Transformation of eye to antenna by misexpression of a single
gene
Hao A. Duong1*,, Cheng Wei Wang2*,, Y. Henry Sun2,#, and Albert J. Courey1,#
1
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA
90095-1569 USA
2
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China
* These authors contributed equally to the paper
#
To whom correspondence should be addressed
Address for correspondence:
Department of Chemistry and Biochemistry
UCLA
607 Charles E. Young Drive, East
Los Angeles, CA 90095-1569
Phone: 310-825-2530
FAX: 310-206-4038
e-mail: courey@chem.ucla.edu
Duong et al.
ABSTRACT
In Drosophila, the eye and antenna originate from the same cluster of cells (the
eye-antennal imaginal disc) and are specified late in development. Illumination of the
mechanisms controlling subdivision of this epithelium into eye and antenna would
enhance our understanding of the mechanisms that restrict stem cell fate. We show here
that Dip3, a transcription factor that is required for eye development, alters fate
determination when misexpressed in the early eye-antennal disc; we have taken
advantage of this observation to gain new insight into the mechanisms controlling the
eye-antennal switch. Early expression of Dip3 yields extra antennae by two distinct
mechanisms: the transformation of the eye disc to an antennal fate, and the splitting of the
antennal field into multiple antennal domains (antennal duplication). Both of these
phenotypes are suppressed by the expression of genes that drive the cell cycle providing
support for tight coupling of cell fate determination and cell cycle control. Antennal
duplication results from under proliferation of the eye disc and concurrent over
proliferation and splitting of the antennal disc. While previous studies have shown that
overgrowth of the antennal disc can lead to antennal duplication, our results show that
overgrowth is not sufficient for antennal duplication, which may require additional
signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from
the combination of activation of antennal selector genes, the repression of eye
determination genes, and perturbation of the cell cycle in the eye disc to reduce disc size.
The finding that this transformation occurs only in the eye disc, and not in other imaginal
discs, suggests a close developmental and therefore evolutionary relationship between
eyes and antennae.
2
Duong et al.
INTRODUCTION
In Drosophila melanogaster, the eye and antenna originate from a cluster of ~23
cells set aside during embryonic development. During the three larval instars, this cell
cluster proliferates continuously and organizes into an epithelial sac termed the eyeantennal imaginal disc. During late larval and pupal development, the anterior lobe of this
epithelium (the antennal disc) gives rise to the antenna, while the posterior lobe (the eye
disc) gives rise to the eye. The eye or antennal identity of these domains is not
determined until mid or late second larval instar with the restricted expression of genes
such as eyeless (ey) in the eye disc and cut (ct) in the antennal disc (Garcia-Bellido and
Merriam, 1969; Kenyon et al., 2003; Kumar and Moses, 2001; Postlethwait and
Schneiderman, 1971).
During the mid to late second larval instar, the core components of the retinal
determination gene network (RDGN), eyeless (ey), eyes absent (eya), sine oculis (so), and
dachshund (dac), are first co-expressed in the eye field (Kenyon et al., 2003; Kumar and
Moses, 2001). Each RDGN gene encodes a conserved transcription factor that is required
for normal retinal development (Bonini et al., 1993; Cheyette et al., 1994; Mardon et al.,
1994; Quiring et al., 1994). Over-expression of each of these genes in other imaginal
discs including the antennal, leg, wing, genital, and haltere discs can induce ectopic eye
development, but only in the presence of the products of all the other RDGN genes.
Furthermore, these genes cross-regulate each other's expression and synergistically
enhance each others ability to induce ectopic eyes (Bonini et al., 1997; Chen et al., 1997;
Halder et al., 1995; Pappu and Mardon, 2002; Pignoni et al., 1997; Shen and Mardon,
1997).
3
Duong et al.
Antennal determination is thought to require homothorax (hth), extradenticle
(exd), and Distal-less (Dll). Loss-of-function mutations in any one of these genes leads
to antenna-to-leg transformation (Casares and Mann, 1998; Cohen et al., 1989; Pai et al.,
1998; Sunkel and Whittle, 1987), while ectopic expression of either hth or Dll produces
ectopic antennae in the head, leg, wing, or genitals, but only in the presence of the
product of the other gene (Casares and Mann, 1998; Dong et al., 2000; Gorfinkiel et al.,
1997). Analysis of the interactions among these genes and their products reveals that Hth
is required for nuclear localization of Exd, and only in the presence of Hth can nuclear
Exd produce ectopic antennae. Dll and Hth, on the other hand, function cooperatively
and in parallel to regulate normal antennal development.
Transdetermination, a process whereby already determined imaginal disc cells
change fate to that of another disc, has been observed in many Drosophila imaginal discs,
giving rise, for example, to eye-to-wing, wing-to-leg, leg-to-antenna, and antenna-towing transformations (Maves and Schubiger, 2003). A hallmark of transdetermination is
the “transdetermination weak point”, a small cell cluster in each imaginal disc that has a
high probability of changing fate in response to fragmentation of the disc through the
weakpoint or misexpression of the Wnt-family signaling protein Wingless (Wg) in the
weakpoint. Cell proliferation has an essential role in this process and cells about to
undergo transdetermination exhibit a distinct cell cycle profile that is not seen in normal
development (Sustar and Schubiger, 2005).
Since the eye and antenna originate from the same cell population and are
specified relatively late in development, it is perhaps not surprising that an antenna can
be regenerated from in vivo culture of an eye disc (Gehring and Schubiger, 1975;
4
Duong et al.
Schubiger and Alpert, 1975). However, neither mechanical disc fragmentation followed
by regeneration nor over-expression of wg, the two treatments that induce other forms of
transdetermination, induce eye-to-antenna transdetermination (Maves and Schubiger,
2003). Furthermore, the conversion of the eye disc to an antennal fate by misexpression
of antennal determination genes such as exd, Dll, or hth has not been previously
demonstrated.
