Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland
** Author for correspondence (e-mail: walter.gehring{at}unibas.ch)
Accepted 21 May 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ey, toy, sey, PD, HD, Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The eye disc is fused to the antennal disc and forms a compound
eye-antennal disc. The eye-antennal disc grows during the three larval stages
and, towards the end of the third instar, the cells of the eye disc start to
differentiate. Differentiation is marked by a wave of morphological changes,
reflected by the morphogenetic furrow (MF), which moves from the posterior to
the anterior of the disc (reviewed by
Wolff and Ready, 1991). Cells
anterior to the furrow are still undifferentiated, whereas cells posterior to
the furrow start to form clusters of hexagonal arrays, which reflect the shape
of an adult ommatidium. Only the undifferentiated cells anterior to the MF
express the two Drosophila Pax6 genes ey and toy,
whereas in the differentiated cells posterior to the MF, both mRNAs seem to be
downregulated (Czerny et al.,
1999
); however, TOY protein is still detected posterior to the MF
(U. Walldorf, personal communication). Although differentiation of the eye
disc only starts at the third larval stage, its cells become determined during
the second instar, when the eye specifying genes ey and toy,
and their early downstream target genes sine oculis (so),
eyes absent (eya) and dachshund (dac), are
expressed (Kumar and Moses,
2001
). Interestingly, during first to mid-second instar stages of
disc development, ey, the selector gene for eye identity, is also
expressed in the antennal disc (Kenyon et
al., 2003
). Because cells specifying the eye-antennal disc
invaginate together and form a compound disc, selector genes are found to be
expressed in both eye and antennal discs at an early stage, and only later do
they become restricted to either the eye or the antennal part when
determination sets in. Consistent with this, recent studies have shown that
ectopic ey is able to repress DLL, an antenna specific gene
(Kurata et al., 2000
).
Furthermore, it has been shown that this repression is mediated by the
homeodomain (HD) of ey (Punzo et
al., 2001
). The ability of selector genes to downregulate each
other can therefore lead to an exclusive territorial expression, and to the
specification of the identity of a given disc
(Benassayag et al., 2003
).
The existence of two Pax6 genes in Drosophila raises the
question of whether they have a redundant function or whether they have
diverged to control different sets of target genes. A recent characterization
of new alleles of ey and toy mutants by Kronham et al.
(Kronham et al., 2002), and by others (S. Flister, U. Kloter, M.S., C.P., L.
Michaut and W.J.G., unpublished), suggests a functional divergence, with a
partial redundancy remaining. Epistasis studies showed that toy lies
upstream of ey, because ectopic toy is capable of inducing
ectopic ey but not vice versa
(Czerny et al., 1999).
Additionally, toy cannot induce ectopic eyes in an
ey2 mutant background whereas ey can
(Czerny et al., 1999
). The
regulation of ey by toy is due to a direct binding of the
TOY-PD to the ey-enhancer, which is located in the second intron of
the ey gene. The EY-PD contains a glycine at position 14, whereas the
TOY-PD has an asparagine at that same position. This difference allows TOY to
regulate ey through the ey-enhancer, whereas EY cannot
regulate itself (Czerny et al.,
1999
). Complementary experiments showed that ectopic expression of
a HD-deleted version of the EY protein did not induce the endogenous
full-length gene, and therefore confirmed the lack of an auto-regulatory
feedback loop for ey (Punzo et
al., 2001
). Endogenous ey can only be induced by
misexpression of the three downstream genes eya, so and dac,
or by toy (Halder et al.,
1998
; Czerny et al.,
1999
). Interestingly, the mouse Pax6 gene sey
also has an asparagine at position 14 of the PD and has, therefore, the same
DNA-binding properties as toy
(Hill et al., 1991
;
Czerny et al., 1999
). Moreover,
sey and toy have multiple stretches of conserved amino acids
in their C termini, whereas the C terminus of ey diverged. Thus,
ectopic sey is able to induce ectopic ey but it does not
induce ectopic toy (Czerny et al.,
1999
). This suggests that the auto-regulatory feedback loop found
in the vertebrate Pax6 gene evolved into a hetero-regulatory
interaction in Drosophila with toy regulating ey
expression (Gehring and Ikeo,
1999
). Overall these data indicate that not only the PDs, but also
the cis-regulatory sequences of the two Drosophila Pax6 genes, have
diverged to control different sets of target genes. This hypothesis is further
supported by the fact that only toy is expressed in the ocelli
territory of the eye disc but both toy and ey regulate the
eye-specific enhancer of the so gene, by binding partly to the same
and partly to different binding sites, and by discriminating between eye and
ocelli development during larval stages
(Punzo et al., 2002
).
