1 Institute of Genetics, National Yang-Ming University, Taipei 111, Taiwan,
Republic of China
2 Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan,
Republic of China
3 Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720-3204, USA
4 Howard Hughes Medical Institute, The Rockefeller University, New York, NY
10021, USA
* Present address: Department of Biology, New York University, 1009 Main, 100
Washington Square East, 10003, New York, New York, USA
Author for correspondence at address2(e-mail:
mbyhsun{at}ccvax.sinica.edu.tw)
Accepted 25 March 2003
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SUMMARY |
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Key words: Drosophila, Eye development, Pax gene, eye gone, eyeless, Morphogenetic furrow
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INTRODUCTION |
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In recent years, a small number of genes, all encoding nuclear proteins,
have been identified as be required and sufficient (in certain contexts) for
the initiation of eye development in Drosophila (for reviews, see
Desplan, 1997;
Treisman, 1999
;
Kumar and Moses, 2001
).
Loss-of-function mutations of toy, ey, dac, eya and so can
lead to reduction or absence of the adult eye, while ectopic expression of
these genes, either alone or in combination, can lead to ectopic eye formation
(Halder et al., 1995
;
Shen and Mardon, 1997
;
Bonini et al., 1997
;
Chen et al., 1997
;
Pignoni et al., 1997
;
Czerny et al., 1999
;
Kronhamn et al., 2002
),
suggesting that they act at the very early steps of eye development. Two other
nuclear factor genes, tsh and optix, can also induce ectopic
eye upon ectopic expression (Pan and
Rubin, 1998
; Seimiya and
Gehring, 2000
) [see also Singh et al.
(Singh et al., 2002
) for an
additional role of tsh].
Epistasis analysis in loss-of-function mutants and in misexpression
situations suggests that toy, ey, so, eya, dac and tsh form
a complex regulatory network (Desplan,
1997; Shen and Mardon,
1997
; Bonini et al.,
1997
; Chen et al.,
1997
; Pignoni et al.,
1997
; Halder et al.,
1998
; Pan and Rubin,
1998
; Czerny et al.,
1999
). toy acts upstream to regulate ey
expression but is not regulated by ey
(Czerny et al., 1999
). Several
lines of evidence suggest that ey acts upstream and regulates the
expression of dac, eya, so and tsh. (1) Its ability to
induce ectopic eyes is the strongest. (2) Its normal expression in the eye
disc starts earlier (in the embryonic eye disc primordia) than eya, so,
dac and tsh. (3) The normal expression of ey does not
require dac, eya and so, while the normal expression of
eya and so requires ey. (4) Ectopic ey
expression can induce the expression of dac, eya, so and
tsh. (5) Ectopic eye induction by ey requires dac,
eya and so. However, the regulation is not a simple linear
pathway, because (1) ectopic expression of dac, eya, so and
tsh can also induce ey expression, at least in the antennal
disc, (2) ectopic expression of dac and eya can induce each
other, and (3) so and eya may up-regulate the expression of
the other (when ectopically induced by ey). These relationships
suggest a positive feedback regulation among these genes. These feedback
regulations are important for their function, because ectopic eye formation by
eya, so/eya and dac/eya also requires the
upstream ey gene (Bonini et al.,
1997
; Pignoni et al.,
1997
; Chen et al.,
1997
).
Although ey has been called a master regulator of eye development
(Halder et al., 1995), eye
development does not occur in every cell induced to express ey or its
downstream genes dac, eya and so
(Halder et al., 1995
;
Shen and Mardon, 1997
;
Bonini et al., 1997
;
Pignoni et al., 1997
;
Chen et al., 1997
;
Halder et al., 1998
). Other
genes must collaborate with ey in the induction of eye development.
For example, DPP and HH signaling collaborates with EY for ectopic eye
induction (Chen et al., 1999
;
Kango-Singh, 2003
). Therefore,
the identification of genes that are required for eye development but not
directly under ey regulation will lead to a better understanding of
the mechanism of eye induction. The optix gene of the Six/so
gene family is such a gene. It is capable of inducing ectopic eye development
but its expression is not under regulation by ey
(Seimiya and Gehring,
2000
).
eye gone (eyg) is another gene required for eye
development. The first mutation identified, eyg1, causes
the eye to become significantly smaller
(FlyBase, 2003). It also shows
genetic interaction with ey, as mutants doubly homozygous for
hypomorphic viable eyg and ey alleles are not viable
(Hunt, 1970
). The lethal
pharate adults have severely reduced head and complete absence of eyes. These
results suggest that eyg may act in the early stages of eye
development and interact with ey.
