments (Fig. 2H; see also Fig. 4A).
These results, in combination with our in-vivo data, strongly suggest that so itself is responsible for the ocellus-specific expression of so10-soAE.
so10-soAE-lacZ and so7-lacZ are not expressed in the ocellar region of so2 mutant flies
To further test this hypothesis, we moved on to a genetic approach.
so2 is a hypomorphic allele that originated as a
spontaneous partial reversion of so1
(Lindsley and Zimm, 1992).
Different from so1 adult flies, which completely lack
compound eyes and ocelli, so2 flies develop compound eyes
that range from normal appearance to slightly reduced shapes but still lack
ocelli completely. In so2/so1 flies,
eyes are of intermediate size (Heitzler et
al., 1993
). Because of the common origin and the genetic
interaction of these two alleles, we tested if there is a mutation in
so2 flies that affects the genomic so9/so10 sequences.
Using PCR on genomic so2 DNA, we found a deletion of 1.2
kb that indeed affected so7. We further confirmed this result by Southern
blotting (data not shown). The deletions of so1 and
so2 partially overlapped
(Fig. 1A) and in
so2, four of the five previously described
Pax6-binding sites (Punzo et al.,
2002
) were missing. In fact, both binding sites exclusively
recognized by TOY were deleted. According to Punzo et al.
(Punzo et al., 2002
), these
toy-specific binding sites within the so10 enhancer fragment are
required for ocellus development. The sequence representing so9, which
contains the soAE fragment, appeared not to be affected by the
so2 deletion.
Next we took advantage of so2 mutant flies to test whether the cis-regulatory potential of soAE depends on SO protein in vivo. Therefore we analysed so7- and so10-soAE-mediated expression in the ocellar region in so2 mutant flies. As expected, so7-lacZ and so10-soAE-lacZ expression was lost in the ocellar region of so2 mutant flies (Fig. 3D), supporting the idea of so being required for the ocellus-specific expression of so7 and so10-soAE further. The absence of reporter gene expression cannot be explained by a loss of ocellus-specific precursor cells, as eya expression, which represents a marker for this specified cell population, was detectable in so2 mutant flies in the prospective ocellar region (Fig. 3E).
Taken together, toy and so binding to so10 and soAE, respectively, seem to cooperatively drive so-expression in the ocellar region of third instar eye discs.
|
These data strongly suggest that feedback of so on its own enhancer is needed for ocellus development.
4xsoAE is not expressed in so3 clones
To assess whether soAE is a target of so also in the compound eye
part of the eye disc, we tested the expression of the 4xsoAE reporter
construct in cells homozygous for so3, a null allele of
so (Cheyette et al.,
1994). so3 mutant cells, however, tend to
overproliferate, fail to differentiate into neurons and subsequently die
(Pignoni et al., 1997
). Hence,
to be able to analyse reporter gene activity in living cells within
so3 clones we tested them for eya expression.
Eya is a suitable marker for viable cells in so3
mutant clones for the following reasons. First, so and eya
are both targets of ey and show the same expression pattern in third
instar eye discs (Halder et al.,
1998
; Niimi et al.,
1999
; Bui et al.,
2000b
). Both are expressed in a few cells anterior to the MF,
within the MF, and in the differentiating photoreceptors posterior to the MF
(Curtiss and Mlodzik, 2000
).
Second, SO and EYA proteins form a complex that works as a transcriptional
activator when the proteins are co-expressed
(Pignoni et al., 1997
;
Silver et al., 2003
). Third,
so1 mutant eye discs still express eya, whereas
in eya1 mutants, expression of so is lost
(Halder et al., 1998
).
Finally, so can be induced by eya in third instar eye
imaginal discs (Curtiss and Mlodzik,
2000
). For these reasons we assume eya-positive-cells of
third instar eye discs also express so during normal development.
