1 Program in Developmental Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030, USA
2 Department of Molecular and Human Genetics, Baylor College of Medicine, One
Baylor Plaza, Houston, TX 77030, USA
3 Department of Pathology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
4 Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
5 Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
6 Department of Neuroscience, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
* Author for correspondence (e-mail: gmardon{at}bcm.tmc.edu)
Accepted 13 April 2005
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SUMMARY |
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Key words: dac, Enhancer, Eye, Drosophila, Retina
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Introduction |
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The adult Drosophila eye develops from an epithelial monolayer
called the eye imaginal disc, which is derived from a group of about 20 cells
set aside during embryonic development
(Garcia-Bellido and Merriam,
1969). Photoreceptor differentiation begins at the posterior
margin of the eye disc in third instar larvae and proceeds anteriorly
following a dorsoventral groove termed the morphogenetic furrow (MF)
(Ready et al., 1976
). The RD
network consists of a series of gene regulatory events, which are initially
linear and then progress to include extensive cross and feedback regulation,
resulting in the conversion of undifferentiated epithelial cells to retinal
cells (Chen et al., 1997
;
Halder et al., 1998
;
Pignoni et al., 1997
). In
addition to the cell-autonomously acting RD genes, extracellular signaling
molecules such as Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) are
also required for coordinating growth, proliferation, patterning and cell fate
specification during retinal morphogenesis in Drosophila
(Baonza and Freeman, 2002
;
Borod and Heberlein, 1998
;
Chanut and Heberlein, 1997
;
Dominguez and Hafen, 1997
;
Heberlein et al., 1995
;
Heberlein et al., 1993
;
Pignoni and Zipursky, 1997b
;
Treisman and Rubin, 1995
).
dac is the most downstream member of the RD network to be
identified in Drosophila (Chen et
al., 1997). dac-null mutants in Drosophila
develop with severely truncated legs and dramatically reduced or absent eyes
(Mardon et al., 1994
). In
addition, dac mutants display defects in genital disc, mushroom body
and antennal development (Dong et al.,
2001
; Dong et al.,
2002
; Kurusu et al.,
2000
; Martini et al.,
2000
; Noveen et al.,
2000
). Misexpression of dac is sufficient to induce
ectopic eye development in non-retinal tissue
(Shen and Mardon, 1997
).
dac encodes a nuclear protein that contains a conserved domain
(Dachshund Domain 1 or DD1) which resembles DNA-binding motifs similar to
those found in the winged helix/forkhead subfamily of helix turn helix
proteins (Kim et al., 2002
).
In addition, a second conserved domain (Dachshund domain 2 or DD2) in Dac can
form a complex with Eya, although recent studies have suggested that DD2 is
largely dispensable for Dac protein function in vivo
(Chen et al., 1997
;
Tavsanli et al., 2004
). Dac is
expressed in multiple tissues during Drosophila development,
including the embryo, eye, leg, wing, antenna, male and female genital discs,
and the mushroom bodies in the brain
(Keisman and Baker, 2001
;
Kurusu et al., 2000
;
Mardon et al., 1994
;
Martini et al., 2000
;
Noveen et al., 2000
). In the
eye disc, Dac is expressed at the posterior margin prior to the initiation of
the MF. After initiation of photoreceptor differentiation, Dac is expressed in
the MF and its expression tapers both anterior and posterior to the furrow
(Mardon et al., 1994
).
Genetic analysis suggests that Dac expression in the eye is controlled by
other members of the RD gene network. Dac expression is lost in eya
or so mutant eye discs, and misexpression of ey or
eya, but not so alone, leads to the inappropriate activation
of Dac expression (Chen et al.,
1997). Moreover, ectopic expression of a combination of
eya and so leads to the synergistic activation of Dac
(Chen et al., 1999
).
Furthermore, dpp signaling can strongly synergize with eya
and so to dramatically activate the expression of Dac in an ectopic
expression assay, and dpp is required for dac expression in
the eye disc (Chen et al.,
1999
). Last, the ability of ey to activate Dac expression
is highly reduced but not completely eliminated in eya2
mutants (Chen et al., 1997
).