In this report, we identify eyg as a Pax gene. It is expressed in the embryonic eye disc primordium and in the larval eye imaginal disc. We characterize its function in eye development and show that ectopic eyg expression can induce ectopic eye formation. eyg is different from eya, so and dac in that its expression is not primarily regulated by ey, nor does it regulate ey expression. Its ability to induce ectopic eyes does not require ey, nor is it required for the ability of ey to induce ectopic eye. Therefore eyg acts neither upstream nor downstream of ey. In addition, coexpression of eyg and ey causes a synergistic ectopic eye formation. Thus, eyg appears to act cooperatively with ey in eye development. As both genes encode Pax proteins with a homeodomain, this is suggestive of a molecular interaction between the two gene products. We also show that the mechanism by which eyg affects eye development may be through suppressing the expression of wg, which is known to inhibit MF initiation.
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MATERIALS AND METHODS |
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Screening and genetic characterization of eyg mutants
Several approaches were used to generate eyg mutations.
-irradiation was used to induce chromosomal aberrations in Eq-1, Eq-2
and Eq-3, which have P-element insertions close to the eyg locus
(Sun et al., 1995
). The males
were irradiated with
-rays (4000R) and mated to w; DH/TM3, Ser
females. The progeny were screened for loss of the Equatorial eye pigmentation
pattern, i.e. loss of the P[lacW] insertion in 69C. C1 was found to be a large
deficiency (69A4-5; 69D4-6; cytology determined by Adelaide Carpenter) and
used as a reference for null. eygM3-12 was generated from
mobilization of P[lacW] from Eq-1. Complementation over the
eyg1 allele gave a strong eyg phenotype.
eygM3-12 homozygotes are pupal lethal. The pharate adults
have a headless phenotype (see text), which is similar to that of
eygM3-12/C1, suggesting that eygM3-12
is close to a functionally null allele. Similarly,
eygEq-2-d2-2, derived from Eq-2 by P mobilization, was
defined as close to a functionally null allele.
In(3LR)gvu/eyg1 flies showed a small eye
phenotype, indicating that In(3LR)gvu (from Bloomington
Stock Center) is a weak eyg allele. EM458 (kindly provided by Leslie
Vosshall, Columbia University) carries a P[GawB] insertion 527 bp upstream of
the first ATG represented in the Lune transcript. It is homozygous viable and
exhibits no apparent phenotype. eyg37-1,
eyg22-2 and eyg94-4 were independently
derived from mobilizing the P[GawB] in EM458. The severity of defect of
eyg alleles was tested over null alleles (C1 or
eygM3-12) and over weak alleles (eyg1
or In(3LR)gvu). eygM3-12 mutant clones
were generated using the hs-FLP/FRT method
(Xu and Rubin, 1993
).
hs-FLP22 and FRT(whs)2A
(FlyBase, 2003
) were used.
Heat shock induction of hs-FLP was at 37°C for 1 hour at the
indicated time after egg laying (AEL). The eyg mutant clones in the
adult are marked by the loss of pigmentation, dependent on the
mini-white marker. In eye discs, the heterozygous cells are marked by
one copy of the Ubi-GFP-nls, which encodes a nuclear GFP
(Davis et al., 1995
). The
mutant cells should have no GFP expression, while the wild-type twin-spot
should have twice the GFP level. However, the twin-spots have a much stronger
GFP expression.
Transgene constructs and germline transformation
The 2.7 kb full-length Lune cDNA was cloned into the NotI site of
the P[CaSpeR-hs] and P[UAST] vectors
(Thummel and Pirrotta, 1992;
Brand and Perrimon, 1993
).
These constructs were used in germline transformation as described previously
(Rubin and Spradling,
1982
).
5'-RACE
Embryos were collected 0-16 hours after egg laying for mRNA purification by
the CLONTECH mRNA purification kit. The 5' end of eyg
transcript was amplified by the SMARTTM RACE cDNA Amplification kit
using two eyg-specific primers for nested PCR. The two primers were
5'-CTAGCAACTTGGAGACAGCTCC-3' and
5'-GCCAGAATTAGCGACAGTAAG-3' respectively. The PCR products were
cloned and multiple clones were sequenced.
Molecular analysis of mutants
For the analysis of eygM3-12 which has a P[lacW]
insertion, plasmid rescue was performed from genomic DNA. A 10 kb
SacII rescued plasmid was sequenced from the termini. The 3'
primer read into an opus retrotransposon [also known as nomad and yoyo
(Whalen and Grigliatti, 1998;
FlyBase, 2003
)], which is
present in the original Eq-1 fly but not in the w1118
parental line. The 5' primer read into a sequence 13 kb downstream of
eyg. Sequence flanking the other side of the P[lacW] in
eygM3-12, was rescued in a 12.5 kb BglII
fragment. The 5' primer read into a sequence 24 bp upstream of the
eyg transcription start site. Thus the eygM3-12
is likely to have a deletion starting 23 bp upstream of eyg and
extending 13 kb downstream of eyg. This was confirmed by genomic
Southern analysis of eygM3-12 homozygotes
(Tb+ larvae and pupae selected from an
eygM3-12/TM6B, Tb stock) with EcoRI digestion.