The PD and the HD are the most conserved regions within the Pax6 proteins,
indicating evolutionary constraints imposed to maintain specific binding to
target genes. We therefore investigated to what extent the PD of TOY and SEY,
which diverged in their DNA binding properties from the PD of EY, were able to
induce the eye developmental pathway independently of ey. Moreover,
we determined whether only the HD of ey was able to downregulate
Dll expression, as previously shown by Punzo et al.
(Punzo et al., 2001), and
whether this function is required during endogenous eye development to specify
the eye territory. We have tested these hypotheses by generating deletion
constructs of sey similar to those described for ey and
toy (Punzo et al.,
2001
; Punzo et al.,
2002
), as well as EY-TOY chimeric proteins, and scored for ectopic
eye formation. Furthermore, we rescued the ey null mutant
eyJ5.71 (Punzo et al.,
2001
) by transferring the genomic region of the ey gene
onto the third chromosome. This allowed us to analyze mutant ey
clones in a wild-type background. We found that both sey and
toy were able to activate eye development in an
ey-independent manner, and that one of the main differences between
toy and ey, besides their DNA-binding properties, lies in
their C-terminal region, and therefore mainly in their transactivation
potential. This suggests that most of the differences reside in their capacity
to interact with different sets of proteins. We also show that only the HD of
ey is able to downregulate DLL expression in an ectopic situation,
and that this downregulation is required during endogenous eye
development.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specific genotypes generated for this publication were: (1)
UAS-sey/UAS-sey; ey2/ey2, (2)
UAS-seyPD/UAS-sey
PD;
ey2/ey2, (3)
UAS-sey
HD/UAS-sey
HD;
ey2/ey2, (4) UAS-toy/UAS-toy;
ey2/ey2, (5)
UAS-toy
PD/UAS-toy
PD;
ey2/ey2, (6)
UAS-toy
HD/UAS-toy
HD;
ey2/ey2, (7) UAS-P35/UAS-P35;
UAS-ey/UAS-ey; ey2/ey2, (8)
UAS-P35/UAS-P35;
UAS-ey
PD/UAS-ey
PD;
ey2/ey2, (9) UAS-P35/UAS-P35;
UAS-ey
HD/UAS-ey
HD;
ey2/ey2, (10) UAS-P35/UAS-P35;
ey2/ey2, (11)
so10-lacZ/so10-lacZ;
TM6Tb/dppblink-Gal4, (12) CyO/so-lacZ;
TM6Tb/dppblink-Gal4;
ey2/ey2, (13) CyO/Dll-lacZ;
TM6Tb/dppblink-Gal4; (14)
ey-Gal4/ey-Gal4; ey2/ey2. For
the clonal analysis the following lines were generated: (15) ywhsflp;
82B FRT/82B FRT;
Dp(1;4)1021y+/Dp(1;4)1021y+, (16)
ywhsflp; 82B FRT/82B FRT; eyJ5.71/ciD,
(17) yw; TM6Tb/82B FRT P(smo+,hsp70-GFP) p6.3-ey;
Dp(1:4)1021y+/Dp(1:4)1021y+, (18)
yw; TM6Tb/82B FRT P(smo+,hsp70-GFP) p6.3ey;
eyJ5.71/ciD. The crosses between the lines 15
and 16, and between the lines 17 and 18, respectively, give the two lines that
were used to cross together for the clonal analysis
(Fig. 7B).
|
Antibody staining on discs was performed according to Halder et al.