In this study we show that misexpression of Dip3, which encodes a MADF/BESS
domain family transcription factor required for cell type specification during late eye
development (Bhaskar and Courey, 2003; Duong et al, manuscript in preparation),
perturbs the eye-antennal decision. By pursuing this observation, we have gained new
insight into the mechanisms that control this switch. Expression of Dip3 in the early eyeantennal disc leads to both eye-to-antenna transformation, in which the eye disc gives rise
to one or more partial or complete antennae, as well as antennal duplication, in which the
antennal disc gives rise to two or more antennae. Both of the phenotypes may result in
part from perturbation of the cell cycle, since expression of cell cycle genes prevents their
appearance. Antennal duplication occurs when cell cycle perturbation leads to underproliferation of the eye disc and concurrent over-proliferation and splitting of the
antennal disc, while eye-to-antenna transformation results from cell cycle perturbation
along with down-regulation of retinal determination genes and concurrent up-regulation
of antennal determination genes in the eye disc. These findings provide support for the
idea that cell fate determination is intimately coupled to the cell cycle. Furthermore, the
ability of Dip3 to reprogram the eye disc, but not other discs, to an antennal fate implies a
close relationship between these two sense organs.
5
Duong et al.
RESULTS
Dip3 misexpression results in antennal duplication and eye-to-antenna
transformation
In a screen for genes that perturb eye development when misexpressed, we
randomly integrated a UAS/promoter-containing P-element (the EP element (Brand and
Perrimon, 1993; Rorth, 1996)) into the genome. An insertion immediately upstream of
the Dip3 coding region was found to result in the appearance of extra antennae when
combined with the ey-Gal4 driver (Fig. 1). Several lines of evidence (see below) lead us
to conclude that these extra antennae are of two distinct origins: some result from
antennal duplication, while others result from eye-to-antenna transformation. In antennal
duplication (Fig. 1B), the extra antennae arise from over-proliferation and splitting of the
antennal disc into multiple domains, each of which gives rise to an antenna. In this case
the extra antennae are located anterior to the antennal foramen (dashed line), where
antennae are normally found. In eye-to-antenna transformation, the extra antennae arise
from the transformed eye disc and are therefore located posterior to the antennal foramen
(Fig. 1C), where eyes are normally found.
In previous cases where extra antennae were initially thought to arise from eye-toantenna transformation, subsequent analysis showed that they were more likely to be the
result of antennal duplication (Kenyon et al., 2003). Evidence that the extra antennae
observed in ey>Dip3 flies do, in some cases, result from the transformation of eye tissue
to an antennal fate comes from our observation of partial eye-to-antenna transformations.
In mild to moderate partial transformations, the eye consists exclusively of ommatidial
units, but bulges out or forms a rod-shaped structure (Fig. 1D, E), suggesting that
6
Duong et al.
although eye tissue identity is intact, the eye is assuming a shape similar to that of an
antenna. In strong partial transformations, the eye domain contains the proximal portion
of an antenna tipped with ommatidia (Fig. 1F). Finally, in complete transformations,
ommatidia are absent and are replaced with a complete antenna posterior to the antennal
foramen (Fig. 1C).
The phenotypes described above are only observed when flies are raised at 18˚,
resulting in low levels of Dip3 expression. At higher temperatures (25˚-29˚) we observe
complete lethality due to deletion of most or all of the head. As will be discussed below,
this is likely due to inhibition of cell proliferation by misexpressed Dip3.
To confirm that the extra antennae are due to Dip3 misexpression, we utilized a
completely independent insertion of the EP element generated by the Drosophila genome
project that maps 21 base pairs upstream of the Dip3 transcriptional start site. This insert
was also found to generate extra antennae when combined with the ey-Gal4 driver (data
not shown). In addition, we created germ-line transformants of a UAS-Dip3 construct and
found that driving expression of this construct with the ey-Gal4 driver also resulted in
extra antennae. This phenotype apparently requires both the MADF and BESS domains
of Dip3 since deletion constructs lacking either domain did not yield extra antennae (data
not shown).
The use of other Gal4 drivers (e.g., dpp-Gal4, C765-Gal4, GMR-Gal4) to direct
Dip3 expression in other tissues or at other times during eye development does not result
in ectopic antennae (data not shown). Thus Dip3 appears to have a specific ability to
produce extra antennae in the eye-antennal disc. This ability is highly sensitive to Dip3
expression level and restricted to a narrow developmental time window. Attempts to
7
Duong et al.
separate the two phenotypes using the temporal and regional gene expression targeting
system (TARGET) (McGuire et al., 2003)were not fruitful due to the narrow
developmental time window separating the two phenotypes.
Molecular evidence for antennal duplication
An examination of the dac expression pattern in the ey>Dip3 eye-antennal discs
reveals two different patterns, one most likely corresponding to antennal duplication and
the other to eye-to-antenna transformation. In wild-type third instar larvae, dac is
expressed in a broad stripe around the morphogenetic furrow in the eye disc, and in a
single circular domain in the antennal disc that constitutes the future A3 antennal
segment (Dong et al., 2002) (Fig. 2A, A’). However, a large proportion of Dip3
misexpressing discs display multiple circular dac expression domains and no stripe.
Some of these discs consist of a single large sac of epithelium, and show expression of
Dll, the product of which normally marks the antennal primordium (Cohen et al., 1989;
Dong et al., 2000; Gorfinkiel et al., 1997), in domains overlapping the extra circular dac
expression domains (Fig. 2E,E’). Similar discs have previously been observed in
response to inhibition of Notch signaling, interference with cell cycle exit, activation of
GTPases (Rac1/Cdc42), or activation of EGFR (Go, 2005; Kenyon et al., 2003; Kumar
and Moses, 2001; Pimentel and Venkatesh, 2005). In agreement with the conclusion from
a previous analysis (Kenyon et al., 2003), we suggest that these discs represent antennal
duplication. In other words, the multiple circular dac/Dll expression domains in these
discs all derive from the antennal region of the eye-antennal imaginal disc.
8
Duong et al.
In support of the idea that supernumerary circular dac/Dll expression domains in
these discs result from splitting of the antennal domain and not from transformation of
the eye disc, these discs often contain a small posterior lobe (Fig. 2F, arrow) in addition
to the large anterior lobe. When such discs are stained with antibodies to Elav (Fig. 2F’),
which marks differentiated photoreceptors, we observe Elav in the small posterior lobe.