For the eyg37-1, eyg22-2 and
eyg94-4 mutants that carried a P[GawB] insert, the genomic
region flanking the P insert was amplified by PCR and analyzed by
sequencing.
In situ hybridization and histochemical staining
Digoxigenin-labeled antisense RNA probes were used for in situ
hybridization experiments as described previously
(Tautz and Pfeifle, 1989).
Alkaline phosphatase histochemical staining was used to visualize in situ
hybridization signals. DNA template for the ey probe was derived from
ey exon 9 and was recovered by PCR from genomic DNA. The eyg
probe was described previously (Jones et
al., 1998
). The toe probe was transcribed from an
EcoRI-linearized toe EST clone pOT2a-toe (kindly
provided by Jyh-Lyh Juang, NHRI, Taiwan). The stringent hybridization
condition excluded cross hybridization. w1118 was used as
the wild-type control. Embryos were photographed using a Leica DMRB microscope
with differential interference contrast (DIC) optics. For the double-labeling
RNA in situ hybridization, the fluorescein-labeled eyg antisense RNA
probes and the biotin-labeled ey antisense RNA probes were
transcribed by the T7, T3 promoter (Boehringer Mannheim). The detection was
first with HRP-conjugated anti-fluorescein antibody (1:200) and amplified by
the Cy-3-tyramides TSA (NEN Life Sciences, UK), followed by inactivation for
15 minutes at 70°C, and then by HRP-conjugated streptavidin and amplified
with FITC-tyramid TSA (NEN Life Sciences, UK). X-gal staining of lacZ
expression was done according to the method of Sun et al.
(Sun et al., 1995
). mAb22C10
(1:100) (Fujita et al., 1982
;
Zipursky et al., 1984
) was
from Seymor Benzer (Caltech). X-gal and antibody double staining was modified
from the procedure of Kobayashi and Okada
(Kobayashi and Okada, 1993
),
namely primary and secondary antibody incubation were performed first,
followed by X-gal staining, then the peroxidase color development.
Incorporation of 5-bromo-2'-deoxyuridine (BrdU) into dividing cells in
imaginal discs was done according to the method of Baker and Rubin
(Baker and Rubin, 1992
).
Acridine Orange staining of apoptotic cells was done according to the method
of Spreij (Spreij, 1971
).
Confocal microscopy was performed on a Zeiss LSM510.
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RESULTS |
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Weak loss-of-function eyg mutations resulted in the reduction or
absence of the adult eyes (Fig.
1A,B). In late third instar larvae of the hypomorphic
eyg1 mutant, the eye discs were significantly reduced in
size, while the antennal discs appeared normal
(Fig. 1E). In strong
loss-of-function mutants, the adults failed to emerge from the pupal case.
Their heads were severely reduced in size, but the appeared normal
(Fig. 1C). In a null mutant
eygM3-12, the adults have a headless phenotype
(Fig. 1D) and all structures
derived from the eye-antennal discs were missing. The prominent remaining
structure was the labellum (Fig.
1D) derived from the labial discs. The fish-trap bristles, derived
from the clypeo-labral disc, were also present (not shown). The headless
phenotype is similar to those reported for ey and toy null
mutants (Jiao et al., 2001;
Kammermeier et al., 2001
;
Kronhamn et al., 2002
). In
flies with eyg alleles of different strengths, the size reduction of
third instar eye discs was proportional to the severity of the adult eye
phenotype. Strong alleles did not affect the morphology and size of the
antennal discs (Fig. 1J).
However, no eye-antennal discs could be found in eygM3-12
larvae. These observations suggested that the antennal disc only requires a
low EYG level or activity, so the antennal phenotype is manifested only in the
null mutant, a situation similar to those of ey and toy
mutants (Kronhamn et al.,
2002
). Alternatively, the effect of eyg may be specific
to the eye disc, and the loss of antennal disc-derived structures in null
mutants may be secondary effect due to the missing eye disc.
|
The small size of early third instar mutant eye discs indicated either that
early cell proliferation is affected, or that there is excessive apoptosis
prior to third instar, or both. Misexpression of the anti-apoptosis
baculoviral protein P35 (Hay et al.,
1994), driven by the dpp-GAL4 or ey-GAL4, failed
to rescue the `no eye' phenotype in the
eyg1/eygM3-12 mutant
(Fig. 1I and data not shown).