(Halder et al., 1998).
ß-Galactosidase staining and antibody stainings on cryosections were
performed as desribed in Ashburner
(Ashburner, 1989
). Dilution of
antibodies was as follows: rabbit
-EY, 1:3000 (for clonal analysis);
mAb
-DLL, 1:20; and
-GFP, 1:1000 (Torrey Pines Biolab).
Cloning procedure and western blots
Both sey PD and HD deletion constructs were made by standard
recombinant PCR techniques, deleting only the PD or the HD, respectively. PCR
was performed directly on the cDNA that was cloned in pUAST
(Halder et al., 1995). The
newly generated sey cDNAs were directly ligated into a PCR cloning
vector, sequenced and then cloned back into pUAST with NotI-Asp718.
All four chimera constructs were made by recombinant PCR techniques, switching
exactly the PD or the C terminus at the end of the HD of the corresponding
protein. PCR was performed directly on the cDNAs, which were cloned in pBSK.
The newly generated cDNAs were directly ligated into pUAST using
BglII and XbaI. Both sites were inserted into the primers
used for the recombinant PCR. All four constructs were confirmed by
sequencing. Detailed descriptions of the primers used are available upon
request. To rescue the ey null mutant eyJ5.71, we
first screened a genomic P-element library with ey cDNA
(Tamkun et al., 1992
).
Positive clones were further analyzed by means of restriction digest and PCR
analysis to confirm the presence of all exons, and to map the length of the
5' and 3' region of the rescue clones. Two clones containing the
entire genomic area were injected: one with a 2 kb extension at the 5'
end of exon 1 and a 15 kb extension at the 3' end of exon 9 (referred to
as clone p6ey), and one with a 5 kb extension at the 5' end of
exon 1 and an 8 kb extension at the 3' end of exon 9 (referred to as
clone p14ey). The cosmid p6ey was able to rescue the
eyJ5.71 mutant. As only one line carrying p14ey
was obtained we cannot exclude at present that this genomic region would also
be sufficient to rescue the eyJ5.71 mutant. Embryonic and
pupal lethality, developmental delay, as well as eye phenotypes were restored
by p6ey. Sterility was restored for males but not for females,
indicating that regulatory sequences required for ey expression in
the female sexual organs were not present within the rescue cosmid
p6ey. Western blot experiments were carried out as described by Punzo
et al. (Punzo et al., 2001
).
The rabbit
-quail-PD (Carriere et
al., 1993
) was used at a dilution of 1:200, the rabbit
-EY
at a dilution of 1:200, in which the antibody was pre-absorbed with larval
tissue. The anti-ß-Galactosidase antibody was used at a dilution 1:2000
(Promega).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We made use of the previously published PD and HD deletion constructs of
ey and toy (Punzo et
al., 2001; Punzo et al.,
2002
), and, in addition, generated a PD- and HD-deleted version of
the mouse Pax6 gene sey. Because sey has the same
in vitro DNA-binding specificity as toy, it is also able to induce
ectopic ey expression and ectopic eyes when misexpressed in a
wild-type background, but it is not able to induce ectopic toy
(Czerny et al., 1999
). We used
the UAS/Gal4 system (Brand and Perrimon,
1993
) to drive our different UAS-deletion constructs by
dppblink-Gal4
(Staehling-Hampton and Hoffmann,
1994
). First, we confirmed that the HD-deleted versions of
toy (toy
HD) and sey
(sey
HD) were still able to induce ectopic eye
development in a wild-type background (data not shown). This result was in
agreement with data showing that the TOY-PD and the SEY-PD regulate
ey through the ey enhancer
(Czerny et al., 1999
).
To test to what extent the PD of sey and toy could still
activate the eye developmental cascade in an ey-independent manner,
we misexpressed both toyHD and
sey
HD in an ey mutant background.