Taken together, these results suggest that dac regulation is under
the control of ey, eya and so coupled with extracellular
inputs from Dpp signaling. Despite a host of genetic data, the exact nature of
the protein complexes that regulate dac expression in the eye are
still unknown. It has been proposed that So acts as the DNA binding unit of a
protein complex that includes Eya, which in turn is thought to act as a
transactivator (Chen et al.,
1997
). Furthermore, the roles of ey and downstream
effectors of dpp signaling in the regulation of dac
expression in the eye remain to be characterized.
The isolation of genomic elements that direct the eye-specific expression
of the RD genes provide important tools for deciphering the molecular
interactions that regulate early eye specification and determination. The eye
enhancers of ey, eya, and so have been defined in some
detail (Bui et al., 2000;
Hauck et al., 1999
;
Niimi et al., 1999
;
Punzo et al., 2002
;
Zimmerman et al., 2000
). These
studies used eye-specific alleles of these genes to identify genomic lesions
that disrupt regulatory elements that direct transcription in the eye.
However, despite multiple attempts, no eye-specific alleles of dac
have been isolated to date. Therefore, we turned to the use of functional
genomics to identify the eye-specific regulatory elements of the dac
gene in Drosophila. We hypothesized that crucial cis-regulatory
non-coding sequences are highly sensitive to mutational changes and remain
largely unaltered over millions of years of evolution. Therefore, significant
conservation in non-coding sequences among evolutionarily disparate species is
a strong indicator of functional constraint and often uncovers
cis-regulatory elements. We compared the sequences of the
40 kb
dac genomic region among five different species of Drosophilids to
uncover highly conserved non-coding sequences (CNCSs). Two such CNCSs define
eye-specific regulatory elements in the dac genomic locus. We
demonstrate that one of these eye enhancers maps to the 3' non-coding
region of the dac locus and is under the genetic control of eya,
so and dpp signaling. Two potential So-binding sites are
embedded within an
40 bp conserved stretch in this 3' eye enhancer
and disruption of these binding sites abolishes enhancer activity in vivo.
Surprisingly, in spite of the 3' eye enhancer being completely deleted
in dac7 homozygotes, these animals develop with only
moderately disrupted eyes. Our genomic analysis identifies a second,
independent 5' eye enhancer that maps to intron 8 of the dac
locus and that acts redundantly and in concert with the 3' eye enhancer.
This 5' eye enhancer is not deleted in dac7 mutants
and is regulated by a combination of ey, eya and so. Our
results highlight the power of functional genomics to uncover genomic
regulatory elements, especially in the absence of tissue-specific genetic
mutants and in cases with redundant enhancers.
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Materials and methods |
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Drosophila genetics
All Drosophila crosses were carried out at 25°C on standard
media. The mad1-2 FRT40A recombinant stock was provided by
Marek Mlodzik (Curtiss and Mlodzik,
2000). The nature of the dac3 and
dac7 mutant alleles were previously described
(Tavsanli et al., 2004
). The
presence of intron 8 in dac7 mutants was confirmed by PCR
on genomic DNA prepared from dac7 homozygotes with intron
8 specific primers. A similar assay was used to demonstrate the deletion of
exon 9, placing the deletion in dac7 beyond intron 8 but
including exon 9 (data not shown). The 30A-GAL4, UAS-ey, UAS-eya and
UAS-so flies were previously described
(Brand and Perrimon, 1993
;
Pignoni et al., 1997
).
UAS-eya and UAS-so stocks were provided by Francesca Pignoni
and Larry Zipursky. All other stocks were obtained from the Bloomington stock
center. Flies containing multiple transgenes were generated by meiotic
recombination using eye color as an initial selection. Polymerase chain
reaction (PCR) with gene-specific primers was used to confirm genotypes.
Ectopic expression followed by antibody staining (where possible) was used to
confirm expression of individual genes from recombinant chromosomes.
P-element vectors and reporter transgene construction
Genomic fragments spanning the dac locus were subcloned into
appropriate P-element reporter vectors using convenient restriction sites.
Three different P-element reporter vectors were used in this study:
pCasper-hs43-AUG-ßGal
(Thummel et al., 1988),
pH-Pelican and pH-Stinger
(Barolo et al., 2000
). The
reporters in pH-Pelican and pH-Stinger are
ß-galactosidase and nuclear GFP, respectively. To generate an HA-dac
version of the enhancer-reporter construct, we deleted the entire GFP-coding
region from the pH-Stinger vector and replaced it with an HA tag in frame with
the dac cDNA. This vector still contains the 390 bp eye enhancer and
a minimal hsp70 TATA promoter. Detailed information about this vector is
available upon request.