The eyg1/eygM3-12 mutant had complete absence
of the adult eyes (Fig. 2H) and
had rudimentary eye discs (not shown). The adult eyes and the larval eye discs
were not rescued by misexpression of P35, suggesting that apoptosis is not the
major cause of the eye phenotype.
|
eyg encodes a Pax protein
A Pax-like cDNA [originally named Lune
(Jun et al., 1998)] that also
mapped at 69C was independently isolated based on the presence of a homeobox
encoding a Paired-type homeodomain (HD) with a characteristic serine at
position 50 (Burglin, 1994
).
This residue is only found when the HD is associated with a paired domain
(PD). The PD is the defining character of Pax proteins
(Noll, 1993
). It consists of
two subdomains, the N-terminal PAI and the C-terminal RED subdomains, that can
both bind DNA (Czerny et al.,
1993
; Epstein et al.,
1994
; Xu et al.,
1995
; Jun and Desplan,
1996
). Interestingly, the PD encoded by the Lune cDNA contains
only a partial PAI subdomain and a complete RED subdomain
(Fig. 2A)
(Jun et al., 1998
).
eyg has an open reading frame of 670 amino acids
(Fig. 2A) rather than the 523
amino acids originally reported (Jun et
al., 1998
). These changes involved both N-terminal and C-terminal
sequences. 5'-RACE identified two splicing isoforms with their 5'
ends identical to that of the Lune cDNA, indicating that the 5' end of
the cDNA clone represents the transcription start site. Thus the Lune cDNA
represents the full-length transcript, and there is no additional upstream
exon to provide a functional PAI subdomain. The two isoforms differ in a 67 bp
segment (intron I; Fig. 2B),
which does not affect the coding region. Five introns were identified
(Fig. 2B). All exon-intron
junctions conform to the consensus splice site.
In situ hybridization showed that in the eye disc of late third instar
larvae, Lune is expressed in the central anterior region, well ahead of the
morphogenetic furrow (Fig. 2C).
Expression is stronger dorsal to the equator. In the early eye disc, the
expression domain is broader (Fig.
2D,E). It is also expressed in the central region of the antennal
disc (Fig. 2C), in the anterior
notum, dorsal hinge and in an arc at the posterior periphery of the wing pouch
of the wing disc (Fig. 2F), and
is weakly expressed in several arcs in the leg discs
(Fig. 2G). In the embryo it is
expressed in the eye-antennal disc primordium
(Fig. 7B)
(Jones et al., 1998), similar
to ey and toy (Quiring
et al., 1994
; Czerny et al.,
1999
). In the embryo, it is also expressed in the antennal organ,
salivary gland, and in a segmentally repeated lateral pattern
(Jones et al., 1998
).
|
A second Pax gene was identified about 30 kb downstream of eyg,
based on the fly genome sequence (Adams et
al., 2000). The predicted gene is represented by an EST clone. Its
encoded protein is most homologous to EYG in its paired domain and
homeodomain, so we named the gene twin of eyg (toe). In the
eye disc, the expression pattern of toe
(Fig. 3B) is very similar to
that of eyg (Fig. 2C,
Fig. 3B). Three independent
lines, 37-1, 22-2 and 94-4, were generated from mobilization of
P[GawB]EM458, which has the insertion 124 bp upstream of
the Lune transcription start site and 527 bp upstream of the first ATG. The
P[GawB] has transposed 8 bp downstream in all three lines, and is accompanied
by an 86 bp, 159 bp and 224 bp deletion, respectively, of the flanking genomic
region (Fig. 3A). Thus
eyg22-2 and eyg94-4 have deletions
extending into the 5' untranslated region. eyg37-1
is homozygous viable and results in a very weak small eye phenotype
(Fig. 3B).
eyg22-2 is homozygous viable and produces a small eye
(about 500 ommatidia) phenotype (Fig.
3B). The eyg94-4 homozygote dies at the
pharate adult stage. The phenotypes of pharates ranged from nearly headless
(Fig. 3B; 25/57 flies=44%), to
complete absence of eye (similar to Fig.
1B; 5/114 eyes=4%), to small eyes (about 300-400 ommatidia; 59/114
eyes=52%). These mutations failed to complement eyg1, so
they are eyg alleles as defined by genetic complementation. In these
mutants, eyg mRNA was strongly reduced in the eye disc and in the
antenna disc, while toe mRNA level was not significantly affected
(Fig. 3B). The
eygM3-12 mutant has a large deletion starting at 23 bp
upstream of the eyg transcription start site and extending to about
13 kb downstream of eyg (Fig.