Unexpectedly, both toy
HD and
sey
HD were able to induce ectopic eye development in
an ey2 and in an ey null
(eyJ5.71) (Punzo et
al., 2001
) (S. Flister, U. Kloter, M.S., C.P., L. Michaut and
W.J.G., unpublished) mutant background
(Fig. 1B,D; data not shown for
eyJ5.71). In addition, in the control cross, in which both
full-length proteins were misexpressed in the same genetic background, ectopic
eyes were also induced (Fig.
1A,C), whereas the PD-deleted versions
(toy
PD and sey
PD) did not
lead to any phenotype in either a wild-type or ey mutant background
(data not shown). This result shows that the induction of ectopic eyes by
toy
HD and sey
HD in an
ey mutant background is not due to the absence of the HD. Moreover,
we conclude that toy and sey can activate all target genes
required for eye development in the absence of endogenous ey, despite
their different DNA-binding properties.
|
rhodopsin 1 has been suggested to be directly regulated by the HD
of ey (Sheng et al.,
1997). We have previously shown for ey that ectopic eye
development is independent of the EY-HD and that ectopic eyes generated in an
ey mutant background still express rhodopsin 1
(Punzo et al., 2001
). The same
was found to be true for toy and sey
(Fig. 1I-L), suggesting that
ectopic eyes induced in an ey mutant background by toy,
toy
HD, sey and sey
HD complete
their developmental program. It further strengthens the hypothesis that
rhodopsin 1 induction is independent of the Pax6 HD, as
observed in mice where Pax6 is not expressed in differentiating rods or cones
(reviewed by Pichaud et al.,
2001
).
Effects of different expression levels of Pax6
We wondered whether the difference observed in our experiments, in which we
were able to induce ectopic eyes in an ey mutant background by
toy and sey and their HD-deleted versions, might be due to
dosage dependence when compared with previously published data
(Czerny et al., 1999). We
noticed in the course of this work that several independent transgenic
deletion lines of ey (ey
PD and
ey
HD) showed strong variations in expressivity when
crossed to dppblink-Gal4
(Fig. 2A,B; data not shown for
ey
PD). Western blot experiments using an
-EY
antibody revealed a clear correlation between the phenotypes induced and the
amount of protein expressed (Fig.
2C). This result shows that not only the presence, but also the
amount of ectopically induced Pax6 protein is crucial to induce ectopic eye
morphogenesis. In order to compare the phenotypes of individual transgenic
lines of either the same or of a homologous protein, the expression levels of
the corresponding proteins in the individual lines have to be compared too. To
re-examine the different results obtained in our experiments and the earlier
ones of Czerny et al. (Czerny et al.,
1999
), we made use of the temperature sensitivity of the Gal4
system (Jarrett, 2000
). We
repeated the misexpression experiment with toy in an
ey2 mutant background using the line that gave us ectopic
eyes and reared the flies at 18°C and 25°C. Flies developing at
18°C showed appendage truncation without ectopic eyes as previously
described by Czerny et al. for misexpression of toy in an
ey2 mutant background
(Czerny et al., 1999
). By
contrast, the flies developing at 25°C exhibited ectopic eyes
(Fig. 2D,F). Western blot
experiments on leg discs of third instar larvae with ey, toy, sey and
their HD-deleted versions confirmed that our ectopically induced proteins were
expressed at the expected molecular weight and at comparable levels
(Fig. 2F). This allows us to
conclude that our toy, toy
HD, sey and
sey
HD lines are able to induce ectopic eyes in an
ey mutant background, when expressed at comparable levels to those
required for ectopic eye induction by ey. Overall, these experiments
show that the ectopically induced protein levels are crucial for inducing
phenotypic changes.
|
|
Dll repression by the HD of ey is required during endogenous eye development
We have previously shown that the HD of ey is involved in
repressing the selector gene Dll, as monitored by antibody staining,
in an ectopic situation leading to leg truncation
(Punzo et al., 2001).
Interestingly, the PD-deleted versions of sey and toy did
not lead to appendage truncation when misexpressed (data not shown).