Sub-fragments of 1 kb or less were obtained by PCR amplification using
appropriate primers with artificial EcoRI-BamHI restriction
site tails. PCR products were digested with EcoRI and BamHI,
and ligated with similarly digested P-element vectors. Positive clones were
sequenced to confirm sequence integrity and orientation. Fragments with
mutated binding sites were obtained by overlap extension PCR as previously
described (Ho et al., 1989).
Subcloned PCR products were sequenced to confirm the sequence and orientation.
Transgenic flies were obtained by standard transgenic injection techniques
(Rubin and Spradling, 1982
). A
minimum of three independent transgenic lines were tested for reporter
activity for each construct.
ß-Galactosidase activity staining
Imaginal discs from second or third instar larvae were dissected into
phosphate buffered saline [PBS; 0.1 M phosphate (pH 7.2), 150 mM NaCl], fixed
for 20 minutes in 1% glutaraldehyde in PBS, and washed three times for 10
minutes each in PBS. The imaginal discs were then incubated in pre-warmed
active staining solution (10 mM Na2HPO4, 10 mM
NaH2PO4, 150 mM NaCl, 1 mM MgCl2, 3 mM
K3[Fe(CN)6], 3 mM K4[Fe(CN)6])
with 0.1% X-gal in N,N-dimethylformamide. The discs were allowed to stain for
appropriate times up to 16 hours and then washed in PBS three times for 10
minutes each wash. The discs were allowed to equilibrate in 80% glycerol in
PBS overnight before they were mounted on glass slides.
Immunohistochemistry and scanning electron microscopy
Primary antibodies used in this study were: monoclonal mouse anti-Dachshund
(mAbdac2-3: 1:200, Developmental Studies Hybridoma Bank), rabbit
anti-ß-galactosidase (1:1000; Cappel), rabbit anti-GFP (Molecular
Probes), chicken anti-GFP (Upstate) and mouse anti-HA (Covance). Conjugated
goat anti-mouse, chicken and rabbit fluorescent secondary antibodies were
ALEXA 488 (Molecular Probes), Cy3 (Jackson Immunochemicals) or Cy5 (Jackson
Immunochemicals), all at 1:600 dilution. HRP-conjugated goat anti-mouse
antibodies were used as previously described
(Mardon et al., 1994). Discs
were then processed as previously described
(Frankfort et al., 2001
).
Fluorescent images were captured with a Zeiss LSM 510 confocal microscope. All
other images were captured on a Zeiss Axioplan microscope with Nomarski
optics. All images were processed with Adobe Photoshop software. Adult flies
were prepared for electron microscopy as previously described
(Kimmel et al., 1990
).
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Results |
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We then used a functional genomics approach to uncover new genomic
non-coding sequences across the entire the dac locus that are
required for tissue specific enhancer activity (see Materials and methods). We
hypothesized that non-coding regions that remain unaltered over the course of
millions of years of evolutionary time are under functional constraint and
define important regulatory protein binding targets. We compared the
conservation of non-coding DNA across the 40 kb dac genomic
locus among five related species of Drosophilids, D. melanogaster, D.
pseudoobscura, D. erecta, D. willistoni, and D. virilis that
represent over 60 million years of evolutionary time (see Materials and
methods). As we were primarily interested in uncovering eye enhancer
fragments, we initially focused on sequences within 3EE1.9
kb. The VISTA output of pairwise comparisons to D.
melanogaster along 3EE1.9 kb is shown in
Fig. 1C
(Mayor et al., 2000
). Six
conserved non-coding sequences (CNCSs) are present in 3EE1.9
kb. To test the correlation of CNCSs with enhancer activity, we
cloned an 850 bp fragment (3EE850 bp) that contains all
six CNCS blocks upstream of a minimal promoter driving expression of a GFP or
ß-galactosidase reporter. Transgenic flies were then tested for reporter
(GFP or ß-galactosidase) expression in the eye. 3EE850
bp, like 3EE1.9 kb, is expressed only posterior
to the furrow (Fig. 1D). However, a smaller 659 bp fragment (3EE659 bp) that
contains only the first four CNCS blocks drives strong expression of GFP in
the eye disc both anterior and posterior to the furrow, similar to endogenous
Dac protein expression (Fig.