3C). The molecular nature of these mutations, together with the
rescue results, strongly suggest that the eyg gene is a Pax gene
represented by the Lune cDNA.
|
Rescue experiments were performed by induced eyg expression to
determine the site of functional requirement for eyg. The
eyg cDNA was cloned into the pUAST vector behind a tandem array of
five GAL4 binding sites (Brand and
Perrimon, 1993). Expression of the UAS-eyg transgene is
dependent on the presence of the yeast GAL4 transcription factor. Expression
of eyg induced by the E132-GAL4 and ey-GAL4
(abbreviated E132>eyg and ey>eyg,
respectively) could partially or fully rescue the strong
eyg1/eygM3-12 mutant: the eyes were
morphologically normal and often of normal size
(Fig. 4B and data not shown).
ey>eyg can rescue the eye in
eyg1/eygM3-12 mutants to medium to
full size with complete penetrance (Fig.
4D). E132 drives GAL4 expression in the center of the
posterior margin in late third instar eye discs
(Fig. 4A)
(Halder et al., 1995
). In
second instar larval eye discs E132 drives expression in a broader
posterior-ventral domain (Fig.
4A inset) (Pignoni and
Zipursky, 1997
). ey-GAL4 drives expression from the
posterior margin to a few rows of cells anterior of the MF in late third
instar eye disc (Fig. 4C), and
in the entire eye disc in the early third and late second instar eye disc
(Fig. 4C inset). These rescue
results suggest that eyg expression in cells common to both E132 and
ey domain in early eye discs is important for eye development.
|
In the eye disc, dpp-GAL4 is expressed along the posterior and
lateral margins (Treisman and Rubin,
1995). The eyg-induced extra eyes were almost always
located ventral to the endogenous eyes. They often appeared linked to the
endogenous eye (Fig. 5A), but
occasionally were well separated and were surrounded by orbital bristles
(Fig. 5B), suggesting that they
represent intact eye fields. The penetrance of extra eye formation is low.
With a strong UAS-eyg line, almost all UAS-eyg/+; dpp-GAL4/+
(abbreviated dpp>eyg) flies have some recognizable defect
in the ventral head: the ventral-posterior rim of the eye was reduced and
replaced by head cuticle, and/or a few extra bristles, only 16% of eyes have
extra ommatidia, and only 38% of these have the extra ommatidia as an isolated
extra eye.
|
eyg and ey are transcriptionally independent
Ectopic expression of eyg and ey with the same
dpp-GAL4 driver produced ectopic eyes at different sites:
eyg-induced eyes occurred in the ventral part of the head, while
ey-induced eyes occurred at the base of antennae, wings, halteres and
on leg segments (Fig. 5E)
(Halder et al., 1995). In
imaginal discs, dpp>ey caused overgrowth and the
formation of MFs and photoreceptors in the antenna, wing, haltere and leg
discs (Fig. 5F, and data not
shown), consistent with the ectopic eyes seen in adults. No extra
photoreceptors were detected at the ventral margin in the eye-antennal disc
(Fig. 5F), in contrast to the
effect of eyg expression. The preferential effect in the ventral side
of the eye disc by dpp>eyg is similar to the effect of
dpp>dac+eya (Chen et
al., 1997
). In the wing disc, dpp>eyg
occasionally caused an extra dpp-lacZ-expressing spot at the
anterior side of the hinge region (not shown). However, the dpp-lacZ
spot does not represent an ectopic MF, as no photoreceptors were detected in
the wing and leg discs. These results suggest that neither eyg nor
ey activate the expression of the other, since their phenotypes are
so distinct. We checked this possibility by analyzing ey and
eyg expression in mutant backgrounds.
eyg expression was examined in the ey2 mutant.
Although ey2 is not a null mutant
(Kronhamn et al., 2002), it
has no detectable ey transcript in the embryonic eye disc primordium
and in the larval eye disc (Quiring et
al., 1994
). ey2 eye phenotype is variable. For
the ey2 stock we used, 40% of the eyes had 300-400
ommatidia, 40% had about 200 ommatidia, and 20% had less than 100 ommatidia.
The ey2 eye disc is also variable in size. However, an
ey2 eye disc of substantial size still has no ey
expression (Quiring et al.,
1994
). So we examined ey2 eye discs that were
clearly reduced in size (to be sure that it was a mutant disc) but had
sufficient eye field present to check for eyg expression. In
ey2 late third instar eye disc, the eyg dorsal
expression was not affected while the ventral expression was reduced
(Fig. 6E). In
ey2 embryos, eyg expression was still present in
the eye disc primordium (Fig.
6B). Similarly, ey expression in the eye disc primordium
(Fig. 6C) was still present in
eyg null mutant embryos (C1/C1)
(Fig. 6D). The presence of
ey expression suggests that the development of the eye disc
primordium is not strictly dependent on eyg function. Halder et al.