Consistent with this observation, the Dll-lacZ enhancer was
downregulated by misexpression of ey
PD only, and not
by misexpression of sey
PD or
toy
PD (data not shown). Although all three proteins
share the same crucial amino acids at positions 50 and 51 of the recognition
helix of their HD, this difference may be attributed either to a specific
interaction of the ey C terminus with other cofactors, or to
protein-protein interactions between the homeodomains of ey and
Dll. Therefore, we asked whether the repression of DLL by EY-HD might
have any endogenous function during eye development. First, we analyzed
Dll expression in third instar eye discs of ey2
mutants, assuming that, if ey is required to repress Dll, a
lack of ey would lead to an activation of Dll. Antibody
staining with an
-DLL antibody did not reveal any DLL expression in
ey2 mutant eye discs (data not shown). However, cell death
is increased in those mutant eye discs because of the absence of EY
(Halder et al., 1998
) and,
therefore, the lack of DLL expression could also be due to apoptosis. To
overcome this problem, we prevented cell death by expressing the
anti-apoptotic protein P35 of baculovirus with the ey enhancer
(ey-Gal4) in ey2 mutant eye discs. Expression of
P35 in the eye discs of ey2 mutants leads to a duplication
of the antennal disc in almost 100% of the offspring and also to a duplication
of the Dll-expressing domain (Fig.
4E,F). Consequently, the adult fly replaces the normal eye with an
additional antenna, resulting in an imago with four antennae
(Fig. 4G,H). In the absence of
ey, when cell death is prevented, DLL remains active and the cells
differentiate into the fate dictated by Dll and form an antenna.
|
To mimic the endogenous situation more closely, we prevented cell death by
expressing the different ey deletion constructs, hence rescuing the
ey2 mutant, instead of expressing the anti-apoptotic
protein P35. We repeated previously published rescue experiments in which
ey, eyPD or ey
HD were
expressed by ey-Gal4 in an ey2 mutant background
(Punzo et al., 2001
) and
checked for DLL expression. Rescuing the ey2 mutant by
ey or ey
HD leads to ectopic DLL induction in
the absence of the HD, whereas in the presence of the HD DLL expression is
completely inhibited (Fig.
5A-C). DLL is induced only in some cells in around 5% of the
discs. This explains why antenna-like outgrowths were obtained in a small
percentage of flies (around 2%) that showed an incomplete rescue with
ey
HD (Fig.
5D); these outgrowths were never observed when ey was
expressed. These results led us to the following interpretation: the EY-PD is
required to induce the eye developmental pathway preventing cell death,
whereas the HD is required, maybe in conjunction with other genes, to enhance
the switch between antennal fate and eye fate by repressing one or several
antennal-specific genes.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As the PD of EY and TOY have diverged in evolution, we have analyzed to
what extent the PD of TOY would be able to initiate eye development in an
ey-independent manner. In the analysis we also included the mouse
Pax6 gene sey, which functionally is more closely related to
toy than to ey. Regardless of the differences in DNA-binding
specificity between toy and sey versus ey, all
three genes retain the ability to induce ectopic eyes, indicating that they
bind to the cis-regulatory elements of Pax6 target genes. This is
supported by our analysis of the sine oculis enhancer so10,
which contains five Pax6-binding sites, all of which bind TOY, but only three
of them also interact with EY (Punzo et
al., 2002).
Dependence on expression levels
In mammals Pax6 mutations are haploinsufficient and in
heterozygotes eye development is critically affected. However, our recent
analysis of ey and toy mutants (ey15.71
and toyG3.39) and deletions indicate that ey and
toy are completely recessive (Flister et al., unpublished).
Nevertheless, overexpression of ey+ in the eye disc leads
to a reduced eye phenotype (Curtiss and
Mlodzik, 2000), indicating that expression levels are
important.
Because of its variable expressivity, the ey2 mutant
has been considered to be a hypomorph. However, we have found that neither
ey mRNA nor EY protein can be detected in ey2 eye
discs and the embryonic eye-anlagen
(Quiring et al., 1994;
Punzo et al., 2001
). These
findings strongly suggest that ey2 is a null mutation with
respect to eye development. Thus, the variable eye size observed in
ey2 flies may be due to redundant functions of ey
and toy. This interpretation is supported by recent analyses of
ey and toy mutants (Kronhem et al., 2002) (Flister et al.,
unpublished), which indicates partial complementation between the two genes.