Thus, the posterior lobe represents a reduced eye disc, while the anterior region
containing the multiple circular dac/Dll expression domains represents an enlarged and
split antennal field. This conclusion is consistent with the observation that the antennal
duplication heads often contain reduced eyes (Fig. 1B). Thus, antennal duplication
appears to result, at least in part, from reduction of the eye field, leading to compensatory
over-proliferation and splitting of the antennal field.
To determine if overgrowth of the antennal disc is sufficient for antennal
duplication, we took advantage of the ability of activated Notch (Nact) to stimulate cell
proliferation. While misexpression of Nact in the antennal disc (using the antennal discspecific OK384-Gal4 driver) induced 2 to 3-fold overgrowth of the antennal disc, we
observed neither duplication of the Dll expression domain in larval discs nor extra adult
antennae (compare Fig. 2G to 2C). This suggests that generation of duplicated antennae
requires not only overgrowth of the antennal disc, but also the reduction of the eye disc.
Thus, active communication between the eye and antennal discs may contribute to fate
determination in the antennal disc.
The formation of the duplicated antenna suggests the existence of an extra
proximal-distal (PD) axis in the duplicated antennal disc. Previous studies have shown
that the formation of the PD axis requires the intersection of domains with high levels of
9
Duong et al.
wg and dpp expression (Brook and Cohen, 1996)(Lecuit and Cohen, 1997)(Jiang and
Struhl, 1996)(Penton and Hoffmann, 1996)(Diaz-Benjumea et al., 1994)(Campbell et al.,
1993). Accordingly, the antennal duplication discs exhibit ectopic wg and dpp expression
domains that intersect at the center of the duplicated Dll expression domain (compare
Fig. 2H to 2D).
Molecular evidence for eye-to-antenna transformation
In contrast to the antennal duplication discs described above, in which the extra
circular dac expression domains are located within the anterior antennal disc, some of the
ey>Dip3 eye-antennal discs contain multiple circular dac expression domains distributed
between the anterior antennal field and the posterior eye field. In these discs, Dll is coexpressed with dac only in the antennal field and not in the eye field (Fig. 3A). We
suggest that these discs represent eye-to-antenna transformations. This interpretation is
supported by the following lines of evidence. First, ct, which encodes a marker of the 2nd
instar antennal disc that can suppress ey expression and transfom eye to partial antenna
(please see the subsequent section for detail), is ectopically expressed in the eye field in
these discs (compare Fig. 3C and 3D). This observation suggested that the eye disc has
been re-programmed to an antennal identity. Furthermore, while Dll is not expressed in
the eye field in these discs, the expression pattern of the antennal determination gene hth
(Casares and Mann, 1998; Pai et al., 1998) in the eye field is altered to resemble its
expression pattern in the antennal field (compare Fig. 3E and 3F). Lastly, in discs likely
to represent partial eye-to-antenna transformations, elav is expressed in the eye field.
However, the expression domain is localized to the center rather than the posterior part of
10
Duong et al.
the field and is surrounded by a circular domain of dac expression (Fig. 3B). This
expression pattern implies that the outer part of the eye field has been transformed to an
antennal identity while the center of the field still retains eye tissue identity.
While previous studies have demonstrated a critical role for Dll in the normal
antennal development program (Casares and Mann, 1998; Cohen et al., 1989; Dong et al.,
2000), the absence of Dll expression in eye discs transformed by Dip3 to an antennal fate
(Figs. 3B’ and F) suggests the existence of a Dll-independent mechanism of antennal
development in the eye disc. Support for this interpretation is provided by the observation
that removal of one copy of Dll does not alter the ey>Dip3 phenotype, whereas removal
of one copy of hth almost completely suppresses the extra antenna phenotype (data not
shown).
Thus, antennal duplication and eye-to-antenna transformation discs display
distinct molecular signatures suggesting that these phenotypes are governed by distinct
mechanisms.
Non-cell-autonomous inhibition of retinal determination genes and activation of
antennal selector genes by Dip3
Transformation of eyes to antennae by Dip3 is likely to require repression of
members of the RDGN such as ey and dac as well as activation of antenna-specific genes
such as ct and hth. To explore this possibility, we examined third instar eye discs
containing clones of Dip3-over-expressing cells. In the wild-type fly, ey is expressed in
the second and third instar eye disc prior to being shut off starting from the posterior of
11
Duong et al.
the disc following passage of the morphogenetic furrow. As mentioned previously, dac is
expressed in a broad stripe straddling the morphogenetic furrow.
Over-expression of Dip3 in clones was found to result in down-regulation of ey
and dac expression. Surprisingly, this effect is non-cell-autonomous as the zone of
RDGN down-regulation extends beyond the borders of the Dip3-over-expressing clone.
The ability of Dip3-over-expressing clones to repress RDGN expression depends on the
location of the clone. In particular, only clones located near the Dorsal-Ventral (DV)
midline (Fig. 4A-4A’’, arrow) lead to decreased RDGN expression. Thus, Dip3 may cooperate with one or more DV midline-localized factors to inhibit RDGN expression.
To examine this position-dependence further, we selectively over-expressed Dip3
along the D-V midline using the eyg-Gal4 driver. In eyg>Dip3 flies, photoreceptor
development is inhibited along the D-V midline, with the strongest inhibition at the
anterior of the eye (compare Fig. 4C to Fig. 4B). Consistent with the idea that retinal cells
are being reprogrammed to antenna, we observe non-cell-autonomous activation of
antennal selector genes such as hth (Fig. 4D-E’) and ct (Fig. 4G, G’) and repression of
retinal determination genes such as ey (Fig. 4F-G’). Therefore, Dip3 appears to elicit one
or more signals that both inhibit RDGN expression and promote antennal selector gene
expression. In addition, eyg>Dip3 also led to the the non-cell-autonomous induction of
wg expression (Fig. 4H-I’). The induction of wg at a position near the dpp-expressing
morphogenetic furrow may create an intersection of high Wg and Dpp signaling, thereby
inducing formation of a proximodistal axis.
ct over-expression can inhibit ey expression and transform eyes to partial antennae.