Therefore, only eya-expressing cells within so3
clones were examined in our assay. In fact, in eya-expressing cells
within so3 clones, expression of the 4xsoAE reporter
construct was lost (Fig. 3F-I,
the clones are negatively marked by the absence of ubiquitin-GFP expression;
Fig. 3G). This strongly
suggests that SO protein in general is required for activation of the soAE
element in the eye field.
|
We induced ectopic eye development by combining a dpp-GAL4 driver with UAS:so, UAS:eya or both of them and tested whether the reporter construct 4xsoAE-lacZ was induced ectopically. As expected, ectopic so alone did not result in reporter gene activity, whereas eya alone or eya combined with so in a synergistic manner was able to activate the reporter construct in wing discs (Fig. 3A-C).
In another in-vitro approach, we took advantage of Drosophila S2 cells to address whether SO and EYA proteins work cooperatively as a complex on soAE DNA to induce transcription. Consistent with the in-vivo data, our in-vitro results using S2-cells showed that SO, which has DNA-binding properties but lacks a transactivation domain, on its own was not able to activate soAE-mediated lacZ expression (Fig. 3J). Likewise, EYA, which contains a transactivation domain but lacks DNA-binding properties, also failed to induce transcription in S2 cells when expressed alone (Fig. 3J). Only when co-expressed, SO and EYA cooperatively worked as transcriptional activators on soAE (Fig. 3J). Interestingly, both SO and EYA mediated weak transactivation when the oligomerized mutated sites mutHD and mutGATA were used (Fig. 3J), despite the fact that these sites do not mediate transgene expression in vivo in the developing ocellus (Fig. 2A,B).
Defining a consensus sequence for SO-DNA interaction
To date, there is only one direct target of so described in
Drosophila, which is the Runx class transcription factor
lozenge (lz) (Yan et
al., 2003). Consistent with a previous invitro study that
addressed the DNA specificity of the SO homeodomain
(Hazbun et al., 1997
), the
authors show that the sequence (C/T)GATA plays a crucial role in SO-DNA
interaction. Another study reports that SO together with EYA is able to
transactivate by binding to an AREC3/Six4-binding site in cell culture. This
motif, however, diverges to some extent from the C/TGATA-motif
(Fig. 4C)
(Silver et al., 2003
).
Our soAE fragment harbours a CGATA motif, which is consistent with the SO-binding consensus of the lz promotor. In our experiments, however, also mutations upstream of this GATA core motif (Fig. 4C nt. 8-11) were able to abolish expression of the reporter construct in vivo and also impaired the capability of SO to shift DNA fragments in the EMSA. This observation suggested that additional sequences upstream of the GATA motif are also necessary for SO binding to its target site.
|
These in vitro experiments revealed a stretch of 13 nucleotides to be important for protein-DNA interaction of SO. There are three nucleotides, G, A, A at positions 1, 4, 9, respectively (Fig. 4A lanes 9, 12, 17 and Fig. 4C nt. 1, 4, 9), that appear to be most important for the interaction. These nucleotides, which show the strongest effects upon mutation, are found in the AREC3/Six4-binding site and are also substituted in the constructs so10-mutHD and so10-mutGATA. This provides strong evidence that these nucleotides are also important for soAE-mediated reporter gene expression in vivo (Fig. 2A,B).
Genome-wide search for potential sine oculis target genes
Combining our in-vitro data on the autoregulatory element with the known
so target sequence of lz and the AREC3/Six4-binding
site, we defined the consensus sequence GTAANYNGANAYC/G as necessary for SO
binding to DNA. This consensus sequence was taken as a basis for scanning the
Drosophila genome for similar sites (see Materials and methods). In
total, 1632 putative so targets emerged from this survey. Out of the
affected genes several candidates are already known to be involved in eye
development. In the following we will describe two of the genes that we picked
for further analysis: ey and hh.
eyeless is a direct target of so
The first soAE similar element that caught our attention was located within
the previously described eye-specific enhancer of the ey gene
(Czerny et al., 1999;
Hauck et al., 1999
). A
positive feedback loop already has been postulated on the basis of the fact
that ey is induced in ectopic eye development upon co-expression of
so and eya (Pignoni et
al., 1997
). Furthermore, the ability of so and
eya to induce ectopic eyes is lost in ey2 mutants
(Pignoni et al., 1997
). In
ey2 mutant flies, the previously mentioned eye-specific
enhancer of ey is disrupted by insertion of a transposable element
(Quiring et al., 1994
) (see
also Fig. 5A). These
experiments genetically show that so and eya are able to
feedback on ey and that this feedback loop relies on the eye-specific
enhancer of the ey gene. However, a direct interaction between SO,
EYA and the ey-enhancer has not been previously demonstrated.