In a previous study, we had observed that toy cannot induce ectopic
eyes in a strongly selected ey2 background with a high
penetrance and expressivity of the eyeless phenotype. However, the data
presented here show that higher expression levels of TOY protein are capable
of inducing ectopic eyes in an ey2 background. This is in
line with our finding that both ey and toy directly activate
sine oculis (so) (Punzo
et al., 2002
) by binding to its so10 enhancer, so that
the prior activation of ey by toy is not absolutely required
for initiation of the genetic cascade leading to eye development.
Finally, we would like to issue a caveat concerning the use of the UAS/Gal4 system for ectopic expression experiments, because it is difficult to ensure that the expression levels are in the range of physiological conditions.
The C terminus of ey and toy
To determine more precisely how the two Drosophila Pax6 proteins
achieve functional specificity, we swapped the PDs and the C termini of both
proteins. We show here that the C-terminal domains of EY and TOY differ
considerably, and imply functional differences between the two proteins. This
is suggested by the fact that we were able to induce ectopic eyes on the
antenna only when the C terminus of ey was present within the Pax6
protein, suggesting that the PD does not play a decisive role in this respect.
Thus, the EY-CT may interact with a different subset of transcription factors
and co-factors to increase DNA-binding specificity, functional activity and
transactivation potential. Interestingly, on the western blot of the chimeric
constructs (Fig. 3B), all of
the proteins harboring the EY-CT show a more diffuse band than the proteins
harboring the TOY-CT. This is typically seen for phosphorylated proteins and
suggests that ey function may also be regulated
post-transcriptionally through multiple phosphorylation sites. At the CT of
Pax6, there are two highly conserved domains
(Glardon et al., 1998) that
are present in SEY and TOY but absent from EY, which may account for the
observed differences in function.
The results obtained on the C terminus complement previous findings on
ey and toy, where it has been shown that the same binding
site (e.g. so10 enhancer) (Punzo
et al., 2002) can be bound by both, but depending either on the
cellular context, the presence of co-factors, protein kinases or phospatases,
the activity of ey and/or toy may be modulated in order to
obtain the correct cellular response.
The Eyeless-homeodomain in endogenous eye development
We have shown previously that eyPD can
downregulate Dll expression at the transcriptional level in an
ectopic situation leading to leg truncation
(Punzo et al., 2001
), whereas
toy
PD and sey
PD do not, even
though all three HDs have the same amino acids at positions conferring
DNA-binding specificity. These functional differences between EY and TOY most
likely reside in the CT of EY, which differs significantly from that of TOY
and SEY. Although our previous findings showed that DNA binding of the HD is
required for the downregulation of DLL
(Punzo et al., 2001
), the C
terminus of EY appears to confer the functional specificity of the
Dll repression.
Several lines of evidence point to the fact that the induction of
Dll is not directly controlled by ey but rather by a
secondary late event of postmitotic differentiation. First, in
ey2 mutants Dll is normally not expressed. Only
in very rare cases do those mutants show a transdifferentiation from eye to
antenna (Lindsley and Zimm,
1992). Second, over expression of P35 in ey2
mutants does not lead to Dll induction until the third larval stage when
differentiation sets in. Those Dll-expressing cells reside at the
posterior tip of the eye disc, where differentiation starts with the onset of
MF movement. Third, rescuing the ey2 mutant by
ey
HD (Fig.