(Halder et al., 1995
)
indicated that ey expression is not affected in eyg mutant
eye discs. These results suggested that, except for a small amount of
eyg expression in the ventral part of the eye disc, the expression of
neither eyg nor ey is strongly dependent on the other.
|
Functional relationship between eyg and ey
We tested whether eyg is functionally dependent on ey,
and vice versa. When E132>eyg induction occurred in an
ey2 mutant, ectopic ventral eyes could still form
(Fig. 7A), suggesting that
ey is not required for eyg function. This is in contrast to
the situation with eya, so, dac and toy: the ectopic eyes
caused by their ectopic expression cannot form in the ey2
mutant (Bonini et al., 1997;
Chen et al., 1997
;
Pignoni et al., 1997
;
Czerny et al., 1999
).
Similarly, dpp>ey induced ectopic eye formation in
eygM3-12 mutants (Fig.
7B), suggesting that eyg is not required for ey
function. Again, this is in contrast to the requirement for eya, so
and dac in ey-induced ectopic eye formation
(Bonini et al., 1997
;
Chen et al., 1997
;
Shen and Mardon, 1997
;
Halder et al., 1998
). These
results suggest that eyg and ey can function independently
to induce eye formation.
While the above experiments show that eyg and ey can independently induce eye formation, their coexpression showed synergistic enhancement of ectopic eye formation. The ectopic eyes in the antenna, wing, haltere and legs are larger (Fig. 5G), similar to the effect of dpp>ey at higher temperatures (due to higher GAL4 activity). The enhancement is only evident with a weak dpp-GAL4 line. With stronger dpp-GAL4 lines, the ectopic eye phenotype is already strong with UAS-ey alone and cannot be enhanced further by adding UAS-eyg. The enhancement is more evident in the imaginal discs than in the adults. The ectopic eyes in the ventral head are not significantly enhanced in the adults, but are clearly enhanced in the eye discs (Fig. 5H). The difference between the strength of phenotypes in adults and the imaginal discs suggest that there may be some regulative mechanism in the eye field that compensates for the ectopic photoreceptors. The synergistic effect of eyg and ey coexpression was also observed when driven by the E132-GAL4 line (not shown).
Expression of ey induced by ey-GAL4 can partially rescue
the eyg1/eygM3-12 mutant eye phenotype
(Fig. 4E), indicating that
ey can functionally substitute for eyg. Since there is
endogenous ey expression, the rescue suggests that EY is required at
a level higher than its endogenous expression level in order to compensate for
the loss of eyg. Reciprocally, we checked whether expression of
eyg could rescue ey mutant phenotype. Since even the
strongest ey alleles result in a variable eye phenotype
(Kronhamn et al., 2002;
Benassayag et al., 2003
), we
used the eyD/ey2 allelic combination, which
results in no eyes and is nearly completely penetrant. eyD
has a chromosomal rearrangement interrupting the ey gene, producing a
truncated protein lacking the homeodomain
(Kronhamn et al., 2002
).
ey2 contains a transposon insertion in an eye-specific
enhancer and has no detectable RNA and protein expression in the larval eye
disc and in the embryonic eye disc primordium
(Quiring et al., 1994
;
Halder et al., 1998
).
dpp>eyg can partially rescue
eyD/ey2 mutants
(Fig. 4F), suggesting that
eyg can functionally substitute for ey.
eyg suppresses wg transcription
The wingless (wg) gene encodes a secreted signaling
protein of the Wnt family. It is expressed in the dorsal and ventral margins
of the eye disc (Fig. 8A), and
acts to inhibit MF initiation from these sites
(Ma and Moses, 1995;
Treisman and Rubin, 1995
). In
eyg1 eye disc, wg expression domain expands
toward the posterior margin (Fig.
8C) (Hazelett et al.,
1998
). However, wg expression is not derepressed in the
central region (Fig. 8C) where
eyg is normally expressed (Fig.
4), probably because eyg1 is a hypomorphic
allele and has sufficient activity in this region to suppress wg.
Because the enhancer trap wg-lacZ reporter was used to monitor
wg expression, the suppression is at the transcriptional level. In
addition, ectopic expression of eyg (dpp>eyg)
suppressed wg-lacZ expression in the dorsal and ventral margins of
the eye disc and in part of the wing disc (compare
Fig. 8B,E with 8A,D).