1E). The smallest active enhancer 3EE194 bp
contains only the third and fourth CNCS blocks, and is expressed only
posterior to the MF in third instar eye discs (data not shown). These results
suggest that 3EE659 bp (expressed both anterior and
posterior to the MF) lacks repressor binding sites contained in the 1.9 kb eye
enhancer that normally inactivate reporter expression anterior to the furrow.
In addition, these results suggest that 3EE194 bp
(expressed only posterior to the MF) further lacks positive regulatory sites
normally present in 3EE659 bp that are required for
reporter expression anterior to the MF. Although the expression of endogenous
Dac protein is decreased posterior to the furrow, GFP expression driven by
3EE659 bp persists all the way to the posterior margin of
the eye disc. To rule out the possibility that the 3' enhancer lacks
repressive elements that normally downregulate dac expression
posterior to the furrow, we generated transgenic flies in which the GFP
reporter was replaced by an HA-dac reporter (see Materials and
methods). HA-Dac reporter expression, visualized using an anti-HA antibody,
reveals a rapid downregulation of HA staining posterior to the MF (see Fig.
S1B-D in the supplementary material). Thus, we conclude that GFP expression
far posterior to the MF in 3EE-GFP transgenic eye discs occurs
because of the perdurance of GFP protein and/or transcript, and not because of
the lack of negative regulatory elements in the 3' enhancer. We
hypothesized that a deletion of the 3' eye enhancer would block
dac expression in the eye, thereby causing eye-specific defects. We
therefore examined known dac mutants to identify an allele that
contains genomic lesions in this 3' eye enhancer but does not affect the
function of the protein.
|
dac7 homozygotes develop with only moderately disrupted eyes
Surprisingly, dac7 homozygotes develop with only
moderately disrupted eyes compared with wild-type adults (compare
Fig. 2C with 2B). By contrast,
dac3 null mutants have no eyes, suggesting that the
dac7 mutant is a hypomorph
(Fig. 2D). We also examined the
expression of Dac protein in the eye imaginal discs of
dac7 homozygous larvae. A monoclonal antibody to Dac
(mabdac 2-3) recognizes an epitope predicted to be present within the
potentially truncated protein encoded by the dac7
transcript. Eye imaginal discs from dac7 larvae are almost
identical to wild-type controls in their Dac protein expression profiles
(compare Fig. 2F to 2E). dac3-null mutants display no detectable Dac protein
(Fig. 2G). As the entire 16.6
kb 3' enhancer is completely deleted in dac7
mutants, these results suggest that additional eye-specific enhancers exist in
the genome, either within the dac locus or outside the genomic
fragments we tested.
A second eye enhancer is present in intron 8 of the dac genomic locus
We next extended our pairwise sequence comparison to the entire
dac genomic locus to identify additional functionally relevant CNCSs.
Multiple regions of significant conservation were found, spread along the
entire locus (data not shown). We used PCR amplification to clone these
CNCS-containing fragments upstream of a ß-galactosidase reporter. One
such fragment contains four CNCS blocks in a 1.7 kb stretch within intron 8 of
the dac locus (called 5' eye enhancer or 5EE;
Fig. 1B). Importantly, this 1.7
kb region is intact in the dac7 allele. We found that
third instar eye discs from 5EE transgenic larvae are positive for
ß-galactosidase activity, which appears to be highest at the posterior
margin of the eye disc (Fig.
2M). Furthermore, late first instar and second instar 5EE
transgenic eye discs also have ß-galactosidase activity, suggesting that
this enhancer is active prior to initiation of the MF
(Fig. 2L; data not shown). A
smaller fragment that contains only the first two CNCS blocks does not have
eye enhancer activity (data not shown). Taken together, these results suggest
that another eye enhancer exists in intron 8 of the dac locus that
perhaps acts redundantly or in concert with the 3' enhancer. We next
tested the response of these putative eye enhancers to known upstream
regulators of dac in the Drosophila eye.