5C) leads to normal eye development and not to uniform
up-regulation of Dll. Only in rare cases was Dll found to be
expressed in those eye discs and in even fewer cases showed antenna like
outgrowth. These results are in line with the clonal analysis, where only a
small percentage of clones show induction of Dll, but no clone
displayed an adult eye phenotype. Thus, only rarely might the size of
Dll-expressing clones be big enough the lead to a
transdifferentiation. Additionally, the ability of toy to function
redundantly to ey (Punzo et al.,
2002
) may account for those observations. Fourth, the
co-expression experiment of the various ey constructs with P35
(Fig. 6) in the
ey2 mutant strongly suggests that only the repression of
Dll is ey dependent, not the induction, as P35 in
conjunction with ey
PD, which does not initiate eye
development, completely abolishes antenna duplication. Antenna duplications
are only observed in those rare cases where P35, in conjuction with
ey
HD, does not rescue eye development and thus fails
to instruct the cells to enter the eye developmental pathway.
Taken together, these findings suggest that expressing a PD-containing Pax6
protein is sufficient to prevent Dll activation. By contrast, the
EY-HD clearly confers downregulation of Dll. A more profound study
with double mutant clones of ey and toy preventing the
presence of any Pax6-PD containing protein may be more conclusive. The
downregulation of Dll by ey may be direct or indirect, but
the activation is ey independent. Recent studies by Kenyon et al.
showed that dpp is required for the activation of Dll in the
antenna primodium (Kenyon et al.,
2003). This may explain why in the absence of ey, Dll is
only activated in cells located in or behind the MF that fail to differentiate
to photoreceptors, cells that have already seen dpp and reside
therefore normally in the posterior part of the eye disc or within the range
of dpp signaling.
Here, we show that Dll repression is required in the normal eye disc to prevent antennal development and to install the eye development program. The failure to repress Dll in the eye primordia leads to a transdetermination from eye to antennal structures, and the formation of an additional antenna in the eye field. The downregulation of Dll in the eye region of the eye-antennal discs depends on the EY-HD and the EY-CT, whereas the EY-PD (and the PD of toy) are required to install the eye development program, mainly by activation of the subordinate target genes.
Divergent functions of EY and TOY
Our results strongly suggest that the functional differences between
ey and toy are not only due to their different DNA-binding
specificities and changes in the cis-regulatory sequences of their PDs, but
also to interactions with different co-factors through their C termini. Recent
studies showed that the transcriptional activator Pax5 is converted
into a repressor by interaction with the groucho protein through its
C terminus and its octapeptide (Eberhard et
al., 2000). Similarly, the EY-CT, which differs strongly from that
of TOY, is likely to interact with a different set of co-factors to confer
specific activation or repression of target genes. This hypothesis is
supported by the analysis of the CT. Only the EY-CT, and not that of TOY, is
capable of inducing ectopic eyes on the antenna, and only the EY-HD with an
EY-CT is able to confer DLL repression, which is required for normal eye
development. Thus, our experiments provide new insights into the evolutionary
divergence of the two Pax6 genes in Drosophila, and their role in eye
and head development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Centre de Biologie du Development, Universite Paul
Sabatier, Bat4R3, 118 Route de Narbonne, 31062 Toulouse Cedex, France
Present address: Graduate Group in Microbiology, University of California
at Berkeley, 401 Barker Hall, Berkeley, CA 94720-3202, USA
Present address: Graduate School of Pharmaceutical Science, Tohoku
University, Aramaki, Aoba-ku, Sendai 980-8578, Japan
¶ Present address: Graduate Program in Genetics, Department of Molecular
Genetics and Microbiology, State University of New York at Stony Brook, NY
11794-5222, USA
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashburner, M. (1989). Drosophila: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Benassayag, C., Plaza, S., Callaerts, P., Clements, J., Romeo,
Y., Gehring, W. J. and Cribbs, D. L. (2003). Evidence for a
direct functional antagonism of the selector genes proboscipedia and
eyeless in Drosophila head development.
Development 130,575
-586.
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.
Carriere, C., Plaza, S., Martin, P., Quantennes, B., Bailly, M., Stehelin, D. and Saule, S. (1993). Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina. Mol. Cell. Biol. 13,7257 -7266.[Abstract]
Casares, F. and Mann, R. S. (1998). Control of antennal versus leg development in Drosophila. Nature 392,723 -726.[CrossRef][Medline]
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.[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.[Medline]
Eberhard, D., Jimenez, G., Heavey, B. and Busslinger, M.