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DISCUSSION |
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In the embryo eyg transcripts appear in the eye-antennal disc primordium beginning at stage 15. It continues to be expressed as the disc cells proliferate during early larval development and then is expressed in an anterior region overlapping the equator of the eye disc as photoreceptor differentiation occurs. eyg is required for eye development, as loss-of-function mutations lead to the reduction or absence of the eye. It appears to be required for the early proliferation of the eye disc cells, as the early third instar eye disc is significantly smaller. The rescue experiments (hs-eyg, ey>eyg and E132>eyg) suggested that the critical time for eyg function is in the late second instar. Excessive apoptosis occurred in the mid-third instar eye disc, but is not the major cause of the eye phenotype because blocking apoptosis did not rescue the eye phenotype. Ectopic expression of eyg can lead to ectopic MF initiation in the ventral side of the eye disc. Thus, the loss-of-function and gain-of-function phenotypes suggest that eyg acts as an important regulator of eye development.
eyg appears to have two major functions. The first is to promote cell proliferation in the eye disc. eyg loss-of-function mutants have reduced eye discs, already apparent in early third instar, before photoreceptor differentiation. In clonal analysis, eygM3-12 mutant clones induced in first or second instar are undetectable in late third instar eye disc (Fig. 1). Ectopic eyg expression caused local overgrowth (Fig. 6C), a phenotype opposite of the loss-of-function phenotype. The overgrowth does not always develop into photoreceptor cells (Fig. 6C). These results indicate that eyg promotes cell proliferation independent of photoreceptor differentiation. The second function of eyg is to promote eye development or MF initiation. If the eyg-induced proliferation occurs at the ventral margin of the eye disc, ectopic MF can initiate (Fig. 6C,D). The induction of ectopic MF is probably mediated by the suppression of wg (see later), which is known to repress MF initiation along the lateral margins.
eyg and ey act cooperatively
Since eyg is a Pax gene that shares sequence similarity with
ey and toy in the PD and HD domains
(Jun et al., 1998), its
relationship with ey is of particular interest. Our results indicate
that eyg and ey are transcriptionally and functionally
independent: (1) except for a small amount of eyg expression ventral
to the equator of the eye disc, eyg and ey do not regulate
each other's expression. In this respect, eyg is different from
dac, so and eya, whose expression is strongly regulated by
ey (and can induce ey expression in some cases). Thus
eyg transcription is neither downstream of ey, nor does
eyg participate in the
ey/eya/so/dac positive feedback loop. This
transcriptional independence is similar to that of optix
(Seimiya and Gehring, 2000
).
(2) eyg and ey can each function (to induce ectopic eyes) in
the absence of the other. Again, this is similar to optix, which can
induce ectopic eyes in ey2 mutant
(Seimiya and Gehring, 2000
).
Whether optix is required for ey function has not been
tested, because of the lack of optix mutants.
However, other evidence indicates that the functions of eyg and
ey must converge at some point in the pathway leading to eye
development: (1) eyg; ey double hypomorphic mutants showed a
much stronger eye-loss phenotype (Hunt,
1970), (2) coexpression of ey and eyg caused
synergistic enhancement of the ectopic eye phenotype, (3) eyg and
ey are able to substitute functionally for each other. Overall, the
results suggest that these two Pax genes may act cooperatively. This genetic
cooperativity might mean that eyg and ey interact and
cooperate as proteins in the same pathway or that they act in parallel
pathways. eyg and ey are coexpressed in the eye disc
primordium in the embryo (Fig.
7). Their expression domain also overlap in the eye disc,
especially in the early eye disc (Fig.
4) when eyg function is critically required. So it is
possible that the two Pax proteins act within the same cell, although we do
not rule out the possibility they act in different cells to achieve a
functional cooperativity.
If eyg and ey are both required for eye development, how could ectopic expression of either one be sufficient for ectopic eye development? One possibility is that the two Pax proteins form heterodimers, directly or indirectly via other proteins, to activate target genes. When the level of either one is low, the target genes that lead to eye formation cannot be induced. However, when either one is strongly expressed ectopically, the high level of homodimer can partially substitute for the heterodimer. Since both genes are required for normal eye formation, this model predicts that the EYG-EY heterodimer is more effective than either homodimer in inducing eye formation. As expected by this model, coexpression of eyg and ey caused enhanced ectopic eye formation.
Possible mechanisms of EYG protein function
The EYG protein has two DNA binding domains: the RED subdomain of its
truncated PD and the Prd-class HD. It probably functions as a transcription
factor by binding DNA targets through these domains, singly or in combination.
In addition, its interaction with other proteins may affect this DNA
binding.
The PD consists of two independent subdomains: the N-terminal PAI and the
C-terminal RED subdomains. Based on crystal structure of the human Pax6 PD,
the linker region connecting the two subdomains also contacts DNA
(Xu et al., 1999). In EYG, the
PAI subdomain is largely missing and most likely cannot bind DNA. One
interesting possibility is that the truncated EYG PD has a dominant negative
effect, competing with other PD proteins. In addition, truncation of the PAI
subdomain in the Pax6-5a and Pax8(S) isoforms probably exposes the RED
subdomain to recognize a distinctly different DNA sequence
(Epstein et al., 1994
;
Kozmik et al., 1998
). Thus the
EYG PD may bind DNA through its RED domain, similarly to the Pax6-5a and
Pax8(S) isoforms (Epstein et al.,
1994
; Kozmik et al.,
1998
) and distinct from the Pax6 PD. This prediction was in fact
proved by site-selection using the EYG RED domain
(Jun et al., 1998
). Through
its RED domain, EYG can probably regulate different target genes than those
regulated by EY. This ey-independent function of eyg is also
shown by its involvement in salivary gland development
(Jones et al., 1998
), and in
bristle formation when ectopically expressed (see Results). Vertebrate
homologs of EYG have not yet been identified. It is possible that EYG plays a
role equivalent to the vertebrate Pax6-5a isoform.