|
Previous studies have shown that dpp signaling acts
synergistically with eya and so, and strongly activate
dac expression in the 30A-Gal4 ectopic expression assay
(Chen et al., 1999). We tested
if 3EE-GFP is also synergistically activated by a combination of
eya, so and dpp in the ectopic wing expression assay. As
with endogenous Dac protein, the expression of 3EE-GFP was strongly
induced in a ring around the wing pouch upon expression of dpp, eya
and so using the 30-Gal4 driver
(Fig. 3C). These results
suggest that the 3' dac eye-specific enhancer may be directly
regulated by a combination of Dpp signaling effector molecules and upstream RD
proteins. Furthermore, these results suggest that 3EE194
bp is sufficient to integrate the input from Dpp signaling with the
tissue-specific factors Eya and So. Interestingly, the intracellular
transducers of Dpp signaling, Mothers against Dpp (Mad) and Medea, do not
bypass the requirement for Dpp in this assay (data not shown). However, a
constitutively active form of the Dpp receptor, Thickveins
(TkvQ253D), was just as effective as Dpp in synergistically
activating GFP expression from the 3' eye enhancer in the presence of Ey
(Lecuit et al., 1996
) (data
not shown). Therefore, we conclude that the ability of Dpp to synergize with
Eya and So to activate 3EE is dependent on downstream signaling
events such as the phosphorylation of Mad. A less probable alternative is that
non-canonical events downstream of Tkv mediate the synergy between Eya, So and
Dpp signaling. We found no evidence for autoregulation of 3EE by Dac
itself, as ectopic expression of Dac with the 30-Gal4 driver does not
activate reporter expression in the wing (data not shown).
ey acts through eya and so to regulate the 3' dac eye enhancer
Results from our ectopic expression analysis suggested that the 3'
dac eye enhancer is regulated by a combination of eya and
so. Consistent with this prediction, 3EE-GFP expression is
completely lost in eya2 and so1
eye-specific mutants (Fig.
4B,C). However, as has been shown previously, endogenous Dac
protein expression is dramatically reduced but not completely eliminated in
eya2 and so1 mutants
(Fig. 4A-C). As our ectopic
expression data suggest that dpp signaling acts in concert with
eya and so to activate 3EE, we tested the
expression of 3EE-GFP in eye imaginal discs cells that have lost the
ability to signal downstream of the dpp receptor tkv. To
disrupt dpp signaling, we induced mad mutant mitotic clones
in the eye disc using a strong hypomorphic allele of mad
(mad1-2). We found that 3EE-GFP expression is
drastically reduced or completely lost from posterior margin
mad1-2 clones (Fig.
4D). These loss- and gain-of-function experiments suggest that
3EE is regulated by a combination of eya, so and
dpp. Coupled with the ectopic expression data, we conclude that
3EE activation is dependent on the canonical dpp signaling
pathway acting synergistically with eya and so.
|
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|
Our ectopic expression analyses suggest that ey is the primary
upstream regulator of 5EE-lacZ. Furthermore, these results suggest
that eya and so are dispensable for 5EE activity in
vivo. Surprisingly, we found that 5EE expression is lost in third
instar so1 and eya2 eye imaginal discs
(Fig. 5G,H). These results
suggest that although eya and so are not sufficient to
regulate 5EE, they are required for ey to regulate this
enhancer. However, as ectopic ey can activate 5EE in
eya2 and so1 mutant wing discs, we
hypothesized that high levels of ey are sufficient to circumvent the
requirement for eya and so. To test this hypothesis
directly, we made use of the unusual nature of the dpp-Gal4 driver to
activate ey expression in the eye. Although normal dpp
expression mirrors movement of the MF, expression of dpp-Gal4 is
limited to the posterior margin of the eye imaginal disc
(Shen and Mardon, 1997). We
used the dpp-Gal4 driver to drive ey expression in
eya or so mutant eye discs. Consistent with ectopic
ey expression in the wing, dpp-Gal4 driven expression of
ey restores 5EE-lacZ expression at the posterior margin of
so1 mutant eye discs
(Fig. 5I). However,
dpp-Gal4 driven expression of ey can induce only weak
expression of 5EE-lacZ in eya2 mutant eye discs
(Fig. 5J). These results
suggest that the activity of the 5' eye enhancer is primarily regulated
by ey. Furthermore, these results suggest that the function of
ey in this context is more sensitive to the levels of eya
than so.
Two conserved So binding sites are essential for normal expression of the 3' eye enhancer
The smallest fragment in the 3' dac eye enhancer that can
respond to dpp, eya and so is 3EE194 bp,
which is centered around two CNCS blocks of 40 bp and 20 bp
(Fig. 1C). These two CNCS
blocks are also common to all active fragments of the 3' eye enhancer.