The fact that the potential so target site within the eye-specific enhancer is perfectly conserved between D. melanogaster, D. pseudoobscura and two other Drosophila species (see Materials and methods), encouraged us to perform additional assays to obtain molecular evidence for a direct interaction.
First we showed that oligonucleotides containing this sequence were strong
competitors for the binding of SO to soAE in EMSA, whereas this competing
potential was lost when the GAT core (Fig.
4C nt. 8-10) of the sequence was mutated
(Fig. 4B, eyeless and
eyeless mut). We then compared the expression pattern of different
mutated versions of a 160 bp fragment, comprising the eye-specific
ey-enhancer, driving a lacZ reporter (sequences shown in
Fig. 5A). The wt enhancer
mediated expression posterior to the MF
(Fig. 5B) (see also
Hauck et al., 1999)
(Fig. 4D). By mutating the
Pax6 sites, expression in the eye disc was reduced to the posterior
margin (Fig. 5C, observed in
all four transgenic lines that were tested) (see also
Hauck et al., 1999
)
(Fig. 4F). Mutating the
so site and the Pax6 sites further reduced expression in the
eye disc (one transgenic line showed no pattern at all, five independent
transgenic lines showed weak activity similar to
Fig. 5D). These data indicate
that so directly regulates ey expression through the
eye-specific enhancer of the ey gene.
|
so and eya also have been shown to be required for
initiation and propagation of the MF
(Pignoni et al., 1997), and
both are expressed at the posterior margin before initiation and later in
front of the MF (Bonini et al.,
1993
; Serikaku and O'Tousa,
1994
). Furthermore, ectopic MFs are found in ectopic eyes induced
by so together with eya
(Pignoni et al., 1997
). These
data suggest that a feedback loop between hh and so/eya
might influence the proper initiation and propagation of the MF. Consistent
with that, hh fulfilled our criteria to be a putative SO target. Both
sites found within the hh locus showed almost perfect conservation
among seven Drosophila species (see Materials and methods) and were
able to compete for SO binding in EMSA
(Fig. 4B,C, hh first,
hh second). In addition, we found these sites to be located within an
area that is deleted in the hh1 (bar-3) mutant
allele, a weak hh allele affecting adult flies. The corresponding
deletion can be found in the first intron of the hh gene
(Mohler, 1988
;
Lee et al., 1992
). The
predominant phenotype of hh1 is a reduction of eye facets.
Therefore, the deletion leading to the hh1 allele may
affect an eye-specific enhancer of hh
(Renfranz and Benzer, 1989
).
This idea is supported by the observation of Kango-Singh et al. that in
hh1 mutant flies targeted expression of ey fails
to induce ectopic eyes (Kango-Singh et
al., 2003
).
We chose to clone 1.4 kb out of the hh1 deletion
encompassing the two so sites and ligated this fragment to the
lacZ reporter gene. Expression of the resulting
hh1-lacZ construct was found exclusively in the
eye disc in cells posterior to the MF (Fig.
6B), in perfect agreement with the observation of Lee et al. that
hh is expressed in differentiating photoreceptor cells
(Lee et al., 1992).
Next we mutated the two SO-binding sites within the
hh1-lacZ construct by replacement of GAG by CCC
(hh first, nt. 8-10 in Fig.
4C) and GAT by CCC (hh second, nt. 8-10 in
Fig. 4C), resulting in the
mutated construct hh1 SOmut-lacZ
(Fig. 6A
hh1 SOmut). In four out of 10 transgenic lines, the
resulting construct had lost its capability to induce lacZ
expression. In six out of 10 transgenic lines, weak expression in the same
pattern as the wt construct was detectable
(Fig. 6C). This residual
activity is probably due to a weak interaction of SO with the mutated binding
sites similar to that seen in our cell culture assays
(Fig. 3J). When we tested a
construct in which the first SO-binding site was deleted and the second was
mutated, this residual expression was lost completely
(Fig. 6A
hh1 5' and
Fig. 6D).