(2000). Transcriptional repression by Pax5 (BSAP) through
interaction with corepressors of the Groucho family. EMBO
J. 19,2292
-2303.
Gehring, W. J. (1966). Uebertragung und aenderung der determinationsqualitäten in antennenscheiben-kulturen von Drosophila melanogaster. J. Embryol. Exp. Morph. 15,371 -377.[Medline]
Gehring, W. J. and Ikeo, K. (1999). Pax-6 mastering eye morphogenesis and eye evolution. Trends Genet. 15,371 -377.[CrossRef][Medline]
Glardon, S., Holland, L. Z., Gehring, W. J. and Holland, N.
D. (1998). Isolation and developmental expression of the
amphioxus Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor
evolution. Development
125,2701
-2710.
Gonzalez-Crespo, S., Abu-Shaar, M., Torres, M., Martinez, A. C., Mann, R. S. and Morata, G. (1998). Antagonism between extradenticle function and Hedgehog signalling in the developing limb. Nature 394,196 -200.[CrossRef][Medline]
Gorfinkiel, N., Morata, G. and Guerrero, I.
(1997). The homeobox gene Distal-less induces ventral appendage
development in Drosophila. Genes Dev.
11,2259
-2271.
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.
Hill, R. E., Favor, J., Hogan, B. L., Ton, C. C., Saunders, G. F., Hanson, I. M., Prosser, J., Jordan, T., Hastie, N. D. and van Heyningen, V. (1991). Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature 354,522 -525.[CrossRef][Medline]
Jarrett, H. W. (2000). Temperature dependence of DNA affinity chromatography of transcription factors. Anal. Biochem. 279,209 -217.[CrossRef][Medline]
Kenyon, L. K., Ranade, S. S., Curtiss, J., Mlodzik, M. and Pignoni, F. (2003). Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head. Dev. Cell 5,403 -414.[Medline]
Kim, J., Sebring, A., Esch, J. J., Kraus, M. E., Vorwerk, K., Magee, J. and Carroll, S. B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382,133 -138.[CrossRef][Medline]
Kronhamn, J., Frei, E., Daube, M., Jiao, R., Shi, Y., Noll, M. and Rasmuson-Lestander, A. (2002). Headless flies produced by mutations in the paralogous Pax6 genes eyeless and twin of eyeless. Development 129,1015 -1026.[Medline]
Kumar, J. P. and Moses, K. (2001). EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification. Cell 104,687 -697.[Medline]
Kurata, S., Go, M. J., Artavanis-Tsakonas, S. and Gehring, W.
J. (2000). Notch signaling and the determination of appendage
identity. Proc. Natl. Acad. Sci. USA
97,2117
-2122.
Lindsley, D. and Zimm, G. (1992). The genom of Drosophila melanogaster. p.1133 . New York, NY: Academic Press.
Methot, N. and Basler, K. (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96,819 -831.[Medline]
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.
Pichaud, F., Treisman, J. and Desplan, C. (2001). Reinventing a common strategy for patterning the eye. Cell 105,9 -12.[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]
Sheng, G., Thouvenot, E., Schmucker, D., Wilson, D. S. and Desplan, C. (1997). Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. Genes Dev. 11,1122 -1131.[Abstract]
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]
Tamkun, J. W., Deuring, R., Scott, M. P., Kissinger, M., Pattatucci, A. M., Kaufman, T. C. and Kennison, J. A. (1992). brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2. Cell 68,561 -572.[Medline]
Wolff, T. and Ready, D. F. (1991). The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave. Development 113,841 -850.[Abstract]
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Zhou, L., Schnitzler, A., Agapite, J., Schwartz, L. M., Steller,
H. and Nambu, J. R. (1997). Cooperative functions of the
reaper and head involution defective genes in the programmed cell death of
Drosophila central nervous system midline cells. Proc. Natl. Acad.
Sci. USA 94,5131
-5136.