12
Duong et al.
While ey is expressed ubiquitously throughout the first instar eye-antennal disc it
is shut off in the antennal primordium in early to mid second instar larvae at
approximately the same time that ct is switched on in this region (Fig. 5A-B; (Kenyon et
al., 2003; Kumar and Moses, 2001). To determine if Ct is sufficient for ey down
regulation in the eye disc, we generated ct over-expressing clones in the eye. Overexpression of ct inhibited ey expression cell-autonomously, suggesting that the reduced
expression of ey in the 2nd instar antennal disc results from up-regulation of ct (Fig. 5CC’).
If the ability of Dip3 to transform eyes to antennae results from its ability to
induce antennal markers such as ct and prevent the expression of retinal determination
genes such as ey, then ct over-expression might be sufficient for eye-to-antenna
transformation. Indeed, ey-Gal4-driven expression of ct leads to partial eye-to-antenna
transformation resembling the partial transformations observed in ey>Dip3 flies (Fig.
5D).
The role of eye disc proliferation in antennal duplication and eye-to-antenna
transformation
The reduced size of the eye field that often results from Dip3 misexpression
suggests that Dip3 either inhibits cell proliferation or induces cell death. To distinguish
these two possibilities, we looked at DNA synthesis and apoptosis in ey>Dip3 discs by
observing the levels of BrdU labeling and activated Drice, respectively. The reduced
discs show a significant reduction in BrdU labeling (Fig. 6A), but no induction of
activated Drice (data not shown). Co-expression with the anti-apoptotic protein p35 did
13
Duong et al.
not rescue the eye defect (data not shown), further confirming the absence of a role for
apoptosis in producing the ey>Dip3 phenotypes.
To explore the role of the cell proliferation defect in generating Dip3
misexpression phenotypes, we co-expressed Dip3 with activated Notch (Nact), which is
known to stimulate cell proliferation (Reynolds-Kenneally and Mlodzik, 2005; Tsai and
Sun, 2004). When Dip3 was expressed alone, 17% of the resulting flies had normal eyes
and antennae, 5% had normal antennae but small eyes, 40% had extra antennae, and 38%
were headless. In flies coexpressing Nact and Dip3, the average severity of the defect was
significantly reduced – 54% had normal eyes, 19% had normal antennae but small eyes,
25% had extra antennae, and 2% were headless. In eye disc development, N promotes
global growth through eyg and upd (Chao et al., 2004). Accordingly, coexpression of
eyg or upd with Dip3 also greatly reduces the severity of the Dip3 misexpression
phenotype (Fig. 6C). Finally, coexpression of Dip3 with CycE, which drives cell
proliferation, also results in suppression of the Dip3 misexpression phenotype (Fig. 6B,
C). In contrast, coexpression of twin of eyeless does not leads to rescue, showing that
rescue is not the consequence of reduced Dip3 expression, which could theoretically
result from the presence of an extra UAS. In conclusion, these findings indicate that both
antennal duplication and eye-to-antennae transformation are due, in part, to the ability of
Dip3 to interfere with cell proliferation in the eye disc.
14
Duong et al.
DISCUSSION
We have discovered that ectopic expression of a single gene in the early eyeantennal disc can lead to both antennal duplication and eye-to-antennal transformation.
Both of these phenotypes appear to result, in part, from inhibition of cell cycle
progression since suppression of the growth defect in these discs by coexpression of
genes that drive cell-cycle progression prevents both phenotypes. Although previous
studies also suggested a link between growth and developmental fate in the eye-antennal
disc, the current study adds a number of new insights: (1) Previous studies showed that
inhibiting growth of the eye disc leads to overproliferation and duplication of the
antennal disc. The current study supports this idea, but also shows that antennal disc
overproliferation is not sufficient for duplication, which may also involve communication
between the eye and antennal discs. (2) This is first study to suggest a connection
between the cell-cycle progression and the eye-antennal decision. In addition, the
observation that this transformation occurs only in the eye disc suggests a close
evolutionary relationship between eye and antenna.
We have recently created loss-of-function alleles of Dip3. While these
demonstrate a role for Dip3 in late eye development, they do not show a role in early fate
determination in the eye-antennal field. This may be due to redundancy as there are 14
other homologous MADF/BESS domain transcription factors encoded in the Drosophila
genome.
Dip3 induces antennal duplication and eye-to-antenna transformation
15
Duong et al.
Antagonism between the N and EGFR signaling pathways influences
developmental fate in the eye-antennal disc leading to a loss of eye tissue and the
appearance of extra antennae (Kumar and Moses, 2001). Although this phenotype was
originally suspected to represent eye-to-antennal transformation, subsequent analysis
suggests that it most likely represents antennal duplication. Specifically, the absence of
the N signal leads to a failure in eye disc proliferation resulting in compensatory overproliferation of the antennal disc and its subdivision into multiple antennae (Kenyon et
al., 2003). Consistent with the idea that the extra antennae result from under-proliferation
of the eye field, it was found that the phenotype was largely suppressed by overexpression of CycE to drive the cell cycle.
In this study, we also find that inhibition of eye disc growth leads to antennal
duplication. But in addition, we show that the same treatment that leads to antennal
duplication can also direct the transformation of eyes to antennae. These two phenotypes
are anatomically distinct. This anatomical distinction is evident in adults: antennae
resulting from antennal duplication are found anterior to the antennal foramen, while the
antennae resulting from eye-to-antenna transformation are found posterior to the antennal
foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication
discs exhibit multiple circular dac expression domains within a single sac of epithelium
(the antennal disc), while eye-to-antennal transformation discs exhibit two or more
circular dac expression domains spread over both the eye and antennal discs. The two
types of discs display distinct molecular signatures as well: the antennal duplication discs
exhibit duplicated Dll expression domains, while the eye discs undergoing transformation
to antennae lack Dll expression.
16
Duong et al.
Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal
transformation is provided by the observation of eyes that are only partially transformed
to antennae. In some cases, we observe proximal antennal segments tipped with eye
tissue. In accord with this phenotype, some third instar larval eye discs display a central
domain of Elav-positive differentiating photoreceptors surrounded by a circular dac
domain.
Antennal disc overgrowth is required but not sufficient for antennal duplication
Our data show that discs undergoing antennal duplication as a result of Dip3
expression are comprised of a severely diminished eye region and an enlarged antennal
region. As shown by BrdU labeling experiments, these antennal duplication discs most
likely result from suppression by Dip3 of cell proliferation in the eye field leading to
overproliferation of the antennal disc. This conclusion is supported by the ability of
factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression
defect.
Many experimental manipulations that reduce the size of the eye disc (e.g.,
surgical excision, induction of cell death, or suppression of cell proliferation) lead to
enlargement and duplication of the antennal primordium (Arking, 1975; Gehring and
Nothiger, 1973; Martin et al., 1977; Russell, 1974; Schubiger and Alpert, 1975). How
might reduction of the eye field lead to antennal field over-growth? One possibility is that
the eye field produces a growth inhibitory signal. Alternatively, the eye field and the
antennal field may compete with each other for limited nutrients or growth factors. In
support of this latter possibility, recent studies of the role of dMyc in wing development
17
Duong et al.
have demonstrated growth competition between groups of imaginal disc cells (de la Cova
et al., 2004; Moreno and Basler, 2004).
While our results imply that antennal disc overgrowth is required for antennal
duplication, we do not believe that overgrowth is sufficient for duplication. This
conclusion derives from experiments in which we used an antennal disc specific driver to
direct over-expression of CycE or Nact (Fig. 2F and data not shown). This resulted in
antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal
duplication was not observed. Thus, in addition to antennal overgrowth, antennal
duplication also appears to require reduction or elimination of the eye disc. Regulatory
signals from the eye disc may act to prevent antennal duplication.
Multiple requirements for eye-to-antenna transformation
The eye and antenna discs differ in several aspects: (1) Specific expression of the
organ-specification genes. The eye disc expresses the RDGN genes, while the antennal
disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern
from that seen in the antennal disc. In the second instar eye disc, hth is expressed
throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell
proliferation (Bessa et al., 2002). The hth expression domain is later retracts to only the
anterior-most region of the eye disc (Caares and Mann, 1998; Pai et al., 1998; Bessa et al.,
2002). This pattern is different from the circular expression pattern observed in the
antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg
is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is
required to specify the proximodistal axis in the leg (Diaz-Benjumea et al., 1996). In the
18
Duong et al.
early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg
and dpp expression domain are separated, so that there is probably no intersection
between high levels of Wg and Dpp signaling (see review by Dominguez and Casares,
2005). (3) Whereas the partial overlap of Dll and hth expression domains in the antennal
disc is important for the proximodistal axis specification (Dong et al., 2000; 2001; 2002),
there is no Dll expression in the eye disc. Dll expression in the center of the antennal and
leg disc is induced by the combination of high levels of Dpp and Wg signaling (DiazBenjumea et al., 1994). Because there is no overlap of Dpp and Wg signaling in the eye
disc, Dll is not induced.
Therefore, efficient transformation of the eye disc into an antennal disc requires at
least three things: (1) repression of the eye fate pathway; (2) Activation the antennal fate
pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in
the antenna and leg disc that induces proximodistal axis formation. Any one of these
three conditions by itself is not sufficient: (1) Loss of the RDGN genes leads only to the
loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the
ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra
antenna are formed (Kurata et al., 2000; Punzo et al., 2004). The induction of Dll is not
ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to
the expression of Dll and the transformation to the antennal fate. (2) Simply expressing
the antennal determining genes Dll or hth in the eye disc does not change the eye fate into
antennal fate. We found that uniform expression of Dll in the eye disc (ey>Dll) resulted
in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll
caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll
19
Duong et al.
and C68a>Dll induced extra antenna but not within the eye field (Gorfinkiel et a., 1997).
Therefore although Dll and hth are important determinant for antennal identity, it is their
specific spatial expression patterns that determine antennal development. (3) Creating the
intersection of Wg and Dpp signaling does not change the eye into antenna. Such
manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc
(Maves and Schubiger, 1998). Therefore, the specific genes induced by Dpp and Wg
signal may depend on disc-specific factors. In the eye disc, turning on Wg signaling in
the Dpp expressing morphogenetic furrow only blocked furrow progression(Treisman
and Rubin, 1995)..
In this study, we found that the ectopic expression of a single gene, Dip3, can
cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1)
Overexpression of Dip3 repressed (non-autonomously) ey and dac. The repression of ey
may be due to the induction of ct. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell
proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and
Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P
border and induced ectopic dpp/wg expression.
Interference with cell cycle progression appears to be a common link between the
two phenotypes described in this study. In the case of antennal duplication, interference
with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for
duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows
the required intersection between Dpp and Wg signaling.
20
Duong et al.
Possible close evolutionary relationship between eye and antenna
The observation that Dip3 misexpression can transform the eye field, but not
other tissues, to an antennal fate suggests a close evolutionary relationship between the
eye and the antenna. Previous studies have emphasized the homology between antennae
and legs (Casares and Mann, 1998; Cohen et al., 1989; Pai et al., 1998). The findings
presented here that misexpression of a single transcription factor, namely Dip3, can
transform eyes to antennae provides support for the notion that the eye and antenna may
also, in some sense, be homologous to one another. Previous evidence in support of this
idea comes from the observation that similar spatial arrangements of Wg and Dpp
signaling along with a temporal cue provided by the ecdysone signal are required for the
formation of the eye and the mechanosensory auditory organ (Johnston’s organ)
associated with the antenna (Niwa et al., 2004). Small mechanosensory sensilla, such as
Johnston’s organ and the chordotonal organs (stretch receptors) are thought to represent
the earliest evolving sense organs. Perhaps the eye resulted from a duplication and
specialization of such a sensillum.