In addition to the PD, many Pax proteins (including EY and EYG) also
contain a Prd-class homeodomain. Two Prd-type HDs can bind cooperatively to a
palindromic site composed of two inverted TAAT motifs separated by 2 or 3 bps
(Wilson et al., 1993). The
Prd-type HD of EYG can form heterodimers with the Prd-type HD of Prd upon
binding to a consensus DNA target (Wilson
et al., 1993
; Wilson et al.,
1996
). It is possible that EY and EYG also form heterodimers via
their HDs. This would be consistent with our findings that they act
synergistically. However, although the HD of EYG is required for its functions
(J. G. Yao and Y.H.S., unpublished), the HD of EY has been shown not to be
required for its function in eye development
(Punzo et al., 2001
). Thus the
HD of EYG is required, not for direct interaction with the HD of EY, but may
be for DNA binding or for interacting with other proteins.
eyg suppresses wg transcription in the eye
disc
Dpp and Wg are two signaling molecules important for the initiation of eye
differentiation: Dpp activates MF initiation while Wg suppresses it
(Heberlein et al., 1993;
Wiersdorff et al., 1996
;
Chanut and Heberlein, 1997
;
Pignoni and Zipursky, 1997
;
Ma and Moses, 1995
;
Treisman and Rubin, 1995
).
Does eyg exert its effect on eye development by activating Dpp
signaling or by suppressing Wg signaling?
dpp is expressed at two stages in the eye disc: an early
expression along the posterior and lateral margins (represented by the
dpp-GAL4), and a later expression in the propagating MF (represented
by the dpp-lacZ). The early expression in the margins is required for
MF initiation (Burke and Basler,
1996; Wiersdorff et al.,
1996
). It was found that dpp expression along the lateral
margins is absent in early third instar eyg1 eye disc
(Hazelett et al., 1998
),
suggesting that dpp expression in the lateral margins is regulated by
eyg. However, activating DPP signaling at the lateral margin did not
rescue the eyg1 phenotype
(Hazelett et al., 1998
),
suggesting that eyg has other functions in addition to activating
dpp expression.
wg is expressed uniformly in the eye disc of second instar larvae
(Royet and Finkelstein, 1997).
In the third instar eye disc, wg is expressed in the lateral margins
and acts to prevent MF initiation from the lateral margins
(Ma and Moses, 1995
;
Treisman and Rubin, 1995
). The
wg-expression domain expands in eyg1 eye discs
(Fig. 8C) (Hazelett et al., 1998
). Our
results further showed that ectopic eyg expression
(dpp>eyg) could suppress wg expression at the
transcriptional level. The suppression of wg is functionally
significant, because expression of the wg-activated omb gene
is similarly suppressed in dpp>eyg (J.-L. Chao and
Y.H.S., unpublished). Hazelett et al.
(Hazelett et al., 1998
) have
shown that blocking of the Wg signaling pathway can partially rescue the
eyg mutant phenotype. These results indicate that the suppression of
wg transcription by eyg may be a major mechanism by which
eyg induces MF initiation, hence eye development. This is consistent
with our finding that ectopic eyg induces ectopic eye formation
primarily in the ventral margin of the eye disc, where wg expression
is weaker (Fig. 8A) and most
easily suppressed by dpp (Pignoni
and Zipursky, 1997
). wg is normally expressed in the
entire eye disc during second instar
(Royet and Finkelstein, 1997
).
It was shown that Wg signaling can suppress the expression of so and
eya (Baonza and Freeman,
2002
). It is possible that in the late second instar eye disc,
eyg expression in the central domain of the eye disc suppresses
wg expression in the central domain, thus allowing the expression of
eya and so, hence eye development.
As predicted by the eyg and ey interaction, ey
also suppresses wg expression (data not shown). Suppression of
wg expression by eyg (and ey) is also seen in the
wing disc (Fig. 8E). However,
suppression does not occur in all cells expressing eyg, suggesting
that additional factors are required for the wg suppression. The
relationship of eyg/ey and wg may be mutually
antagonistic, since ectopic ey cannot induce eya and
so expression in regions of high wg expression
(Halder et al., 1998).
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ACKNOWLEDGMENTS |
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