These results show that the two SO-binding sites within the first intron of the hh gene are functional in vivo and sufficient to mediate expression, reflecting the known hh expression pattern in the eye part of late third instar eye imaginal discs. This strongly suggests that hh is directly regulated by so.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As SO binds to its own enhancer and autoregulation cannot initiate
expression of a gene, the initiation of so expression in the ocellar
region must be triggered by other means. We propose the following model.
Initiation of so expression in early third instar eye discs is
mediated by ey and toy throughout the eye disc, including
the ocellar precursors. Later, after this first induction, so
cooperatively with eya can maintain its own expression in the ocellar
region by a positive autoregulatory feedback. Thus, the initiation of
so expression is mediated by so10, whereas for the maintenance of
so, soAE is required. This is supported by the observation of Punzo
et al. that so10, which is activated by ey and toy mediates
expression in early third instar larvae all over the eye disc and only later
gets restricted to the compound eye part
(Punzo et al., 2002).
In this model the specificity of so expression for ocellar
precursor cells is provided by the expression pattern of eya; EYA
protein can be found only in the ocellar region itself, where it specifically
interacts with SO, and no EYA is present in the proximity of these cells. The
importance of eya is further strengthened by the fact that
eya4 mutants show an eyeless and ocelliless phenotype
(Zimmerman et al., 2000).
Therefore, to elucidate the mechanisms that control gene expression
specifically in ocellar precursor cells, additional studies on eya
are required.
Direct feedback regulation of eyeless by sine oculis in eye development
Positioned at the top of the hierarchy of the retinal determination
network, ey is a potent inducer of ectopic eyes and is able to
directly induce so and eya. Like ey, so and
eya are able to induce ectopic eyes but only when co-expressed;
so alone fails to do so.
To accomplish this induction, eya and so need to feed back on ey, obviously by binding to the eye-specific enhancer of ey. In an ectopic situation, the feedback of so/eya on ey is strong enough to induce ey for ectopic eye formation.
The function of this feedback loop in normal eye development remains to be
elucidated. so and eya are both expressed posterior to the
furrow and are important for neuronal development
(Pignoni et al., 1997).
Nevertheless, ey is tuned down posterior to the MF. The activity of
the so-binding site in the ey gene might, therefore, be
suppressed by other factors or by so itself during cellular
differentiation posterior to the furrow. As co-expression of ey, so
and eya is elevated only in a few cells in front of the MF and within
the MF, a possible role for this feedback loop might be to boost ey
expression in front of and within the furrow, which leads to a strengthening
of so and eya expression in just a few cell rows.
For proper eye development, a well-balanced expression level of the genes
belonging to the retinal determination network is crucial. Loss-of-function
mutations, as well as overexpression of the eye specification genes ey,
eya, so or dac during eye development, impede proper
determination of the organ and result in a reduction in eye size
(Halder et al., 1998;
Curtiss and Mlodzik, 2000
).
Therefore, we hypothesize that a feedback loop of so on ey
is also important for the fine-tuning of ey expression during normal
eye development. Due to its previously proposed ability to activate as well as
to repress the expression of genes (Silver
et al., 2003
), so is a potent regulator in this
context.
Linking the transcriptional cascade to signal transduction by hedgehog
decapentaplegic (dpp) signalling plays an important role
in the complex regulatory network of eye development. In dpp mutant
eye discs, so, eya and dac are not expressed
(Chen et al., 1999), whereas
dpp is able to initiate ectopic expression of so and
dac when expressed at the anterior margin of the eye disc
(Chanut and Heberlein, 1997
;
Pignoni and Zipursky, 1997
).