We scanned these two evolutionarily conserved stretches for known, genetically
upstream transcription factor binding sites. We found that the 40 bp conserved
stretch contains two putative consensus So-binding sites, S1-5'-CGATAT
and S2-5'-CGATAC, compared with the consensus 5'-(C/T)GATA(C/T)
described previously (Hazbun et al.,
1997
; Yan et al.,
2003
) (Fig. 6A). We
mutated each of these putative So-binding sites in 3EE individually
and in combination to test their requirement for normal enhancer activity in
vivo (Fig. 6A). Mutation of
individual So-binding sites causes a severe reduction, but not complete
elimination, of enhancer activity in vivo
(Fig. 6C,D). However,
simultaneous mutation of both So binding sites completely abolishes enhancer
activity in vivo (Fig. 6E).
These results, coupled with loss-and gain-of-function analyses with dpp,
eya and so, suggest that So binds to the 3' eye enhancer
directly and nucleates a protein complex that includes Eya to regulate
3EE. However, despite much effort using a wide variety of binding
conditions, we have been unable to demonstrate specific, direct binding of So
protein to oligos that contain these So-binding sites. We also scanned the
5' eye enhancer, which has four CNCS blocks, for potential upstream
transcription factor binding sites and found no strong candidate binding sites
within the CNCS blocks.
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Discussion |
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Dual regulation of dac expression: the roles of the 5' and 3' eye enhancers
Loss- and gain-of-function analyses with the two eye enhancers suggest that
each enhancer is regulated by a distinct set of protein complexes. The
5' eye enhancer is activated by a combination of ey, eya and
so, but is not activated by Dpp signaling. 5EE is activated
by ectopic ey expression even in eya and so
mutants, suggesting that it is regulated exclusively by ey. However,
somewhat paradoxically, 5EE expression is lost in eya and
so mutants even though ectopic expression of a combination of
dpp, eya and so does not activate this enhancer.
Furthermore, driving high levels of ey in so1
mutant eye discs restores 5EE-lacZ expression. Coupled together,
these results suggest that 5EE is primarily regulated by ey
but that the regulation of 5EE by ey also requires
eya and so.
By contrast, the 3' dac eye enhancer is regulated by a combination of eya, so and dpp signaling, but is not directly dependent on ey. 3EE-GFP expression is lost in eya2 and so1 mutant eye discs, and in posterior margin mad1-2 mutant clones. Furthermore, ey cannot bypass the requirement for eya and so to activate 3EE. Conversely, 3EE is strongly induced by co-expression of eya and so. Moreover, dpp signaling via the tkv receptor can synergize with eya and so to induce 3EE in ectopic expression assays. Furthermore, we find that neither Mad nor Medea, the intracellular transducers of Dpp signaling, is sufficient to bypass the requirement for activation of the Dpp receptor Tkv in these assays (data not shown). Thus, we conclude that events downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are essential for the synergistic activation of the 3' dac eye enhancer by eya and so. Taken together, these results suggest that there are distinct requirements for the activation of the 5' and 3' dac eye enhancers. However, the exact nature of the protein complexes that regulate 5EE and 3EE remain to be determined.
Initiation versus maintenance of dac expression: the roles of the 5' and 3' eye enhancers
MF initiation is completely blocked in posterior margin
dac3-null mutant clones. However, dac3
clones that do not include any part of the posterior margin develop do not
prevent MF progression, but cause defects in ommatidial cell number and
organization (Mardon et al.,
1994). This dichotomy in dac function is reflected in the
two eye enhancers we have characterized in this study. Our analysis of
dac7 homozygotes demonstrates that the 3' eye
enhancer is dispensable for MF initiation and progression. We propose that in
dac7 mutants, the intact 5EE enhancer is
sufficiently activated by ey to drive high enough levels of
dac expression to initiate and complete retinal morphogenesis.
However, dac7 mutants have readily observable defects in
ommatidial organization. Thus, we further propose that this lack of normal
patterning in dac7 mutants is most likely due to the loss
of 3EE, which normally acts in concert with 5EE after MF
initiation, to integrate patterning inputs from extracellular signaling
molecules such as Dpp with tissue-specific upstream regulators such as ey,
eya and so. However, we do not know if the 3' eye enhancer
is sufficient to initiate dac expression in the absence of the
5' eye enhancer.