21
Duong et al.
EXPERIMENTAL PROCEDURES
Misexpression screening
704 EP lines (Rorth, 1996) (generated and generously provided by Dr. Cheng-ting
Chien, Institute of Molecular Biology, Academia Sinica, Taiwan) were crossed to the eyGal4 driver line (Quiring et al., 1994). In the F1 progeny from such crosses, one line
(C00-008) with an EP insertion 198 bp upstream of the Dip3 translation start site,
displayed the antenna duplication, eye-to-antenna transformation, and eye reduction
phenotypes.
Mitotic Clones
Positively labeled flip-out clones expressing Dip3 were generated by crossing EPDip3 flies to hs-FLP22; Act5C>y+>GAL4 UAS-GFPS65T (Ito et al., 1997). Heat-shock
induction of hs-FLP22 was at 37°C for 30 minutes at 24-48 hours after egg laying.
Immunohistochemistry
Antibody staining was performed as described previously (Wolff, 2000). Rabbit
anti-Dll antibody (Dong et al., 2000) was provided by G. Panganiban. Guinea pig antiHth antibody (Casares and Mann, 1998) was provided by R.S. Mann. Rat anti-BrdU
antibody was provided by U. Banerjee. Rabbit anti-Ey antibody (Halder et al., 1998) was
provided by U. Walldorf. Rabbit anti-activated Drice antibody (Yoo et al., 2002) was
provided by B.A. Hay. Rat anti-Elav, mouse anti-Eya, mouse anti-Dac and mouse antiCut antibodies were provided by the Developmental Studies Hybridoma Bank (DSHB) .
ACKNOWLEDGEMENTS
22
Duong et al.
We thank Gerold Schubiger, Raghavendra Nagaraj, and Girish Ratnaparkhi for
valuable discussion; Preeta Guptan, Sudip Mandal and Laurent Bentolila for technical
assistance; and Justin P. Kumar, Francesca Pignoni, Richard S. Mann, Grace Panganiban,
Ulrich Walldorf, Bruce A. Hay and the Developmental Studies Hybridoma Bank for
reagents. Confocal images were obtained in the UCLA CNSI Advanced Light
Microscopy/Spectroscopy Shared Facility and the Confocal Facility in IMB, Academia
Sinica. This work was supported by an NIH grant (GM44522) to A.J.C., and by an NSC
grant (NSC 93-2312-B-001-016) to Y.H.S.
23
Duong et al.
REFERENCES
Arking, R. (1975). Temperature-sensitive cell-lethal mutants of drosophila: isolation and
characterization. Genetics, 519-37.
Bonini, N. M., Bui, Q. T., Gray-Board, G. L. and Warrick, J. M. (1997). The
Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved
between flies and vertebrates. Development 124, 4819-26.
Bonini, N. M., Leiserson, W. M. and Benzer, S. (1993). The eyes absent gene: genetic
control of cell survival and differentiation in the developing Drosophila eye. Cell 72,
379-95.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering
cell fates and generating dominant phenotypes. Development 118, 401-15.
Brook, W. J. and Cohen, S. M. (1996). Antagonistic interactions between wingless and
decapentaplegic responsible for dorsal-ventral pattern in the Drosophila Leg. Science
273, 1373-7.
Campbell, G., Weaver, T. and Tomlinson, A. (1993). Axis specification in the
developing Drosophila appendage: the role of wingless, decapentaplegic, and the
homeobox gene aristaless. Cell 74, 1113-23.
Casares, F. and Mann, R. S. (1998). Control of antennal versus leg development in
Drosophila. Nature 392, 723-6.
Chao, J. L., Tsai, Y. C., Chiu, S. J. and Sun, Y. H. (2004). Localized Notch signal acts
through eyg and upd to promote global growth in Drosophila eye. Development 131,
3839-47.
Chen, R., Amoui, M., Zhang, Z. and Mardon, G. (1997). Dachshund and eyes absent
proteins form a complex and function synergistically to induce ectopic eye development
in Drosophila. Cell 91, 893-903.
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, 977-96.
Cohen, S. M., Bronner, G., Kuttner, F., Jurgens, G. and Jackle, H. (1989). Distal-less
encodes a homoeodomain protein required for limb development in Drosophila. Nature
338, 432-4.
de la Cova, C., Abril, M., Bellosta, P., Gallant, P. and Johnston, L. A. (2004).
Drosophila myc regulates organ size by inducing cell competition. Cell 117, 107-16.
Diaz-Benjumea, F. J., Cohen, B. and Cohen, S. M. (1994). Cell interaction between
compartments establishes the proximal-distal axis of Drosophila legs. Nature 372, 175-9.
Dong, P. D., Chu, J. and Panganiban, G. (2000). Coexpression of the homeobox genes
Distal-less and homothorax determines Drosophila antennal identity. Development 127,
209-16.
Dong, P. D., Dicks, J. S. and Panganiban, G. (2002). Distal-less and homothorax
regulate multiple targets to pattern the Drosophila antenna. Development 129, 1967-74.
Garcia-Bellido, A. and Merriam, J. R. (1969). Cell lineage of the imaginal discs in
Drosophila gynandromorphs. J Exp Zool 170, 61-75.
Gehring, W. J. and Nothiger, R. (1973). The imaginal discs of Drosophila. In
Developmental Systems: Insects, (ed. S. Counce and C. Waddingtion), pp. 211-290.
London: Academic Press.
24
Duong et al.
Gehring, W. J. and Schubiger, G. (1975). Expression of homeotic mutations in
duplicated and regenerated antennae of Drosophila melanogaster. J Embryol Exp
Morphol 33, 459-69.
Go, M. J. (2005). Activation of Rac1 or Cdc42 during early morphogenesis of eye discs
induces ectopic antennae in Drosophila. Dev Growth Differ 47, 225-31.
Gorfinkiel, N., Morata, G. and Guerrero, I. (1997). The homeobox gene Distal-less
induces ventral appendage development in Drosophila. Genes Dev 11, 2259-71.
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, 2181-91.
Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by
targeted expression of the eyeless gene in Drosophila. Science 267, 1788-92.
Jiang, J. and Struhl, G. (1996). Complementary and mutually exclusive activities of
decapentaplegic and wingless organize axial patterning during Drosophila leg
development. Cell 86, 401-9.
Kenyon, K. L., 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, 403-14.
Kumar, J. P. and Moses, K. (2001). EGF receptor and Notch signaling act upstream of
Eyeless/Pax6 to control eye specification. Cell 104, 687-97.
Lecuit, T. and Cohen, S. M. (1997). Proximal-distal axis formation in the Drosophila
leg. Nature 388, 139-45.
Mardon, G., Solomon, N. M. and Rubin, G. M. (1994). dachshund encodes a nuclear
protein required for normal eye and leg development in Drosophila. Development 120,
3473-86.
Martin, P., Martin, A. and Shearn, A. (1977). Studies of l(3)c43hs1 a polyphasic,
temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc
defects. Dev Biol 55, 213-32.
Maves, L. and Schubiger, G. (2003). Transdetermination in Drosophila imaginal discs:
a model for understanding pluripotency and selector gene maintenance. Curr Opin Genet
Dev 13, 472-9.
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and Davis, R. L. (2003).
Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 1765-8.
Moreno, E. and Basler, K. (2004). dMyc transforms cells into super-competitors. Cell
117, 117-29.
Niwa, N., Hiromi, Y. and Okabe, M. (2004). A conserved developmental program for
sensory organ formation in Drosophila melanogaster. Nat Genet 36, 293-7.
Pai, C. Y., Kuo, T. S., Jaw, T. J., Kurant, E., Chen, C. T., Bessarab, D. A., Salzberg,
A. and Sun, Y. H. (1998). The Homothorax homeoprotein activates the nuclear
localization of another homeoprotein, extradenticle, and suppresses eye development in
Drosophila. Genes Dev 12, 435-46.
Pappu, K. and Mardon, G. (2002). Retinal Specification and Determination in
Drosophila. In Results and Problems in Cell Differentiation, vol. 37 (ed. K. Moses), pp.
5-20. Berlin: Springer.
Penton, A. and Hoffmann, F. M. (1996). Decapentaplegic restricts the domain of
wingless during Drosophila limb patterning. Nature 382, 162-4.
25
Duong et al.
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. (1997).
The eye-specification proteins So and Eya form a complex and regulate multiple steps in
Drosophila eye development. Cell 91, 881-91.
Pimentel, A. C. and Venkatesh, T. R. (2005). rap gene encodes Fizzy-related protein
(Fzr) and regulates cell proliferation and pattern formation in the developing Drosophila
eye-antennal disc. Dev Biol.
Postlethwait, J. H. and Schneiderman, H. A. (1971). A clonal analysis of development
in Drosophila melanogaster: morphogenesis, determination, and growth in the wild-type
antenna. Dev Biol 24, 477-519.
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, 785-9.
Reynolds-Kenneally, J. and Mlodzik, M. (2005). Notch signaling controls proliferation
through cell-autonomous and non-autonomous mechanisms in the Drosophila eye. Dev
Biol 285, 38-48.
Rorth, P. (1996). A modular misexpression screen in Drosophila detecting tissuespecific phenotypes. Proc Natl Acad Sci U S A 93, 12418-22.
Russell, M. A. (1974). Pattern formation in the imaginal discs of a temperature-sensitive
cell-lethal mutant of Drosophila melanogaster. Dev Biol 40, 24-39.
Schubiger, G. and Alpert, G. D. (1975). Regeneration and duplication in a temperature
sensitive homeotic mutant of Drosophila melanogaster. Dev Biol 42, 292-304.
Shen, W. and Mardon, G. (1997). Ectopic eye development in Drosophila induced by
directed dachshund expression. Development 124, 45-52.
Sunkel, C. and Whittle, J. (1987). Brista: A gene involved in the specification and
differentiation of distal cephalic and thoracic structures in Drosophila melanogaster.
Rouxs Arch Dev Biol, 124-132.
Sustar, A. and Schubiger, G. (2005). A transient cell cycle shift in Drosophila imaginal
disc cells precedes multipotency. Cell 120, 383-93.
Treisman, J. E. and Rubin, G. M. (1995). wingless inhibits morphogenetic furrow
movement in the Drosophila eye disc. Development 121, 3519-27.
Tsai, Y. C. and Sun, Y. H. (2004). Long-range effect of upd, a ligand for Jak/STAT
pathway, on cell cycle in Drosophila eye development. Genesis 39, 141-53.
Wolff, T. (2000). Histological Techniques for the Drosophila Eye. In Drosophila
Protocols, (ed. W. A. Sullivan, M.; Hawley, R.S.). Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Yoo, S. J., Huh, J. R., Muro, I., Yu, H., Wang, L., Wang, S. L., Feldman, R. M.,
Clem, R. J., Muller, H. A. and Hay, B. A. (2002). Hid, Rpr and Grim negatively
regulate DIAP1 levels through distinct mechanisms. Nat Cell Biol 4, 416-24.
26
Duong et al.
FIGURE LEGENDS
Figure 1: Dip3 misexpression produces antennal duplication as well as eye-to-antenna
transformation phenotypes.
Scanning electron micrographs of a wildtype fly head (A) or of heads from flies in which
Dip3 was misexpressed using the ey-Gal4 driver (B-F). (B) An antenna duplication fly
head. This fly exhibits two reduced eyes (e), and three antennae (a), all located anterior to
the antennal foreman (dashed line). (C) A complete eye-to-antenna transformation fly
head. This fly has one eye (e), two normal antennae (a), and one extra antenna (arrow)
located posterior to the antennal foreman where the missing eye should be. (D-F) Partial
eye-to-antenna transformation fly heads of increasing severity. In mild or moderate
partial transformations, the eye consists exclusively of ommatidia but bulges out (arrow
in D) or forms a rode shape structure (arrow in E). In a nearly complete transformation,
the eye is replaced with a structure consisting of the proximal portion of an antenna,
tipped with ommatidia (arrow in F).