Conversely, dpp expression is patchy in eye discs of eya and
so loss-of-function mutants, suggesting that eya and
so are required for either initiation or maintenance of dpp
at the posterior disc margin before MF initiation
(Pignoni et al., 1997
;
Hazelett et al., 1998
).
hh is required for dpp expression at the posterior margin
before MF initiation (Borod and Heberlein,
1998), and dpp expression is induced by hh in
the MF (Heberlein et al.,
1993
), supporting the assumption that dpp is downstream
of hh signalling. As dpp alone is not able to rescue
posterior margin clones of hh, there have to be more eye-relevant
target genes of hh signalling during third instar larval development.
dpp in combination with eya can restore photoreceptor
differentiation in posterior margin clones lacking smoothened
(smo) expression (smo is a cell-autonomous receptor of
hh signalling). This shows that dpp, in combination with
eya, is able to bypass the requirement of hh during eye
development (Pappu et al.,
2003
). Taken together, it is evident that hh is necessary
for proper eya and dpp expression, both of which can induce
so, and it contains two so target sites. We therefore
hypothesized that the transcriptional complex consisting of EYA and SO, as
with ey might also feed back on hh in order to drive the
furrow during late eye development. In this model the genetic cascade starts
with hh, which induces dpp and eya, moves on to
so and through the SO/EYA complex feeds back to hh in order
to maintain hh expression as a driving force of the MF.
The impact of these so-binding sites in the hh enhancer on eye development becomes evident from the fact that hh1 (bar-3) mutant flies have smaller eyes. The severity of the hh1 mutant phenotype is probably diminished by an additional putative SO-binding site that resides outside the area covered by the hh1 deletion (Fig. 6A, SO-binding motifs). If functional, this region (5' to the hh1 deletion) might mediate a residual hh-expression that overcomes the loss of the other sites to some extent. Another possible explanation for the rather weak hh1 phenotype might be that the feedback of so on hh is not crucial for MF initiation but still might be of importance for the well-balanced expression of hh during MF propagation.
A general theme of Six-gene target sites
so belongs to the Six gene family. All Six proteins are
characterized by a Six domain and a Six-type homeodomain, both of which are
essential for specific DNA binding and protein-protein interaction. Based on
the amino acid sequence of their homeodomain and Six domain, the Six genes
were divided into three subgroups. Each of the three Drosophila
homologues can be assigned to one of these subgroups: so is mostly
related to Six1/2, optix to Six3/6 and DSix4 to
Six4/5 (reviewed by Kawakami and
Kobayashi, 1998).
Promoter analyses of the mouse Six genes (Six1/2, Six4/5) revealed
similar target sequence specificities for these mammalian counterparts of
so. Six2, Six4/AREC3 and Six5 effectively bind to the same
target sequence in a DNA fragment called ARE (Atpla1 regulatory element) that
can be found in the Na,K-ATPase 1 subunit gene
(Fig. 4C, ARE fragment)
(Suzuki-Yagawa et al., 1992
;
Kawakami et al., 1996a
;
Kawakami et al., 1996b
;
Harris et al., 2000
).
Six1 and Six4 have been shown to bind to MEF3 sites in the
myogenin and in the aldolase A muscle-specific (pM) promoters
(Fig. 4C, MEF3 site)
(Spitz et al., 1998
).
Recently, mammalian Six4 has been shown to bind additionally to the
transcriptional regulatory element X (TreX) within the muscle creatine kinase
(MCK) enhancer (Fig. 4C, Trex)
(Himeda et al., 2004
).
Comparison of all these sites confirmed that the three nucleotides we
suggest are the most important for SO-DNA interaction are present and
conserved within these motifs (nt. 1, 4 and 9 in
Fig. 4C). In the case of the
MEF3 site, which comprises seven nucleotides that include only two of the
nucleotides important for SO-DNA interaction (nt. 4 and 9 in
Fig. 4C), we looked up the
original publications to check if the third conserved nucleotide is also
present, and in most of the cases were able to verify its conservation
(Hidaka et al., 1993;
Spitz et al., 1998
;
Himeda et al., 2004
). In fact,
there is only one exception published in a study that describes two
Six2 target sites (Brodbeck et
al., 2004
).
Nevertheless, by combining the vast majority of previous studies describing protein-DNA interaction of Six genes and our study of SO-DNA interaction, we infer that SO, Six1, Six2, Six4 and Six5 have very similar DNA-binding properties. In the case of so, we propose that the consensus sequence GTAANYNGANAY(C/G) marks a good starting point for the identification of additional targets of SO, thereby helping to unravel the complex genetic interactions that orchestrate the development of the visual systems of Drosophila.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bonini, N. M., Leiserson, W. M. and Benzer, S. (1993). The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72,379 -395.[CrossRef][Medline]
Bonini, N. M., Bui, Q. T., Gray-Board, G. L. and Warrick, J.