Based on our results, we propose a two-step model for the regulation of dac expression in the eye. First, the initiation of dac expression in the eye disc is dependent on Ey binding to 5EE. However, Ey is fully functional only when So and Eya are present. It is possible that Ey recruits So and Eya to 5EE, but we favor a model in which Ey bound to 5EE cooperates with an So/Eya complex bound to 3EE to initiate dac expression in the eye. After initiation of the MF, dac expression is maintained by an Eya and So complex bound to 3EE. In addition, 3EE can integrate patterning information received via dpp signaling, thereby allowing the precise spatial and temporal expression of dac in the eye. This two part retinal enhancer ensures that dac expression is initiated only after ey activates eya and so expression. Thus, the dac eye enhancers provide a unique model with which the sequential activation of RD proteins allows the progressive formation of specialized protein complexes that can activate retinal specific genes.
The redundancy in dac enhancer activity also explains our inability to isolate eye-specific alleles of dac, despite multiple genetic screens (K.S.P., E.J.O. and G.M., unpublished). The modular nature of the two enhancers and their potential ability to act independently or in concert suggest that both enhancers must be disrupted to block high levels of transcription of dac. Thus, two independent hits in the same generation, a phenomenon that occurs infrequently in genetic screens, would be required to obtain an eye-specific allele in dac.
The dac eye enhancers provide powerful tools with which to study RD protein function
Despite much investigation, very few direct targets of RD proteins,
especially for Eya and So, have been identified. One study suggests that So
can bind to and regulate an eye-specific enhancer of the lz gene
(Yan et al., 2003). However,
lz is not expressed early during eye development and is required only
for differentiation of individual cell types
(Daga et al., 1996
). Our
results suggest that regulation of dac expression occurs via the
interaction of two independent eye enhancers that are likely to be bound by
Ey, Eya and So, and respond to dpp signaling. Our analysis of the
3' eye enhancer suggests that two putative conserved So-binding sites
are essential for 3EE activity in vivo. Mutation of individual
So-binding sites dramatically reduces, but does not completely eliminate,
reporter expression in the eye. Mutating both predicted So-binding sites
completely blocks enhancer activity in vivo. Thus, we conclude that So binds
to 3EE via these conserved binding sites. However, we have not been
able to demonstrate a direct specific interaction of either So alone or a
combination of Eya and So with oligos that contain these putative So-binding
sites in vitro. It is possible that other unidentified proteins are required
for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that
responds to ectopic expression of dpp, eya, and so contains
no conserved or predicted Mad-binding sites. This raises the intriguing
possibility that dpp signaling activates other genes, which then
directly act with eya and so to regulate the 3' eye
enhancer. Alternatively, a large complex that includes Eya, So and the
intracellular transducers of dpp signaling, such as Mad and Medea,
may be responsible for activation of 3EE. Similarly, our results
suggest that the 5' eye enhancer is regulated primarily by ey.
However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey
is fully functional only in the presence of Eya and So. Thus, Ey either
independently recruits Eya and So into a 5' complex or is activated by
virtue of its proximity to the So/Eya complex bound to the 3' enhancer
or both.
The exact order and dynamics of protein complex assembly at 5EE
and 3EE requires further investigation. However, the two dac
eye enhancers are extremely useful tools with which to investigate fundamental
issues about the mechanism of RD protein action. One significant issue
concerns the mechanism of Eya function during eye development. Eya consists of
two major conserved domains, an N-terminal domain that has phosphatase
activity in vitro and a C-terminal domain that can function as a
transactivator in cell culture assays
(Rayapureddi et al., 2003;
Silver et al., 2003
;
Tootle et al., 2003
). So
contains a conserved Six domain and a DNA binding homeodomain
(Cheyette et al., 1994
;
Kawakami et al., 2000
).
However, it is unclear if Eya provides phosphatase activity, transactivator
function, or both, in this complex. Characterization of the components of the
protein complexes that regulates dac expression may uncover the
targets of Eya phosphatase activity during eye development. Thus, the
isolation of two eye enhancers with distinct regulation provides very useful
tools with which to study protein complex formation and function during
Drosophila retinal specification and determination.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/12/2895/DC1
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