Figure 2: Antennal duplication results from over-proliferation and duplication of the
antennal disc at the expense of the eye disc.
Third instar larval eye-antennal imaginal discs were stained with indicated antibodies.
(A, A’, B, B’) Wild-type discs. At this stage, the eye-antennal disc consists of two
distinct epithelial lobes, the anterior antennal disc and the posterior eye disc. (A, A’)
Double staining with antibodies against Dac and Dll shows that dac is expressed in a
circular domain in the antennal disc and in a stripe along the morphogenetic furrow in the
eye disc, while Dll is only expressed in the antennal disc. (B, B’) Double staining with
27
Duong et al.
Dac and Elav antibodies shows that Elav is expressed only posterior to the
morphogenetic furrow of the eye disc. (E, E’, F, F’) Discs from larvae in which Dip3 was
misexpressed using the ey-Gal4 driver. (E, E’) Double staining of an antennal
duplication disc with Dac and Dll antibodies shows that dac and Dll are expressed in two
circular domains contained within a single epithelial lobe. (F, F’) Double staining of an
antennal duplication disc with Dac and Elav antibodies shows the presence of a reduced
eye domain. This disc contains a small posterior lobe (arrow), in addition to a large
anterior duplicated antennal disc. Expression of Elav in the small lobe indicates that it is
a reduced eye domain. (G) Disc from a larva in which Notch was over-expressed using
the antennal disc-specific OK384-Gal4 driver. This yields two to threefold overgrowth of
the antennal disc (compare to wild-type disc (C) shown at the same magnification), but
no apparent antennal duplication as revealed by staining with Dll antibody. (D,G) Triple
staining for WG, Dll and dpp-lacZ showed the formation of an extra proximal-distal axis,
where ectopic WG and Dpp expression domains intersect, in antennal duplication discs
(compare Fig. 2H to 2D).
Figure 3: Eye-to-Antenna Transformation discs.
Eye-antennal imaginal discs were stained with antibodies against the indicated markers.
(A, B, D, F) Discs from larvae in which Dip3 was misexpressed using the ey-Gal4 driver.
(C, E) Wild-type discs. (A) An eye-to-antennal transformation third instar disc double
stained with Dac and Dll antibodies. The anterior antennal disc contains a normal single
circular dac/Dll expressing domain, while the transformed posterior eye disc has split
into two antennal domains as revealed by the two circular dac expression domains. Dll is
28
Duong et al.
not expressed in the transformed eye disc (compare to Figure 2A, A’). (B) A third instar
larval eye-antennal disc stained with antibodies against Dac and Elav. In this partial eyeto-antenna transformation disc, the Elav expression domain in the eye disc is surrounded
by a dac expression domain, implying that the outer region of the eye disc has assumed
an antennal identity (compare to Figure 2B, B’). (C, D) Discs from second instar larval
eye-antennal discs stained with anti-Ct antibody. In the wildtype disc (C), Ct is only
expressed in the antennal field , while in an ey>Dip3 disc (D), Ct is ectopically expressed
in the eye field suggesting an eye-to-antenna transformation. (E, F) Third instar larval
eye-antennal discs stained with Dll and Hth antibodies. In the ey>Dip3 disc (F), the hth
expression pattern in the eye domain is transformed to an antenna-like pattern, while the
Dll expression pattern is unchanged relative to the wild-type disc (E).
Figure 4: Dip3 inhibits expression of the retinal determination gene network and activate
antennal selector genes.
(A-A’’) Third instar larval eye disc containing two Dip3 over-expressing clones marked
with GFP. The disc was double stained with antibodies against Ey and Dac. ey and dac
expression are inhibited in and around the clone positioned along the D-V midline
(arrow), but not in the clone located outside of that region (arrowhead). (B-C) Light
images of adult eye showing the inhibition of photoreceptors development along the D-V
midline (compare B to C) when Dip3 was over-expressed in that region by the eyg-Gal4
driver. This inhibition is strongest at the anterior edge of the eye. (D-I) third instar larval
eye discs staining for the expression of hth (D,E), ct and ey (F,G) or wg (H,I). hth, ct and
wg were ectopically expressed while ey was inhibited when Dip3 was over-expressed in
29
Duong et al.
that region (compare E to D, G to F or I to H). E’, G’ and I’ are high magnified section of
E, G and I, respectively.
Figure 5: Ct over-expression leads to partial eye to antenna transformation.
(A) Wildtype first instar larval eye-antennal disc stained with Ey antibody showing that
Ey is expressed throughout the disc. (B) Second instar larval eye-antennal discs double
stained with Ct and Ey antibodies showing that, by this stage, Ct is coming on while Ey is
being shut off in the antennal disc. (C, C’) A second instar disc containing Ct overexpressing clones marked with GFP. Staining of the disc with Ey antibodies shows Ey
downregulation in the Ct-expressing clones suggesting that Ct can block Ey expression.
(D) Lateral view of an adult fly head in which UAS-ct was misexpressed using the eyGal4 driver (anterior to the left) showing the transformation of an eye to the first and
second antennal segments (arrow).
Figure 6: Driving cell proliferation prevents antennal duplication and eye-to-antenna
transformation
(A) Eye-antennal disc from early third instar larva in which Dip3 was misexpressed using
the ey-Gal4 driver. The disc was double stained with antibodies against BrdU to reveal
cell proliferation and the antennal disc marker Cut (A). Lack of BrdU incorporation in
the eye disc indicates that the undergrowth of the eye disc is due to a failure of cell
proliferation rather than increased apoptosis. (B) Eye-antennal disc from early third instar
larva in which Dip3 and CycE were expressed using the ey-Gal4 driver. The disc was
double stained with antibodies against BrdU and Dac. CycE over-expression prevents the
30
Duong et al.
cell proliferation defect that normally results from Dip3 misexpression. (C) Phenotypes
of flies expressing Dip3 and coexpressing CycE, Eyg, Upd, or Nact. These factors, which
all drive cell proliferation in the eye disc, significantly reduce the severity of the Dip3
misexpression defect. n is the number of flies examined.
31