M. (1997). The Drosophila eyes absent gene directs
ectopic eye formation in a pathway conserved between flies and vertebrates.
Development 124,4819
-4826.
Borod, E. R. and Heberlein, U. (1998). Mutual regulation of decapentaplegic and hedgehog during the initiation of differentiation in the Drosophila retina. Dev. Biol. 197,187 -197.[CrossRef][Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Brodbeck, S., Besenbeck, B. and Englert, C. (2004). The transcription factor Six2 activates expression of the Gdnf gene as well as its own promoter. Mech. Dev. 121,1211 -1222.[CrossRef][Medline]
Bui, Q. T., Zimmerman, J. E., Liu, H. and Bonini, N. M.
(2000a). Molecular analysis of Drosophila eyes absent
mutants reveals features of the conserved Eya domain.
Genetics 155,709
-720.
Bui, Q. T., Zimmerman, J. E., Liu, H., Gray-Board, G. L. and Bonini, N. M. (2000b). Functional analysis of an eye enhancer of the Drosophila eyes absent gene: differential regulation by eye specification genes. Dev. Biol. 221,355 -364.[CrossRef][Medline]
Chanut, F. and Heberlein, U. (1997). Role of
decapentaplegic in initiation and progression of the morphogenetic furrow in
the developing Drosophila retina. Development
124,559
-567.
Chao, J. L., Tsai, Y. C., Chiu, S. J. and Sun, Y. H.
(2004). Localized Notch signal acts through eyg and upd to
promote global growth in Drosophila eye.
Development 131,3839
-3847.
Chen, R., Amoui, M., Zhang, Z. and Mardon, G. (1997). Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila.Cell 91,893 -903.[CrossRef][Medline]
Chen, R., Halder, G., Zhang, Z. and Mardon, G.
(1999). Signaling by the TGF-beta homolog decapentaplegic
functions reiteratively within the network of genes controlling retinal cell
fate determination in Drosophila. Development
126,935
-943.
Cheyette, B. N., Green, P. J., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S. L. (1994). The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12,977 -996.[CrossRef][Medline]
Curtiss, J. and Mlodzik, M. (2000).
Morphogenetic furrow initiation and progression during eye development in
Drosophila: the roles of decapentaplegic, hedgehog and eyes absent.
Development 127,1325
-1336.
Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W. J. and Busslinger, M. (1999). twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Mol. Cell. 3, 297-307.[CrossRef][Medline]
Dominguez, M., Ferres-Marco, D., Gutierrez-Avino, F. J., Speicher, S. A. and Beneyto, M. (2004). Growth and specification of the eye are controlled independently by Eyegone and Eyeless in Drosophila melanogaster. Nat. Genet. 36, 31-39.[CrossRef][Medline]
Duchek, P., Somogyi, K., Jekely, G., Beccari, S. and Rorth, P. (2001). Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell 107, 17-26.[CrossRef][Medline]
Epstein, J., Cai, J., Glaser, T., Jepeal, L. and Maas, R.
(1994). Identification of a Pax paired domain recognition
sequence and evidence for DNA-dependent conformational changes. J.
Biol. Chem. 269,8355
-8361.
Greenwood, S. and Struhl, G. (1999).
Progression of the morphogenetic furrow in the Drosophila eye: the
roles of Hedgehog, Decapentaplegic and the Raf pathway.
Development 126,5795
-5808.
Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267,1788 -1792.[Medline]
Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U.
and Gehring, W. J. (1998). Eyeless initiates the expression
of both sine oculis and eyes absent during Drosophila compound eye
development. Development
125,2181
-2191.
Harris, S. E., Winchester, C. L. and Johnson, K. J.
(2000). Functional analysis of the homeodomain protein SIX5.
Nucleic Acids Res. 28,1871
-1878.
Hauck, B., Gehring, W. J. and Walldorf, U.
(1999). Functional analysis of an eye specific enhancer of the
eyeless gene in Drosophila. Proc. Natl. Acad. Sci. USA
96,564
-569.
Hazbun, T. R., Stahura, F. L. and Mossing, M. C. (1997). Site-specific recognition by an isolated DNA-binding domain of the sine oculis protein. Biochemistry 36,3680 -3686.[CrossRef][Medline]
Hazelett, D. J., Bourouis, M., Walldorf, U. and Treisman, J.
E. (1998). decapentaplegic and wingless are regulated by eyes
absent and eyegone and interact to direct the pattern of retinal
differentiation in the eye disc. Development
125,3741
-3751.
Heberlein, U., Wolff, T. and Rubin, G. M. (1993). The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina. Cell 75,913 -926.[CrossRef][Medline]
Heberlein, U., Singh, C. M., Luk, A. Y. and Donohoe, T. J. (1995). Growth and differentiation in the Drosophila eye coordinated by hedgehog. Nature 373,709 -711.[CrossRef][Medline]
Heitzler, P., Coulson, D., Saenz-Robles, M. T., Ashburner, M.,
Roote, J., Simpson, P. and Gubb, D. (1993). Genetic and
cytogenetic analysis of the 43A-E region containing the segment polarity gene
costa and the cellular polarity genes prickle and spiny-legs in Drosophila
melanogaster. Genetics 135,105
-115.
Hidaka, K., Yamamoto, I., Arai, Y. and Mukai, T. (1993). The MEF-3 motif is required for MEF-2-mediated skeletal muscle-specific induction of the rat aldolase A gene. Mol. Cell. Biol. 13,6469 -6478.[Abstract]
Himeda, C. L., Ranish, J. A., Angello, J. C., Maire, P.,
Aebersold, R. and Hauschka, S. D. (2004). Quantitative
proteomic identification of six4 as the trex-binding factor in the muscle
creatine kinase enhancer. Mol. Cell. Biol.
24,2132
-2143.
Jun, S., Wallen, R. V., Goriely, A., Kalionis, B. and Desplan,
C. (1998). Lune/eye gone, a Pax-like protein, uses a partial
paired domain and a homeodomain for DNA recognition. Proc. Natl.
Acad. Sci. USA 95,13720
-13725.
Kango-Singh, M., Singh, A. and Henry Sun, Y. (2003). Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction. Dev. Biol. 256,49 -60.[CrossRef][Medline]
Kawakami, K. and Kobayashi, M. (1998). [Structure and function of novel homeobox gene family six and implications in development and differentiation]. Tanpakushitsu Kakusan Koso 43,2120 -2125.[Medline]
Kawakami, K., Ohto, H., Ikeda, K. and Roeder, R. G.
(1996a). Structure, function and expression of a murine homeobox
protein AREC3, a homologue of Drosophila sine oculis gene product,
and implication in development. Nucleic Acids Res.
24,303
-310.
Kawakami, K., Ohto, H., Takizawa, T. and Saito, T. (1996b). Identification and expression of six family genes in mouse retina. FEBS Lett. 393,259 -263.[CrossRef][Medline]
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell 71,33 -50.[CrossRef][Medline]
Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W. et al. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426,247 -254.[CrossRef][Medline]
Lindsley, D. L. and Zimm, G. G. (1992).The Genome of Drosophila melanogaster . San Diego: Academic Press.
Mardon, G., Solomon, N. M. and Rubin, G. M.
(1994). dachshund encodes a nuclear protein required for normal
eye and leg development in Drosophila. Development
120,3473
-3486.
Markstein, M., Markstein, P., Markstein, V. and Levine, M.
S. (2002). Genome-wide analysis of clustered Dorsal binding
sites identifies putative target genes in the Drosophila embryo.
Proc. Natl. Acad. Sci. USA
99,763
-768.
Mohler, J. (1988). Requirements for
hedgehog, a segmental polarity gene, in patterning larval and adult
cuticle of Drosophila. Genetics
120,1061
-1072.
Muller, B., Hartmann, B., Pyrowolakis, G., Affolter, M. and Basler, K. (2003). Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. Cell 113,221 -233.[CrossRef][Medline]
Newsome, T. P., Asling, B. and Dickson, B. J.
(2000). Analysis of Drosophila photoreceptor axon
guidance in eye-specific mosaics. Development
127,851
-860.
Niimi, T., Seimiya, M., Kloter, U., Flister, S. and Gehring, W.
J. (1999). Direct regulatory interaction of the eyeless
protein with an eye-specific enhancer in the sine oculis gene during eye
induction in Drosophila. Development
126,2253
-2260.
Pappu, K. S., Chen, R., Middlebrooks, B. W., Woo, C., Heberlein,
U. and Mardon, G. (2003). Mechanism of hedgehog signaling
during Drosophila eye development.
Development 130,3053
-3062.
Pignoni, F. and Zipursky, S. L. (1997).
Induction of Drosophila eye development by decapentaplegic.
Development 124,271
-278.
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. (1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91,881 -891.[CrossRef][Medline]
Punzo, C., Kurata, S. and Gehring, W. J.
(2001). The eyeless homeodomain is dispensable for eye
development in Drosophila. Genes Dev.
15,1716
-1723.
Punzo, C., Seimiya, M., Flister, S., Gehring, W. J. and Plaza,
S. (2002). Differential interactions of eyeless and twin of
eyeless with the sine oculis enhancer. Development
129,625
-634.
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265,785 -789.[Medline]
Renfranz, P. J. and Benzer, S. (1989). Monoclonal antibody probes discriminate early and late mutant defects in development of the Drosophila retina. Dev. Biol. 136,411 -429.[CrossRef][Medline]
Sambrook, J. and Russel, D. (2001). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Serikaku, M. A. and O'Tousa, J. E. (1994). sine
oculis is a homeobox gene required for Drosophila visual system
development. Genetics
138,1137
-1150.
Silver, S. J., Davies, E. L., Doyon, L. and Rebay, I.
(2003). Functional dissection of eyes absent reveals new modes of
regulation within the retinal determination gene network. Mol.
Cell. Biol. 23,5989
-5999.
Spitz, F., Demignon, J., Porteu, A., Kahn, A., Concordet, J. P.,
Daegelen, D. and Maire, P. (1998). Expression of myogenin
during embryogenesis is controlled by Six/sine oculis homeoproteins through a
conserved MEF3 binding site. Proc. Natl. Acad. Sci.
USA 95,14220
-14225.
Staehling-Hampton, K. and Hoffmann, F. M. (1994). Ectopic decapentaplegic in the Drosophila midgut alters the expression of five homeotic genes, dpp, and wingless, causing specific morphological defects. Dev. Biol. 164,502 -512.[CrossRef][Medline]
Stark, W. S., Sapp, R. and Carlson, S. D. (1989). Ultrastructure of the ocellar visual system in normal and mutant Drosophila melanogaster. J. Neurogenet. 5, 127-153.[Medline]
Suzuki-Yagawa, Y., Kawakami, K. and Nagano, K. (1992). Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind. Mol. Cell. Biol. 12,4046 -4055.[Abstract]
Tootle, T. L., Silver, S. J., Davies, E. L., Newman, V., Latek, R. R., Mills, I. A., Selengut, J. D., Parlikar, B. E. and Rebay, I. (2003). The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426,299 -302.[CrossRef][Medline]
Treisman, J. E. and Heberlein, U. (1998). Eye development in Drosophila: formation of the eye field and control of differentiation. Curr. Top. Dev. Biol. 39,119 -158.[Medline]
Wolff, T. and Ready, D. F. (1993). Pattern formation in the Drosophila retina. In The Development of Drosophila Melanogaster. Vol. 2.2 (ed. M. Bate and A. Martinez Arias). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Yan, H., Canon, J. and Banerjee, U. (2003). A transcriptional chain linking eye specification to terminal determination of cone cells in the Drosophila eye. Dev. Biol. 263,323 -329.[CrossRef][Medline]
Zimmerman, J. E., Bui, Q. T., Liu, H. and Bonini, N. M.
(2000). Molecular genetic analysis of Drosophila eyes
absent mutants reveals an eye enhancer element.
Genetics 154,237
-246.